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What is NAD+? 

Nicotinamide adenine Dinucleotide is an essential molecule that participates in energy exchanges and metabolism throughout our body. It is a building block of energy exchange in a healthy body and brain. 

Why is NAD+ Important? 

NAD+ levels decline with age as well as in stressful conditions such as depression, addiction, infections, and other medical illness. This results in poor neurological and physical functioning seen in aging. Replacement of NAD+ increases clarity of mind, focus, concentration, improved energy, elevated mood, decreased anxiety, and fewer cravings.  

What does NAD + do in the Body? 

NAD+ is used in the production of energy as an electron transporter in metabolism. 

DNA Repair requires NAD+ to activate PARPs, which detect and repair damaged DNA. 

Gene Expression: Sirtuins are NAD dependent enzymes that regulate gene expression, increase metabolism, extend cell life, and prevent neurodegeneration. 

NAD+ also functions as a neurotransmitter for cell-to-cell communications in smooth muscle and brain cells. 

NAD+ and NADH are used in alcohol metabolism and metabolism of lactate. 

What is NAD Therapy? 

High dose NAD + delivered IV bypasses the liver and digestive system and enters cells to increase gene expression and help repair DNA. Treatment lasts 3 to 8 hours per session and may require multiple sessions depending on the protocol and condition. 

Conditions Treated by NAD+ Therapy: 

  • Substance Abuse 
  • Addiction – Alcoholism | Opioid Abuse Disorders 
  • Stress 
  • Depression 
  • PTSD 
  • Neurodegenerative Disorders | Anti-aging | Parkinson’s Disease 
  • Chronic fatigue 
  • Brain Fog 

Benefits of NAD + Therapy: 

  • Increased Energy 
  • Improved Mood 
  • Increased mental clarity 
  • Increased focus and concentration 
  • Reduced cravings and addiction 

                                             More About NAD+ 

NAD+ is short for Nicotinamide Adenine Dinucleotide. NAD+ is an essential coenzyme in the body that regulates cellular energy metabolism and mitochondrial function. The mitochondria are the powerhouse of the cell where energy is made and regulated. 

Mitochondrial health equals overall health and increasing NAD+ levels result in more efficient gene transcription, energy regulation, DNA repair, gene expression, and cell signaling. Improving NAD+ levels are now considered the pivotal process for improving cardiovascular health, weight management, anti-aging, cognitive function, and neuroprotection. NAD+ is the most important cellular co-factor for improvement of mitochondrial performance and energy. 

Chronic illness and diseases of aging result in depression, worsened stress, loss of health-span, and hopelessness. NAD+ therapy has been shown to be capable of dramatically reducing symptoms of these illnesses by boosting the body’s natural cell repair. In addition to chronic illness, NAD+ has been used to treat chronic stress, depression, and anxiety. 

Dr. Sinclair published a groundbreaking paper which found that increasing NAD+ levels in older mice made the mitochondria of a 2 years old mice resemble those of a 6-month-old mouse when evaluated for critical biochemical markers of muscle health. Similar results of improved markers of aging have been seen in humans infused with NAD+, in which mitochondria appear to be rejuvenated. Scientists have shown that mice fed a high fat diet and then had their levels of NAD+ increased gained 60% less weight than mice on the same high fat diet without the NAD+ levels increased. 

As organisms age, their NAD+ levels decline. Dysfunctional cellular energy metabolism in mitochondria is increasingly implicated in diseases of aging, autoimmune disease, muscle wasting, neuropathies and other conditions. NAD+ therapy seems to reverse these conditions of aging by allowing Sirtuins to access DNA transcription and repair.  

NAD+ has a favorable profile on enhanced mitochondrial functioning and protects against age related axonal degeneration. This may be part of the reason for the positive impact in the treatment of patients with Reflex Sympathetic Dystrophy, Parkinson’s Disease and Alzheimer’s Disease. 

                    What Is NAD Therapy and Why It May Work For You 

NAD+ is short for Nicotinamide Adenine Dinucleotide. NAD+ is an essential coenzyme in the body that regulates cellular energy metabolism and mitochondrial function. The mitochondria are the powerhouse of the cell where energy is made and regulated. We now understand that mitochondrial health equals overall health. Increasing NAD+ levels results in more efficient gene transcription, energy regulation, DNA repair, gene expression, and cell signaling. Improving NAD+ levels is now considered the pivotal process for improving cardiovascular health, weight management, anti-aging, cognitive function, and neuroprotection. NAD+ is the most important cellular co-factor for improvement of mitochondrial performance and energy. Chronic illness is a rampant medical condition plaguing millions. These conditions typically do not have a cure and are managed through medication and lifestyle changes. For many people, it is difficult to do the things they enjoy and often leads to depression and a feeling of hopelessness. NAD+ therapy has been shown to be capable of dramatically reducing symptoms of these illnesses by boosting the body’s natural cell repair. In addition to chronic illness, NAD+ has been used to treat chronic stress, depression, and anxiety. Long-term stress depletes our mental and physical health in tremendous ways. It impedes our sleep, digestion, healing and cognitive abilities. Chronic stress can be an underlying contributor to autoimmune diseases, heart disease, osteoarthritis, obesity, depression, anxiety and more. Many mental health conditions cannot be explained by a chemical imbalance or deficiency alone, but experts are learning that NAD+ levels do play a role. If your depression or anxiety is caused by stress, medications, or mood disorders, we believe that it may benefit you to incorporate NAD+ into your treatment plan. 

NOVA Health Recovery prescribes Nicotinamide Adenine Dinucleotide (NAD+) for intravenous administration. NAD+ treatments are performed in our outpatient infusion rooms. While each infusion session typically takes a full 3-8 hours, the duration of NAD+ treatment typically requires multiple sessions to ensure complete results. At the end of treatment, patients report a great reduction or complete absence of symptoms. There is an increase in clarity with improved focus, higher energy levels, and improvement in mood.  

NAD+ treatment is dramatically effective for drug and alcohol disorders, and we’re now finding that it can be a very effective treatment for Alzheimer’s Disease, Parkinson’s Disease, migraine headaches, PTSD, depression and RSD (Reflex Sympathetic Dystrophy). NAD+ has already shown promise as a therapy for several neurodegenerative conditions. 

As we age, our NAD+ levels decline. This leads to a decline in mitochondrial health which contributes to age related health issues. Sirtuins are a group of enzymes which play key roles in longevity. NAD+ control the access of sirtuins to transcription of genes. When the sirtuins are protected, aging slows and in some cases even seems to be reversed. 

                    Nicotinamide Adenine Dinucleotide IV Therapy (NAD) 

What does it treat? 

  • Alcohol and Opioid Detox Therapy 
  • Depression and Anxiety 
  • Parkinson’s Disease 
  • RSD (CRPS)  Reflex Sympathetic Dystrophy 
  • Migraine Headaches 
  • Alzheimer’s Disease 
  • Anti-aging therapy 
  • Low energy and fibromyalgia 
  • Brain Fog 

How is it administered? 

NAD is administered as a 3 to 8 hour infusion. Several infusions in a row may be needed to be for some conditions. Single infusions can be used for anti-aging purposes, brain fog, and maintenance therapy. 

What are the side effects during treatment? 

Primarily the main side effect is an elevated heart rate during the infusion that can easily be monitored and controlled.  

What are the outcomes? 

  • Rapid improvement from depression, anhedonia, and anxiety. 
  • Anti-aging strategy  
  • Improvement of Parkinson’s Symptoms 
  • Improvement of cognition in Alzheimer’s Disease 
  • Decreases pain in CRPS/RSD 
  • Migraine prevention 
  • Increased energy, performance, and less fibromyalgia 

                                 NAD Related Articles 

NAD+ is part of more chemical reactions in the body than any other vitamin-derived molecule.

How do your cells produce energy?

Almost every cell in your body uses nicotinamide adenine dinucleotide (NAD) to help fuel reactions that produce energy. Think of your cells passing an electron like a hot potato to NAD+ to form NADH. Cells accumulate NADH through different metabolic pathways, including glycolysis and the citric acid cycle. Cells use NADH in the electron transport chain to produce energy through adenine triphosphate (ATP).  The delicate ratio of NAD+:NADH will determine if your cell has enough energy to survive and function normally.

Think of your cells as a representation of your house. When the electricity fluctuates during a storm, or when your mitochondria produces less ATP, the lights in your house flicker and possibly go out. The dimmed lights won’t impact you personally, but in reality the lack of electricity may shut down your wifi. Or the unstable supply of energy will lower the temperature in your refrigerator causing your food to spoil. You may not notice the fluctuating levels of ATP in your mitochondria, but it will disturb the maintenance of your cells resulting in a slow decline in health.

How do you boost NAD+ levels?

Wouldn’t it be nice if we could take a pill to ensure our cells never run out of energy? Unfortunately, NAD+ through pill form is not an effective option because your body is unable to absorb it through the digestive system. The enzymes that live in the lining of your digestive tract need to break down NAD+ several times in order for it to be absorbed. Eventually nicotinamide (NAM) is the end product of NAD+ digestion, which can be directly absorbed into the body. The digestion of NAD+ is a lengthy process, and it’s not as efficient as other methods of supplementation.

You can assist your cells to produce more NAD+ by supplementing with the four major precursors:
  1. Tryptophan is an amino acid that has a bad rap for making you tired after Thanksgiving turkey dinner, but tryptophan is also required for the production of serotonin.
  2. Nicotinic acid (NA)
  3. Nicotinamide (NAM)
  4. Nicotinamide riboside (NR)

NA, NAM and NR are all different forms of Vitamin B3, and vary slightly in chemical structure. A deficiency in Vitamin B3 is famously associated with pellagraLow levels are also associated with decreased metabolism, cold intolerance, and delayed brain development.

Tryptophan, nicotinamide, nicotinic acid, and nicotinamide riboside all have pathways to NAD.

Meat and fish are the main sources of tryptophan and Vitamin B3. Other vegetarian sources of Vitamin B3 include spirulina, California avocados, sprouted kidney beans, buckwheat and asparagus. It is important to note that the cooking process will deplete some of the bioavailable Vitamin B3. Eating sprouted grains and legumes will have more nutrient density than cooked foods

Sleep, Exercise and NAD+

Every health practitioner agrees that quality sleep and moderate exercise can have beneficial effects on your health, and can help reduce your risk to certain types of diseases. There is no doubt that sleep and exercise will boost your metabolism, but did you know it has the ability to boost your NAD+ levels as well?

While you’re fast asleep dreaming, your body is actively repairing itself. At the same time your cells are producing tremendous amounts of NAD+.  And if you’re thinking about having that after dinner nightcap, you might want to think again. The metabolism of alcohol significantly reduces NAD+ because it’s used to break down ethanol. Those two glasses of wine you had with dinner will also have an impact on your quality of sleep, further depleting your ability to replenish NAD+ levels.

When you go to the gym, or go outside for a walk, your skeletal muscles use the ATP for energy. Studies have found that moderate exercise increases your mitochondrial energy and the production of NAD+. On the other hand, without sufficient amounts of NAD+ your muscles will start to deteriorate.

You don’t have to rely solely on food

Supplement companies sell various forms of B vitamins, but which supplement is the best for NAD+ production?

If you’ve ever shopped for B vitamins before, you might have felt overwhelmed by all the different types. Vitamin B3 is the main precursor to NAD+, but there are several different forms of Vitamin B3 to be aware of.  You’ve probably heard of niacin (NA or nicotinic acid), and possibly even niacinamide (NAM or nicotinamide). Both of these forms will most likely be available at your local health food store. Remember NR (nicotinamide riboside) also leads to the production of NAD+, which is bioavailable and more efficient as a precursor. Oral supplementation of NR has been proven to raise NAD+ levels to a degree. You can find various oral NR supplements online.

The best way to replenish your cells with NAD+/NADH is intravenously (IV). IV vitamin therapy bypasses your digestive system and delivers nutrients straight into the bloodstream.

There are many internal and external ways our DNA gets damaged. Some examples include spontaneous conversions, replications errors, exposure to free radicals, etc. This damage contributes to aging and puts us at high risk for developing various cancers. DNA repair is the most important factor for cell survival and cancer prevention. But unfortunately, as we age our cells become less efficient at correcting DNA damage. Currently, scientists don’t understand why DNA repair declines with age.

But we have exciting news! A recent scientific discovery reveals a promising solution to this critical issue with aging. Scientists at Harvard Medical School have found a way to reverse the accumulation of DNA damage. An article in the latest issue of Science explains how NAD+ regulates protein-protein interactions involved in DNA repair.

“We recently showed that raising NAD levels in mice can reverse aspects of aging within just one week of treatment.” said David Sinclair, Ph.D., professor of genetics at Harvard University.

The Key Players
The Science
Research reveals NAD+ has crucial role in DNA repair

This study examined how PARP1 is inhibited by DBC1 by locking together. When NAD+ levels are optimal, NAD+ is able to bind to DBC1 and prevent the PARP1-DBC1 partnership. This study found replenishing the cells with NAD+ through NMN supplementation releases PARP1, so it is able to repair DNA.

The scientists at Harvard Medical School performed experiments by putting drops of NMN into the water consumed by mice for one week. NMN is required for the synthesis of NAD+and is thought to be more effective than it’s sister molecule nicotinamide riboside (which has gained popularity over the past couple of years). Oral supplements have to battle the digestive system, resulting in limited absorption and nutrient delivery to cells. Intravenous NAD+ therapy is the most effective way to increase NAD+ levels because it bypasses the digestive tract.

Before the experiment, scientists found low levels of NAD+ in older mice and also higher PARP1-DBC1 complexes. This information suggests that PARP1 is unable to repair DNA damage. After NMN supplementation, NAD+ concentrations were higher. There was also increased activity of PARP1,  indicating NAD+ plays a critical role in PARP1 activity.

NAD+ and the framework for anti-aging

Accelerated by the publication in December 2013 of a seminal paper by David Sinclair and his US and Australian colleagues(ref), there has been increasing interest and excitement about the prospects of discovering means for offsetting age–related declines in NAD+ as a strategy for disease prevention and life extension.  Actually, similar proposals have appeared in the literature for several years.  However, for lay people and even many scientists, the scientific discussions of NAD+ are so complicated that they seem to be virtually unfathomable.  In fact, understanding NAD+ and its roles requires fathoming a large number of related actors and their roles – complex metabolic pathways, an alphabet soup collection of enzymes and gene activation cofactors, the family of Sirtuins, biological processes in different cell compartments and organs, and multiple pathological and health-producing concomitants.  The complex of related actors has come to be called the NAD World.   It is not surprising that many intelligent  people are left confused about it. {Anti-aging firewall}

These entries lay out reasons why simply enhancing NAD+ levels, say by oral consumption of nicotinamide riboside, might or might not work to increase health or longevity.  Nonetheless, my perception is that a big new area of longevity science is opening up, one that could offer new practical interventions that lead to longer healthier lives.

Haijun Shao et al. from the Department of Anesthesiology at the Rujin Hospital in the School of Medicine at Shanghai Jiao Tong University determined that the activation of a histone deacetylase diminishes neuropathic pain in mice. Neuropathic pain involves damaged nerves, which can be caused by many diseases, infections and injuries including diabetes and multiple sclerosis. This type of pain causes symptoms such as hyperalgesia (an increased sensitivity to pain), allodynia (an increased sensitivity to touch) and paresthesia (tingling). Researchers in the past have speculated that neuropathic pain stems from gene modifications, specifically abnormal histone acetylation.

Histone acetylation is a fancy way of describing relaxed DNA that is available to be transcribed. DNA is wrapped around histones, which are made of eight proteins and can also be referred to as octamer. Looking like spools of thread, the thread-like DNA circles around each histone. Gene modification can affect how close the spools of thread are to each other.  Acetylation adds an acetyl group to each histone creating more space between threads allowing transcription and gene expression to occur. Conversely, histone deacetylases remove acetyl groups and tighten the DNA coils, limiting DNA transcription. The absence of the enzyme silent information regulator 1 (SIRT1), a histone deacetylase found in the spinal cord, may be the culprit for the development of neuropathic pain.

Haijun Shao et al. conducted a study to identify the presence of SIRT1, the co-enyzme nicotinamide adenine dinucleotide (NAD), as well as the byproduct of NAD metabolism, nicotinamide (NAM), in the spinal cords of mice after chronic constriction injury or sham injury. They also tested the effects of NAD on hyperalgesia and mechanical allodynia by injecting this coenzyme into the spinal cords of the mice. The researchers additionally determined if a SIRT1 inhibitor, EX-527, could reverse the anti-nociceptive effect of NAD and resveratrol.

It was found that the amount of spinal SIRT1 expression decreased, and hyperalgesia and allodynia increased after chronic constriction injury versus the sham group, which had no change in spinal SIRT1 levels. They also found that NAD levels were decreased, whereas NAM was increased in chronic constriction injured mice compared to the sham group. This shows that chronic constriction surgery decreases SIRT1 activity and depletion of NAD in the spinal cords of mice.

Furthermore, they tested whether NAD outside of the cell could weaken neuropathic pain. NAD injections, one hour before and on the first day after injury showed brief reduction in pain for 48 hours compared to the chronic constriction injured mice treated with saline. The data suggests NAD injected into the spinal cord regulated pain through histone deacetylation with SIRT1.

In order to prove that neuropathic pain was resolved through the activation of SIRT1 by NAD, Haijun Shao et al. used a SIRT1 inhibitor, EX-527. Injecting EX-527 one hour before NAD administration blocks the pain preventing benefits of NAD. This evidence demonstrates that SIRT1 consumes the NAD producing an anesthetic effect.

The data presented by Haijun Shao et al. implicates the crucial role of NAD activated sirtuins enzymes in neuropathic pain. More research should be conducted in human models to evaluate the effectiveness of intravenous NAD for neuropathic pain.

If you are unfamiliar with mitochondrial dysfunction, you are not alone. Emerging research surrounding mitochondrial dysfunction, also referred to as mitochondrial disease, did not gain full momentum until the last few years. But, in fact, the first mitochondrial disorder was diagnosed as far back as the late 1950’s. However, a full understanding of the domino effect caused by mitochondrial dysfunction was delayed until recently due to modern advances in molecular medicine.

Mitochondrial dysfunction affects nearly every organ and system within the body, and is tied to various chronic conditions and symptoms.

“If your mitochondria stop working, even for just about six seconds, you die, because those mitochondria are that active.”
Dave Asprey, Author of Headstrong
Mithochodrial Dysfunction

Mitochondria’s “Kryptonite”

Your mitochondria are susceptible to defects and damage for various reasons. Mitochondria are structurally unique because they were once a type of bacteria that invaded the cell. But through the course of evolution and time, mitochondria have made a permanent home in our cells. Unlike nuclear DNA, often seen in the shape of an ‘x’, mitochondrial DNA is always in the shape of a ring. While this allows the DNA to be transcribed easily, it also makes it vulnerable to damage.

Chronic stress, disease, certain prescribed drugs, and opiates interfere with NAD+ metabolism, thus short circuiting the energy production of cells. This is like Superman trying to fly with kryptonite in his pocket.

Oxidative Stress

Cells rely on oxygen and nitrogen for metabolism, but sometimes these elements can cause damage in the form of reactive oxidative species (ROS) and reactive nitrogen species (RNS). Cells have natural defense mechanisms to protect against ROS and RNS damage, but sometimes these defense mechanisms can fail. Damage to cell membranes, DNA, and proteins from ROS and RNS can severely impact the vitality of the cell.

DNA Damage

Cells could not grow, divide or even survive without well preserved DNA. Key enzymes, known as sirtuins and PARPs, help repair DNA damage that has occurred from oxidative stress. These protective enzymes need NAD+ to repair the DNA damage. With excess DNA repair, mitochondria have little NAD available for energy production. Remember, NAD is used throughout biology to create energy within the cell.

Poor Nutrition

This may seem obvious, but poor nutrition has serious implications on your mitochondrial health. Mitochondria depend on vitamins and minerals to function properly. Many of our diets are insufficient in minerals, trace elements, and vitamins that are necessary for our metabolic processes.

Aluminium

This heavy metal is known to interfere with ATP production by disrupting the cytoskeleton in the cell, which is similar to the support beams in your house. Think of the mitochondria as one room within a house. Mitochondrial dysfunction is a result of poor structural support. The main sources of aluminium are found in cosmetic products, over the counter medication and some pharmaceuticals. Even some of our food and water have trace amounts of aluminium. Other toxins and heavy metals poison the body leading to disease and dysfunction.

Prescription Drugs

There is no doubt that prescription medications have saved countless lives. If it wasn’t for recent advances in medicine, we would not be able to battle infections and control blood sugar levels. Alternatively, however, some medications can significantly affect our mitochondrial health.


Therapies for Mitochondrial Dysfunction

  • Exercise. The most economical way to increase mitochondrial function is through exercise. Incorporating an exercise routine may seem like a daunting task to some, thus it is best to take small and gradual steps towards a type of exercise you find rewarding. One study evaluated the effects of exercise and observed improvements in mitochondrial function by 50%. This study evaluated mitochondrial DNA expression, energy production (electron transport chain), and membrane structure through cardiolipin.
     
  • Sleep. The number one hack to help restore energy levels and mitochondrial function is getting enough sleep. Mitochondria are replenished while you sleep at night during your circadian cycle. A key enzyme called NAMPT is regulated by sleep. Furthermore, this enzyme becomes less active by eating and sedentary lifestyle. It is important to get quality sleep and exercise regularly in order to maintain healthy mitochondria.

  • NAD+ Therapy. Nicotinamide adenine dinucleotide (NAD+) is a crucial coenzyme for energy synthesis in the mitochondria. Additionally NAD+ is also a key activator of protective enzymes, including sirtuins and PARPs, which help repair DNA and protect the cell from dying. When cellular NAD+ levels fall past a particular threshold, the mitochondria are signaled to induce apoptosis, or programmed cell death.

Research has recently shown NAD+ intravenous therapy as a promising treatment for several different neurological conditions, including CTE. Studies have shown increasing NAD+ levels can promote neurogenesis, or the growth of new brain cells, even after trauma.

In CTE, there are holes in the brain, dead areas or areas where abnormal plaques develop. A lack of NAD+ seems to promote neurons to go “off-line” in all aging brains. Even though the brain cells are present, they don’t conduct electrical impulses as efficiently and stop reaching out to other brain cells, thus resulting in impaired brain function. NAD+ encourages new connections in the brain and the connections may bypass injured or non-functional areas of the brain, resulting in increased senses and clarity of thought.

During our earliest years in life, the nervous system matures by seeking new connections and cementing new pathways. Over time, these pathways are either reinforced or are no longer utilized due to injury and/or fluctuations in energy. When NAD+ is replenished, existing brain cells seem to come back online almost immediately. 

NAD+ encourages new connections in the brain resulting in heightened senses and clarity of thought.

It is suggested that NAD+ has the ability to repair and regenerate neuronal cells through several mechanisms of action. NAD+ encourages the mitochondria (cellular power plants) to communicate with the cell nucleus. This causes the cell to kick back into function by increasing the energy and robustness, as well as increasing sirtuin production. 

A class of enzymes, known as sirtuins, have been associated with the growth and extension of new neurons. Sirtuins are also known to protect neurons from oxidative stress and can inhibit cell death. Improving brain energy metabolism through increasing available NAD+ has been shown to help degrade dysfunctional proteins associated with neurodegeneration.

A 2017 report from the Harvard Medical School Genetics laboratory found that NAD+ can help restore youthful levels of DNA repair and even reverse the effects of radiation. NAD+ activates the enzyme responsible for DNA repair, PARP1, which is compromised by low levels of NAD+ associated with aging and disease. 

Conventional Treatments for Depression

A combination of cognitive behavioral therapy and/or prescription medication is the typical treatment for depression. In patients who receive both types of therapy, 30 percent fail to achieve remission of depressive symptoms. Most antidepressant medications target neurotransmitters within the brain, including serotoninacetylcholinecortisol. and dopamine. Unfortunately, these types of medications are only effective with long term use in 50-70 percent of patients.

Nutrition Therapy for Depression

Nutritional neuroscience is an emerging field that identifies how nutrition is related to human cognition, behavior, and emotion. The most common nutritional deficiencies found in patients with mental disorders are B vitamins, omega-3 fatty acids, minerals, and amino acids. Many clinicians are starting to understand the importance of nutrition when it comes to treating depression, thus adding intravenous nutrition therapy to conventional treatment.

IV NAD+ Therapy

One effective form of nutrition therapy is intravenous nicotinamide adenine dinucleotide (IV NAD+), which is a derivative of vitamin B3. NAD+ is used as coenzyme for various cellular functions including energy production, DNA repair, regulating the immune system, and reducing inflammation. Sirtuins are only one of the enzymes that depend on NAD+ in order to be activated.

Functions of Sirtuins

Sirtuins (SIRT) are enzymes found throughout the cell. They function to reduce inflammation, inhibit protein aggregation, and reduce oxidative damage. The different roles and actions sirtuins play help protect the nervous system and brain from deterioration. Decreased levels of sirtuins have been found in subjects with mood disorders.

Research is starting to emerge on the various roles sirtuins play in depression and brain health.

  • SIRT1 activates MAO-A, an enzyme that metabolizes neurotransmitters in the brain that can mediate anxiety and depressive symptoms.
  • SIRT1 has been linked to increased expression of BDNF, thus protecting your neurons against damage.
  • SIRT1 can also promote the growth of new neurons.
  • SIRT2 can reduce inflammation in microglial cells (the cells surrounding the neurons).
  • SIRT2 can restore neurogenesis from stress induced damage.
  • SIRT3 protects neurons from oxidative stress.

INTRODUCTIONS TO THE MAIN ACTORS AND THEIR RELATIONSHIPS —  THE NAD WORLD

It was suggested back in 2009 that NAD and the molecules and pathways it dances with constitute a world for explaining metabolism and aging.

The 2009 publication The NAD world: A new systemic regulatory network for metabolism and aging-Sirt1, systemic NAD biosynthesis, and their importance related: “For the past several years, it has been demonstrated that the NAD-dependent protein deacetylase Sirt1 and nicotinamide phosphoribosyltransferase (Nampt)-mediated systemic NAD biosynthesis together play a critical role in the regulation of metabolism and possibly aging in mammals. Based on our recent studies on these two critical components, we have developed a hypothesis of a novel systemic regulatory network, named “NAD World”, for mammalian aging. Conceptually, in the NAD World, systemic NAD biosynthesis mediated by intra- and extracellular NAMPT functions as a driver that keeps up the pace of metabolism in multiple tissues/organs, and the NAD-dependent deacetylase Sirt1 serves as a universal mediator that executes metabolic effects in a tissue-dependent manner in response to changes in systemic NAD biosynthesis. This new concept of the NAD World provides important insights into a systemic regulatory mechanism that fundamentally connects metabolism and aging and also conveys the ideas of functional hierarchy and frailty for the regulation of metabolic robustness and aging in mammals.”

Here is a handy reference guide to some of the main molecular actors that will be showing up in the following discussions and their significance – a cast of main characters in the NAD World but by no means all of them.

NAD – Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells. The compound is a dinucleotide, since it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine base and the other nicotinamide. Nicotinamide adenine dinucleotide exists in two forms, an oxidized and reduced form abbreviated as NAD+ and NADH respectively.(ref)  As we will see, it is a molecule of central importance to metabolic and other key biological processes in humans.

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NAD+ – oxidized form of NAD, very important as a cofactor for health and longevity-related processes including DNA repair and mitochondrial operability as will be discussed.  The central actor in our current drama, it acts as a biological oxidizing agent.  Declines sharply with age negatively impacting on DNA repair and mitochondrial health and benefits conveyed by sirtuins.

NADH – reduced form of NAD. Acts as a biological reducing agent.  Cycled back in the body to NAD+ by what are known as the NAD salvage pathways described below.

NA  – Nicotinic acid (aka Niacin aka Vitamin B3) – Niacin –” is an organic compound with the formula C6H5NO2 and, depending on the definition used, one of the 20 to 80essential human nutrients.(ref)”  Important in the current drama as a precursor to Nicotinamide which is a precursor to NAD. Unfortunately, consuming niacin is generally not a good way to enhance body levels of NAD since its metabolite niacinamide inhibits the sirtuins.

NAM – Nicotinamide (also known as niacinamide and nicotinic amide), “is the amide of nicotinic acid (vitamin B3 / niacin). Nicotinamide is a water-soluble vitamin and is part of the vitamin B group. Nicotinic acid, also known as niacin, is converted to nicotinamide in vivo.”(ref).  It is a precursor of NAD but there is a catch to simply supplementing with it.  It inhibits the sirtuins and their health-producing properties.

NR – Nicotinamide riboside – a precursor of NAD, and is a source of Vitamin B3.   Available as a commercial dietary supplement thought to promote NAD+.  One of the possible approaches to enhancing body levels of NAD+(ref)

NAD Salvage Pathways –  “Besides assembling NAD+ de novo from simple amino acid precursors, cells also salvage preformed compounds containing nicotinamide. Although other precursors are known, the three natural compounds containing the nicotinamide ring and used in these salvage metabolic pathways are nicotinic acid (Na), nicotinamide (Nam) and nicotinamide riboside (NR).[2] These compounds can be taken up from the diet, where the mixture of nicotinic acid and nicotinamide are called vitamin B3 or niacin. However, these compounds are also produced within cells, when the nicotinamide moiety is released from NAD+ in ADP-ribose transfer reactions. Indeed, the enzymes involved in these salvage pathways appear to be concentrated in the cell nucleus, which may compensate for the high level of reactions that consume NAD+ in this organelle.[26]  Cells can also take up extracellular NAD+ from their surroundings[27](ref).”

blogp2

Image and legend source is the 2002 publication Manipulation of a Nuclear NAD+ Salvage Pathway Delays Aging without Altering Steady-state NAD+ Levels:   ” Model for life span extension via increased flux through the NAD+ salvage pathway. – Type III histone deacetylases such as Sir2 and Hst1–4 catalyze a key step in the salvage pathway by converting NAD+ to nicotinamide. Additional copies of PNC1, NPT1, NMA1, and NMA2 increase flux through the NAD+salvage pathway, which stimulates Sir2 activity and increases life span. Additional copies of QNS1 fail to increase silencing. Unlike other steps in the pathway, its substrate cannot be supplied from a source outside the salvage pathway and is therefore limiting for the reaction. Abbreviations: NAD +, nicotinamide adenine dinucleotide; NaMN, nicotinic acid mononucleotide; NaAD, desamido-NAD+(ref).”  A number of important matters are not shown in this simplified diagram, such as ATP (required as an energy source in the lower loop to make NAD), the key roles of SIRT1, and CD38 which eats up NAD+.

NADPH – is the reduced form of NADP+. “NADPH provides the reducing equivalents for biosynthetic reactions and the oxidation-reduction involved in protecting against the toxicity of ROS (reactive oxygen species), allowing the regeneration of GSH (reduced glutathione).[3] NADPH is also used for anabolic pathways, such as lipid synthesis, cholesterol synthesis, and fatty acid chain elongation. — The NADPH system is also responsible for generating free radicals in immune cells. These radicals are used to destroy pathogens in a process termed the respiratory burst.[4] It is the source of reducing equivalents for cytochrome P450 hydroxylation of aromatic compoundssteroidsalcohols, and drugs(ref).”

NAADP – Nicotinic acid adenine dinucleotide phosphate “(NAADP) is one of the most potent stimulators of intracellular Ca2+ release known to date. The role of the NAADP system in physiological processes is being extensively investigated at the present time. Exciting new discoveries in the last 5 years suggest that the NAADP-regulated system may have a significant role in intracellular Ca2+ signaling. The NAADP receptor and its associated Ca2+ pool have been hypothesized to be important in several physiological processes including fertilization, T cell activation, and pancreatic secretion(ref).”

NADPH oxidase – “Nicotinamide adenine dinucleotide phosphate-oxidase is a membrane-bound enzyme complex. It can be found in the plasma membrane as well as in the membranes of phagosomes used by neutrophil white blood cells to engulf microorganisms. — NADPH oxidase generates superoxide by transferring electrons from NADPH inside the cell across the membrane and coupling these to molecular oxygen to produce superoxide anion, a reactive free-radical. Superoxide can be produced in phagosomes, which contain ingested bacteria and fungi, or it can be produced outside of the cell. In a phagosome, superoxide can spontaneously form hydrogen peroxide that will undergo further reactions to generate reactive oxygen species (ROS)(ref).”  Plays important roles in generating ROS to deal with pathogens.

NAMPT – “Nicotinamide phosphoribosyltransferase (NAmPRTase or NAMPT) also known as pre-B-cell colony-enhancing factor 1 (PBEF1) or visfatin is an enzyme that in humans is encoded by the PBEF1 gene.[1] This protein has also been reported to be a cytokine (PBEF) that promotes B cell maturation and inhibits neutrophil apoptosis. — This enzyme participates in nicotinate and nicotinamide metabolism.(ref)”  NAMPT is the rate-limiting enzyme of the NAD(+) salvage pathway and enhances SIRT1 activity by increasing the amount of NAD+.

NMNAT – Nicotinamide/nicotinic acid mononucleotide adenylyltransferase  An enzyme that plays a role in NAD synthesis. “–  a rate-limiting enzyme present in all organisms, reversibly catalyzes the important step in the biosynthesis of NAD from ATP and NMN. NAD and NADP are used reversibly in anabolic and catabolic reactions(ref).”

SIRTUINS (Silent Information Regulators  SIRT1 – SIRT7)  Sirtuins extend the lifespans of lower ife forms including yeast, nematodes and drosphila, and appear to have pluripotent health effects in humans.  “Sirtuin or Sir2 proteins are a class of proteins that possess either mono-ADP-ribosyltransferase, or deacylase activity, including deacetylase, desuccinylase, demalonylase, demyristoylase and depalmitoylase activity.[2][3][4][5] Sirtuins regulate important biological pathways in bacteriaarchaea and eukaryotes. The name Sir2 comes from the yeast gene ‘silent mating-type information regulation 2’,[6] the gene responsible for cellular regulation in yeast. Sirtuins have been implicated in influencing a wide range of cellular processes like agingtranscriptionapoptosis, inflammation[7] and stress resistance, as well as energy efficiency and alertness during low-calorie situations.[8] Sirtuins can also control circadian clocks and mitochondrial biogenesis(ref).”  Sirtuins convert NAD(+) into nicotinamide (NAM).  The sirtuins vary in their functions and in general serve as deacetylases. We have written about them a number of times in this blog(ref), and we will comment further here on some of their specific functions.  SIRT1, in particular, the mammalian counterpart of SIR2, both depends on the presence of NAD+ for its activation and has important regulatory functions in the NAD salvage pathway.  “SIRT1 is required to regulate adaptive responses to acute and chronic energy limitations, such as fasting and dietary restriction.  In general, the evolutionary role of SIR2 ones is thought to be that they provide mechanisms through which an organism can adopt to changes in environment and diet. An example of the possible role of SIRT6 could be to allow animals to switch between vegetarian and meat diets. Sirtuins are completely dependent on the availability of NAD+ for their formation and thus are central actor’s in the NAD World. An important negative consequence of insufficient levels of NAD+ is insufficient levels of sirtuins and compromise of their important biological activities.”  A table showing the deacetylase and deacelase activities of the seven sirtuin sisters can be found on this site.

CCAR2 (Cell Cycle And Apoptosis Regulator 2, also known as DBC1) An important negative regulator of SIRT1 (ref) and a consumer of NAD+.

CD38  – CD38 (cluster of differentiation 38), also known as cyclic ADP ribose hydrolase is a glycoprotein[1] found on the surface of many immune cells (white blood cells), including CD4+CD8+B lymphocytes and natural killer cells. — CD38 is a multifunctional ectoenzyme that catalyzes the synthesis and hydrolysis of cyclic ADP-ribose (cADPR) from NAD+ to ADP-ribose. These reaction products are essential for the regulation of intracellular Ca2+[5] (ref).”  CD38 can be a major consumer of NAD as a substrate.

MNA – Methylnicotinamide –  ” Methylnicotinamide is a metabolite of nicotinamide and is produced primarily in the liver. It has anti-inflammatory properties (PMID 16197374 ). It is a product of nicotinamide N-methyltransferase [EC 2.1.1.1] in the pathway of nicotinate and nicotinamide metabolism (KEGG). 1-Methylnicotinamide may be an endogenous activator of prostacyclin production and thus may regulate thrombotic as well as inflammatory processes in the cardiovascular system(ref).”

PARP – “Poly (ADP-ribose) polymerase (PARP) is a family of proteins involved in a number of cellular processes involving mainly DNA repair and programmed cell death. — The PARP family comprises 17 members (10 putative). They have all very different structures and functions in the cell.– PARP is found in the cell’s nucleus. The main role is to detect and signal single-strand DNA breaks (SSB) to the enzymatic machinery involved in the SSB repair. PARP activation is an immediate cellular response to metabolic, chemical, or radiation-induced DNA SSB damage. Once PARP detects a SSB, it binds to the DNA, and, after a structural change, begins the synthesis of a poly (ADP-ribose) chain (PAR) as a signal for the other DNA-repairing enzymes such as DNA ligase III (LigIII), DNA polymerase beta (polβ), and scaffolding proteins such as X-ray cross-complementing gene 1 (XRCC1). After repairing, the PAR chains are degraded via Poly(ADP-ribose) glycohydrolase(PARG).[1]  — It is interesting to note that NAD+ is required as substrate for generating ADP-ribose monomers. The overactivation of PARP may deplete the stores of cellular NAD+ and induce a progressive ATP depletion and necrotic cell death, since glucose oxidation is inhibited. In this regard, PARP is inactivated by caspase-3 cleavage (in a specific domain of the enzyme) during programmed cell death. — PARP enzymes are essential in a number of cellular functions,[2] including expression of inflammatory genes:[3] PARP1 is required for the induction of ICAM-1 gene expression by smooth muscle cells, in response to TNF[4] (ref).”

ATP – ” Adenosine triphosphate (ATP) is a nucleoside triphosphate used in cells as a coenzyme, often called the “molecular unit of currency” of intracellular energy transfer.[1]  — ATP transports chemical energy within cells for metabolism(ref).”

ADP – Adenosine diphosphate, abbreviated ADP, is an important organic compound in metabolism and is essential to the flow of energy in living cells. — The cleavage of a phosphate group from ATP results in the coupling of energy to metabolic reactions and a by-product, a molecule of ADP.[1] Being the “molecular unit of currency”, ATP is continually being formed from lower-energy molecules of ADP and AMP(ref).”.

AMP – Adenosine monophosphate,  NAD can be synthesized from it(ref),

“AMP, ADP, ATP – ATP consists of three phosphate groups attached in series to the 5’ carbon location, whereas ADP contains two phosphate groups attached to the 5’ position, and AMP contains only one phosphate group attached at the 5’ position. Energy transfer used by all living things is a result of dephosphorylation of ATP by enzymes known as ATPases. The cleavage of a phosphate group from ATP results in the coupling of energy to metabolic reactions and a by-product, a molecule of ADP.[1] Being the “molecular unit of currency”, ATP is continually being formed from lower-energy molecules of ADP and AMP. The biosynthesis of ATP is achieved throughout processes such as substrate-level phosphorylationoxidative phosphorylation, and photophosphorylation, all of which facilitating the addition of a phosphate group to an ADP molecule.”(ref)

AMPK – ” AMP-activated protein kinase (AMPK) plays a key role as a master regulator of cellular energy homeostasis. The kinase is activated in response to stresses that deplete cellular ATP supplies such as low glucose, hypoxia, ischemia, and heat shock(ref).”

cAMP – “AMP can also exist as a cyclic structure known as cyclic AMP (or cAMP). Within certain cells the enzyme adenylate cyclase makes cAMP from ATP, and typically this reaction is regulated by hormones such as adrenaline or glucagon. cAMP plays an important role in intracellular signaling. (ref)

CREB – cAMP response element-binding protein[1] – is a cellular transcription factor. It binds to certain DNA sequences calledcAMP response elements (CRE), thereby increasing or decreasing the transcription of the downstream genes.[2] CREB was first described in 1987 as a cAMP-responsive transcription factor regulating the somatostatin gene[3] (ref)”

PGC1 alpha  – “PGC-1-alpha (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha)  “is a protein that in humans is encoded by thePPARGC1A gene.[1] The protein encoded by this gene is a transcriptional coactivator that regulates the genes involved in energy metabolism. This protein interacts with the nuclear receptor PPAR-gamma, which permits the interaction of this protein with multiple transcription factors. This protein can interact with, and regulate the activities of, cAMP response element binding protein (CREB) and nuclear respiratory factors (NRFs). It provides a direct link between external physiological stimuli and the regulation of mitochondrial biogenesis, and is a major factor that regulates muscle fiber type determination. This protein may be also involved in controlling blood pressure, regulating cellular cholesterol homoeostasis, and the development of obesity[2] (ref).”  We have discussed PGC-1-alpha several times in this blog (ref).

TOP LEVEL POINTS

My approach in this blog entry will be top-down, starting with general points that establish the importance of the topic to health and longevity – and finishing with some of the more-technical detail that conveys so much richness to the topic. Here is a list of top-level points that will be detailed in the following citations:

  1.  NAD levels decline precipitously with aging in mammals and humans, and are strongly negatively affected by multiple disease and stress processes including obesity. At advanced ages these levels are a tiny fraction of what they are in young people.
  2. Maintaining a high level of constitutive NAD+ in the body is critical for maintaining health and functionality, for averting or reversing a number of deleterious disease phenomena, for energy and vitality, and possibly for extending life spans in humans.
  3. NAD+ is a key substrate for the production on SIRT1 and other sirtuins needed for proper histone deacetylation and gene regulation in response to changing conditions. Its availability is also critical for preventing a state of pseudohypoxia in cell nuclii and proper generation of mitochondrial proteins required for efficient electron transfer chain operation.
  4. Inadequate levels of NAD can negatively impact on DNA repair, runaway inflammation, normal metabolism, responses to oxidative stress, mitochondrial health and electron transfer chain efficiency in mitichondria, and lead to the Warburg Effect of pathological metabolism.
  5. NAD+ is needed for responsiveness to oxidative stresses and for control of inflammatory processes
  6. NAD+ deficiency thus impairs a. Proper DNA damage repair, b. Production of sirtuins and processes that depend on sirtuins for homeostasis and health, c. Control of gene activation, d. Mitochondrial functionality and viability, e. Capacity to deal with injuries and pathological situations, and f. Ability to control excess inflammation.
  7. The rate of DNA damage increases significantly with aging, increasing demand for NAD+ for DNA repair. However in general, the reasons for age-related decline in NAD are not well understood.
  8. As a consequence of the above, inadequate levels of NAD can lead to a laundry list of disease processes, including type 2 diabetes, Alzheimer’s disease, and cancer. And to acceleration of aging.  Indeed, I venture an opinion that there is an unvirtuous cyclic process of interaction of the NAD-related feedback loops that manifests itself in the progressive acceleration of the processes of aging with aging itself – why we generally age faster and faster as we get older.
  9. There is a great deal of research knowledge about what generates NAD, what runs it down, its relationship to metabolism and other metabolic factors, the biological processes affecting it or affected by it, and how it’s expression relates to multiple disease processes.
  10. It has long been known that many deleterious disease phenomena can be prevented or reversed by promoting higher levels of NAD in animal models. There appears to be ample evidence that this approach is generally safe. 
  11.  For a number of years it has been proposed in research publications that promoting NAD levels in humans could be an effective preventative and/or therapeutic strategy.for multiple disease processes  Also, it is a proven strategy for extending the lifespans of c-elegans worms. However, this strategy has never entered our mainline medical or health maintenance system.
  12. Multiple strategies have been investigated for enhancing human levels of NAD, including enhancing its original synthesis in the body, downregulating biological processes that consume it, ingesting precursor molecules such as NMN or NR, and direct introduction of NAD into the bloodstream via IV.  At present, we do not know which of these approaches will be most efficacious and cost-effective. 

I will amplify on these points by citing selections from many relevant publications that describe aspects of the NAD World.  After this, I go on to second-level of selected key points that relate to the biological mechanisms involved – for example, the key systematic roles that NAD plays in regulating metabolism and aging,  and how NAD is consumed as an important substrate for DNA repair processes. Topics 11 and 12 above will mainly be discussed in further blog entries in this series.

RESEARCH RESULTS

The 2013 publication The NAD+/sirtuin pathway modulates longevity through activation of mitochondrial UPR and FOXO signaling reports: “NAD+ is an important co-factor regulating metabolic homeostasis and a rate-limiting substrate for sirtuin deacylase. We show that NAD+ levels are reduced in aged mice and C. elegans and that decreasing NAD+ levels results in a further reduction in worm lifespan. Conversely, genetic or pharmacological restoration of NAD+ prevents age-associated metabolic decline and promotes longevity in worms. These effects are dependent upon the protein deacetylase sir-2.1 and involve the induction of mitonuclear protein imbalance as well as activation of stress signaling via the mitochondrial unfolded protein response (UPRmt) and the nuclear translocation and activation of FOXO transcription factor DAF-16. Our data suggest that augmenting mitochondrial stress signaling through the modulation of NAD+ levels may be a target to improve mitochondrial function and prevent or treat age-associated decline. — Alterations in NAD+ levels have a powerful metabolic impact, since it serves as an obligatory substrate for the deacetylase activity of the sirtuin proteins (Guarente, 2008Haigis and Sinclair, 2010Houtkooper et al., 2010a). The best-characterized mammalian sirtuin is SIRT1, which controls mitochondrial function through the deacetylation of targets that include PGC-1α and FOXO (Chalkiadaki and Guarente, 2012Houtkooper et al., 2012). The administration of NAD+ precursors, such as nicotinamide mononucleotide (Yoshino et al., 2011) or nicotinamide riboside (NR) (Canto et al., 2012), has proven to be an efficient way to increase NAD+ levels and SIRT1 activity, improving metabolic homeostasis in mice. Furthermore, the NAD+-consuming poly(ADP-ribose) polymerase proteins—with PARP1 and PARP2 representing the main PARP activities in mammals—were classically described as DNA repair proteins (Gibson and Kraus, 2012Schreiber et al., 2006), but recent studies have linked these proteins to metabolism (Asher et al., 2010Bai et al., 2011aBai et al., 2011bErener et al., 2012). Indeed, genetic or pharmacological inactivation of PARP1 increased tissue NAD+ levels and activated mitochondrial metabolism (Bai et al., 2011b). An association between PARPs and lifespan has been postulated (Grube and Burkle, 1992Mangerich et al., 2010), but a causal role remained unclear. A final line of evidence in support of a role for NAD+ in metabolic control came from the deletion of an alternative NAD+-consuming protein, CD38, which also led to NAD+ accumulation and subsequent SIRT1 activation in mice, and proved protective against high-fat diet-induced obesity (Barbosa et al., 2007). — Considering the intimate link between metabolism and longevity (Guarente, 2008Houtkooper et al., 2010b), we hypothesized that increasing NAD+ levels may be sufficient to increase mitochondrial activity and extend lifespan (Houtkooper and Auwerx, 2012). Here we show how supplementation of PARP inhibitors or NAD+ precursors led to improved mitochondrial homeostasis through the activation of the worm sirtuin homolog, sir-2.1” 

Because the NAD World pathways are evolutionarily conserved in mammals and humans, there is reason to believe these mechanisms apply to us humans as well.

The 2013 publication The importance of NAMPT/NAD/SIRT1 in the systemic regulation of metabolism and ageing reports: “Ageing is associated with a variety of pathophysiological changes, including development of insulin resistance, progressive decline in β-cell function and chronic inflammation, all of which affect metabolic homeostasis in response to nutritional and environmental stimuli. SIRT1, the mammalian nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase, and nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting NAD biosynthetic enzyme, together comprise a novel systemic regulatory network, named the ‘NAD World’, that orchestrates physiological responses to internal and external perturbations and maintains the robustness of the physiological system in mammals. In the past decade, an accumulating body of evidence has demonstrated that SIRT1 and NAMPT, two essential components in the NAD World, play a critical role in regulating insulin sensitivity and insulin secretion throughout the body. In this article, we will summarize the physiological significance of SIRT1 and NAMPT-mediated NAD biosynthesis in metabolic regulation and discuss the ideas of functional hierarchy and frailty in determining the robustness of the system. We will also discuss the potential of key NAD intermediates as effective nutriceuticals for the prevention and the treatment of age-associated metabolic complications, such as type 2 diabetes.”

As mentioned above, sirtuins depend on the availability of NAD+ as a substrate and some of the negative consequences of insufficient NAD+ are associated with surtuin insufficiency.  Likewise, there is evidence that high expression of sirtuins associated with high levels of NAD+, SIRT1 and the others as well, convey a variety of longevity benefits.

One way this shows up is slowing or averting stem cell senescence.  The 2014 publication SIRT1 ameliorates age-related senescence of mesenchymal stem cells via modulating telomere shelterin reports: “Mesenchymal stem cells (MSCs) senescence is an age-related process that impairs the capacity for tissue repair and compromises the clinical use of autologous MSCs for tissue regeneration. Here, we describe the effects of SIRT1, a NAD(+)-dependent deacetylase, on age-related MSCs senescence. Knockdown of SIRT1 in young MSCs induced cellular senescence and inhibited cell proliferation whereas overexpression of SIRT1 in aged MSCs reversed the senescence phenotype and stimulated cell proliferation. These results suggest that SIRT1 plays a key role in modulating age-induced MSCs senescence. Aging-related proteins, P16 and P21 may be downstream effectors of the SIRT1-mediated anti-aging effects. SIRT1 protected MSCs from age-related DNA damage, induced telomerase reverse transcriptase (TERT) expression and enhanced telomerase activity but did not affect telomere length. SIRT1 positively regulated the expression of tripeptidyl peptidase 1 (TPP1), a component of the shelterin pathway that protects chromosome ends from DNA damage. Together, the results demonstrate that SIRT1 quenches age-related MSCs senescence by mechanisms that include enhanced TPP1 expression, increased telomerase activity and reduced DNA damage.”

The 2011 publication Dissecting systemic control of metabolism and aging in the NAD World relates “Accumulating bodies of evidence have suggested that SIRT1 plays an important role in retarding age-associated pathophysiological changes and preventing from diseases of aging, such as type 2 diabetes, Alzheimer’s disease, and cancer. Whole-body SIRT1-overexpressing transgenic mice show significant protection from the adverse effects of high-fat diet or aging on glucose metabolism [9] and [10]. SIRT1-activating compounds are also able to improve glucose homeostasis and insulin sensitivity in diet-induced and genetic type 2 diabetes animal models [9] and [10]. ”

From the same publication:  “It has recently been demonstrated that SIRT1 prevents two critical pathological aspects of Alzheimer’s disease: Aβ amyloid deposition and tauopathy. SIRT1 decreases the production of Aβ amyloid by deacetylating the retinoic acid receptor β and thereby up-regulating ADAM10, a major component of α-secretase [19]. SIRT1 also promotes degradation of phosphorylated tau by deacetylating it and prevents tau-mediated neurodegeneration [20]. Furthermore, SIRT1 regulates memory and synaptic plasticity, providing insight into potential intervention against age-associated cognitive disorders [21] and [22]. It has also been reported that SIRT1 transgenic mice show a lower incidence of spontaneous carcinomas and sarcomas and a reduced susceptibility to high-fat diet/carcinogen-induced liver tumors, compared to wild-type control mice [23]. These findings provide strong support for the importance of SIRT1 in the prevention of major age-associated diseases.”

The 2011 publication  NAD+ treatment decreases tumor cell survival by inducing oxidative stress reports: “NAD+ plays important roles in various biological processes. It has been shown that NAD+ treatment can decrease genotoxic agent-induced death of primary neuronal and astrocyte cultures, and NAD+ administration can reduce ischemic brain damage. However, the effects of NAD+ treatment on tumor cell survival are unknown. In this study we found that treatment of NAD+ at concentrations from 10 micromolar to 1 mM can significantly decrease the survival of various types of tumor cells such as C6 glioma cells. In contrast, NAD+ treatment did not impair the survival of primary astrocyte cultures. Our study has also indicated that oxidative stress mediates the effects of NAD+ on the survival of tumor cells, and P2X7 receptors and altered calcium homeostasis are involved in the effects of NAD+ on the cell survival. Collectively, our study has provided the first evidence that NAD+ treatment can decrease the survival of tumor cells by such mechanisms as inducing oxidative stress. Because NAD+ treatment can selectively decrease the survival of tumor cells, NAD+ may become a novel agent for treating cancer.”

The 2012 publication Age-associated changes in oxidative stress and NAD+ metabolism in human tissue reports: “Nicotinamide adenine dinucleotide (NAD(+)) is an essential electron transporter in mitochondrial respiration and oxidative phosphorylation. In genomic DNA, NAD(+) also represents the sole substrate for the nuclear repair enzyme, poly(ADP-ribose) polymerase (PARP) and the sirtuin family of NAD-dependent histone deacetylases. Age associated increases in oxidative nuclear damage have been associated with PARP-mediated NAD(+) depletion and loss of SIRT1 activity in rodents. In this study, we further investigated whether these same associations were present in aging human tissue. Human pelvic skin samples were obtained from consenting patients aged between 15-77 and newborn babies (0-1 year old) (n = 49) previously scheduled for an unrelated surgical procedure. DNA damage correlated strongly with age in both males (p = 0.029; r = 0.490) and females (p = 0.003; r = 0.600) whereas lipid oxidation (MDA) levels increased with age in males (p = 0.004; r = 0.623) but not females (p = 0.3734; r = 0.200). PARP activity significantly increased with age in males (p<0.0001; r = 0.768) and inversely correlated with tissue NAD(+) levels (p = 0.0003; r = -0.639). These associations were less evident in females. A strong negative correlation was observed between NAD(+) levels and age in both males (p = 0.001; r = -0.706) and females (p = 0.01; r = -0.537). SIRT1 activity also negatively correlated with age in males (p = 0.007; r = -0.612) but not in females. Strong positive correlations were also observed between lipid peroxidation and DNA damage (p<0.0001; r = 0.4962), and PARP activity and NAD(+) levels (p = 0.0213; r = 0.5241) in post pubescent males. This study provides quantitative evidence in support of the hypothesis that hyperactivation of PARP due to an accumulation of oxidative damage to DNA during aging may be responsible for increased NAD(+) catabolism in human tissue. The resulting NAD(+) depletion may play a major role in the aging process, by limiting energy production, DNA repair and genomic signalling.”

What eats up and depletes NAD?

In the normal operation of the NAD salvage cycle, NAD is conserved, shuttled back and forth between its two forms NAD+ and NADH.  In this process, NAD acts as a cofactor in a major metabolic cycle.  In other vital processes, however, NAD acts as a substrate and is consumed. It is quite possibly the case that age-related decline in levels of NAD are mainly associated with increased demand by those substrate processes with aging. Here I discuss consumption of NAD by 1. PARPS, 2. Production of sirtuins, and 3. CD38  I also mention 4. CCAR2, a negative regulator of SIRT1 which can nullify health benefits of the availability of NAD+.

1.  PARPS are major consumers of NAD+, using it for DNA repair.

Probably they are the largest consumer of NAD+, especially in older people where the need for DNA repair accelerates with age.  As a matter of fact, one strategy for increasing levels of NAD in mice is to inhibit PARPs,  Although this strategy seems to increase lifespans of mice by making more NAD available to mitochondria, I suspect it is a shortsighted approach when it comes to human health and longevity,  ” Nuclear poly(ADP ribose) polymerase 1 (1) generates polyADP ribose and probably accounts for the majority of nuclear NAD+ degradation. Direct interaction with NMNAT1 facilitates NAD+ supply to activated PARP1.(ref)”

The 2011 publication PARP-1 inhibition increases mitochondrial metabolism through SIRT1 activation reports: “SIRT1 regulates energy homeostasis by controlling the acetylation status and activity of a number of enzymes and transcriptional regulators. The fact that NAD(+) levels control SIRT1 activity confers a hypothetical basis for the design of new strategies to activate SIRT1 by increasing NAD(+) availability. Here we show that the deletion of the poly(ADP-ribose) polymerase-1 (PARP-1) gene, encoding a major NAD(+)-consuming enzyme, increases NAD(+) content and SIRT1 activity in brown adipose tissue and muscle. PARP-1(-/-) mice phenocopied many aspects of SIRT1 activation, such as a higher mitochondrial content, increased energy expenditure, and protection against metabolic disease. Also, the pharmacologic inhibition of PARP in vitro and in vivo increased NAD(+) content and SIRT1 activity and enhanced oxidative metabolism. These data show how PARP-1 inhibition has strong metabolic implications through the modulation of SIRT1 activity, a property that could be useful in the management not only of metabolic diseases, but also of cancer.”

2.  Sirtuins also draw down on NAD+ as a substrate, although the exact mechanisms of sirtuin activation are not clear to me.

From the 2011 publication Mammalian Sirtuins and Energy Metabolism “Despite diverse subcellular localizations and a broad range of substrate specificities, the activity of all sirtuins is directly controlled by cellular NADlevels, which is an indicator of cellular metabolic status. The activity of these enzymes is also inhibited by their common enzymatic product, nicotinamide (19), and possibly by NADH (20). It is therefore not surprising that the activity of sirtuins changes in response to environmental cues that impact cellular metabolic state.”

The 2011 publication Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats reports: “The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats.  Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio in all organs by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These changes in [NAD(H)] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in total antioxidant capacity in these organs.  An age dependent increase in DNA damage (phosphorylated H2AX) was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein.  Reduced mitochondrial activity of complex I-IV was also observed in aging animals, impacting both redox status and ATP production.  The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor.

The 2012 publication The NAD+-dependent protein deacetylase activity of SIRT1 is regulated by its oligomeric status reports “SIRT1, a NAD+-dependent protein deacetylase, is an important regulator in cellular stress response and energy metabolism. While the list of SIRT1 substrates is growing, how the activity of SIRT1 is regulated remains unclear. We have previously reported that SIRT1 is activated by phosphorylation at a conserved Thr522 residue in response to environmental stress. Here we demonstrate that phosphorylation of Thr522 activates SIRT1 through modulation of its oligomeric status. We provide evidence that nonphosphorylated SIRT1 protein is aggregation-prone in vitro and in cultured cells. Conversely, phosphorylated SIRT1 protein is largely in the monomeric state and more active. Our findings reveal a novel mechanism for environmental regulation of SIRT1 activity, which may have important implications in understanding the molecular mechanism of stress response, cell survival, and aging.”

3.  CD38 is a key enzyme sitting on the outside of the cell surface that is a major consumer of NAD that plays an important role in cell calcium homeostasis

From Mouse embryonic fibroblasts from CD38 knockout mice are resistant to oxidative stresses through inhibition of reactive oxygen species production and Ca(2+) overload: “CD38 is a multifunctional enzyme that has both ADP-ribosyl cyclase and cADPR hydrolase activities, being capable of cleaving NAD(+) to cyclic ADP ribose (cADPR) and hydrolyzing cADPR to ADPR. It has been reported that there is markedly a reduction of cADPR and elevation of NAD in many tissues from CD38 knockout (CD38(-/-)) mice. Cyclic ADPR is a potent second messenger for intracellular Ca(2+) mobilization, and NAD is a key cellular metabolite for cellular energetic and a crucial regulator for multiple signaling pathways in cells. We hypothesize that CD38 knockout may have a protective effect in oxidative stresses through elevating NAD and decreasing cADPR. In the present study, we observed that the mouse embryonic fibroblasts (MEFs) from CD38(-/-) mice were significantly resistant to oxidative stress such as H(2)O(2) injury and hypoxia/reoxygenation compared with wild type MEFs (WT MEFs). We further found that production of reactive oxygen species (ROS) and concentrations of intracellular Ca(2+) ([Ca(2+)](i)) in CD38(-/-) MEFs were markedly reduced compared with WT MEFs during hypoxia/reoxygenation. Coincidence with these results, a remarkably lower mRNA level of Nox1, one of the enzymes responsible for ROS generation, was observed in CD38(-/-) MEFs. Furthermore, we found that transcription of Nox1 mRNA in WT MEFs could be elevated by calcium ionophore ionomycin in a dose-dependent manner, indicating that the expression of Nox1 mRNA can be regulated by elevation of intracellular [Ca(2+)]. Therefore we concluded that CD38(-/-) MEFs are resistant to oxidative stresses through inhibiting intracellular Ca(2+) overload and ROS production which may be regulated by Ca(2+)-mediated inhibition of Nox1 expression. Our data should provide an insight for elucidating the roles of CD38 in oxidative stresses and a novel perspective of dealing with the ischemia/reperfusion-related diseases.”

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Image and legend source    “CD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca2+ signalling molecules and triggers proliferation and immune responses in lymphocytes —  These results indicate that CD38 has a key role in neuropeptide release, thereby critically regulating maternal and social behaviours, and may be an element in neurodevelopmental disorders.”

NAD+ can be pumped out of cells to make CD38

The 2011 publication Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport reports:  “The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. —  Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.”

CD38 and its NAD+ and SIRT1 reducing effects can be inhibited by the phytosubstances quercetin and apigenin

The 2013 pubication Flavonoid apigenin is an inhibitor of the NAD+ ase CD38: implications for cellular NAD+ metabolism, protein acetylation, and treatment of metabolic syndrome reports: “Metabolic syndrome is a growing health problem worldwide. It is therefore imperative to develop  new strategies to treat this pathology. In the past years, the manipulation of NAD(+) metabolism has emerged as a plausible strategy to ameliorate metabolic syndrome. In particular, an increase in cellular NAD(+) levels has beneficial effects, likely because of the activation of sirtuins. Previously, we reported that CD38 is the primary NAD(+)ase in mammals. Moreover, CD38 knockout mice have higher NAD(+) levels and are protected against obesity and metabolic syndrome. Here, we show that CD38 regulates global protein acetylation through changes in NAD(+) levels and sirtuin activity.  In addition, we characterize two CD38 inhibitors: quercetin and apigenin. We show that pharmacological inhibition of CD38 results in higher intracellular NAD(+) levels and that treatment of cell cultures with apigenin decreases global acetylation as well as the acetylation of p53 and RelA-p65. Finally, apigenin administration to obese mice increases NAD(+) levels, decreases global protein acetylation, and improves several aspects of glucose and lipid homeostasis. Our results show that CD38 is a novel pharmacological target to treat metabolic diseases via NAD(+)-dependent pathways.”

 4.  CCAR2  can be an important negative regulator of SIRT1

CCAR2 was until very recently called DBC1 after what was originally thought to be its main property (deleted in breast cabcer), so most of the research literature relating to it talks only about DBC1.

The 2012 publication Role of deleted in breast cancer 1 (DBC1) protein in SIRT1 deacetylase activation induced by protein kinase A and AMP-activated protein kinase  reports: “The NAD(+)-dependent deacetylase SIRT1 is a key regulator of several aspects of metabolism and aging. SIRT1 activation is beneficial for several human diseases, including metabolic syndrome, diabetes, obesity, liver steatosis, and Alzheimer disease. We have recently shown that the protein deleted in breast cancer 1 (DBC1) is a key regulator of SIRT1 activity in vivo. Furthermore, SIRT1 and DBC1 form a dynamic complex that is regulated by the energetic state of the organism. Understanding how the interaction between SIRT1 and DBC1 is regulated is therefore essential to design strategies aimed to activate SIRT1. Here, we investigated which pathways can lead to the dissociation of SIRT1 and DBC1 and consequently to SIRT1 activation. We observed that PKA activation leads to a fast and transient activation of SIRT1 that isDBC1-dependent. In fact, an increase in cAMP/PKA activity resulted in the dissociation of SIRT1 and DBC1 in an AMP-activated protein kinase (AMPK)-dependent manner. Pharmacological AMPK activation led to SIRT1 activation by a DBC1-dependent mechanism. Indeed, we found that AMPK activators promote SIRT1-DBC1 dissociation in cells, resulting in an increase in SIRT1 activity. In addition, we observed that the SIRT1 activation promoted by PKA and AMPK occurs without changes in the intracellular levels of NAD(+). We propose that PKA and AMPK can acutely activate SIRT1 by inducing dissociation of SIRT1 from its endogenous inhibitor DBC1. Our experiments provide new insight on the in vivo mechanism of SIRT1 regulation and a new avenue for the development of pharmacological SIRT1 activators targeted at the dissociation of the SIRT1-DBC1 complex.”

The 2008 publication DBC1 is a negative regulator of SIRT1  reports: “The NAD-dependent protein deacetylase Sir2 (silent information regulator 2) regulates lifespan in several organisms1, 2, 3. SIRT1, the mammalian orthologue of yeast Sir2, participates in various cellular functions4, 5, 6, 7 and possibly tumorigenesis8. Whereas the cellular functions of SIRT1 have been extensively investigated, less is known about the regulation of SIRT1 activity. Here we show that Deleted in Breast Cancer-1 (DBC1), initially cloned from a region (8p21) homozygously deleted in breast cancers9, forms a stable complex with SIRT1. DBC1 directly interacts with SIRT1 and inhibits SIRT1 activity in vitro andin vivo. Downregulation of DBC1 expression potentiates SIRT1-dependent inhibition of apoptosis induced by genotoxic stress. Our results shed new light on the regulation of SIRT1 and have important implications in understanding the molecular mechanism of ageing and cancer.”

The DBC1–SIRT1 interaction increases following DNA damage and oxidative stress

The 2012 publication Regulation of SIRT1 activity by genotoxic stress reports: “SIRT1 regulates a variety of cellular functions, including cellular stress responses and energy metabolism. SIRT1 activity is negatively regulated by DBC1 (Deleted in Breast Cancer 1) through direct binding. However, how the DBC1–SIRT1 interaction is regulated remains unclear. We found that the DBC1–SIRT1 interaction increases following DNA damage and oxidative stress. The stress-induced DBC1–SIRT1 interaction requires the ATM-dependent phosphorylation of DBC1 at Thr 454, which creates a second binding site for SIRT1. Finally, we showed that the stress-induced DBC1–SIRT1 interaction is important for cell fate determination following genotoxic stress. These results revealed a novel mechanism of SIRT1 regulation during genotoxic stress.”  So, there is another unvirtuous loop:  if there is insufficient NAD+, the PARP DNA repair machinery will not work well creating greater oxidative stress which down-regulates SIRT1 further impairing the NAD salvage cycle.

 Interestingly, it apears that resveratrol can block the SIRT1 down-regulating effect of DBC1/CCAR2.

The 2014 publication Resveratrol delays Wallerian degeneration in a NAD(+) and DBC1 dependent manner  reports: “Axonal degeneration is a central process in the pathogenesis of several neurodegenerative diseases. Understanding the molecular mechanisms that are involved in axonal degeneration is crucial to developing new therapies against diseases involving neuronal damage. Resveratrol is a putative SIRT1 activator that has been shown to delay neurodegenerative diseases, including Amyotrophic Lateral Sclerosis, Alzheimer, and Huntington’s disease. However, the effect of resveratrol on axonal degeneration is still controversial. Using an in vitro model of Wallerian degeneration based on cultures of explants of the dorsal root ganglia (DRG), we showed that resveratrol produces a delay in axonal degeneration. Furthermore, the effect of resveratrol on Wallerian degeneration was lost when SIRT1 was pharmacologically inhibited. Interestingly, we found that knocking out Deleted in Breast Cancer-1 (DBC1), an endogenous SIRT1 inhibitor, restores the neuroprotective effect of resveratrol. However, resveratrol did not have an additive protective effect in DBC1 knockout-derived DRGs, suggesting that resveratrol and DBC1 are working through the same signaling pathway. We found biochemical evidence suggesting that resveratrol protects against Wallerian degeneration by promoting the dissociation of SIRT1 and DBC1 in cultured ganglia. Finally, we demonstrated that resveratrol can delay degeneration of crushed nerves in vivo. We propose that resveratrol protects against Wallerian degeneration by activating SIRT1 through dissociation from its inhibitor DBC1.”

Other relevant publications on DBC1/CCAR2 include:

Age-related decline in NAD+

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Image and legend source  Correlation between NAD+ levels and Age in (A) Males (B) Females.. From: Age-Associated Changes In Oxidative Stress and NAD+ Metabolism In Human Tissue .
(A) NAD+ concentrations decline with age in males. NAD+ levels decreased significantly in males aged between 0–77 years (line a; p = 0.0007; n = 27). Pearson’s correlation coefficient for a normally distributed population, r = −0.769. The post-pubescent data for male subjects also showed a decline in NAD+ levels with age (line b; r = −0.706; p = 0.0001; n = 19). An exponential (first-order) least squares fit was used to generate the nonlinear trend lines (line a and b). (B)NAD+ concentrations decreased significantly with age (36–76) in post-pubescent females (p = 0.01; n = 22).Pearson’s correlation coefficient for a normally distributed population,r = −0.537. An exponential (first-order) least squares fit was used to generate the nonlinear trend line.”

NAD SYNTHESIS

in general, there are two major pathways via which NAD can be synthesized in the body: the de-novo pathway for synthesizing NAD from raw materials, and the salvage pathway which involves recycling NAD between its two states NAD+ and NADH.

The de-novo pathway.

Again from Dissecting Systemic Control of Metabolism and Aging in the NAD World: The Importance of SIRT1 and NAMPT-mediated NAD Biosynthesis:   “NAD is synthesized from three major precursors—tryptophan, nicotinic acid, and nicotinamide [38] and [39]. Lower eukaryotes and invertebrates, such as yeast, worms, and flies, use nicotinic acid, a form of vitamin B3, as a major NAD precursor, whereas mammals predominantly use nicotinamide, another form of vitamin B3, for NAD biosynthesis. In mammals, NAMPT initiates the major NAD biosynthesis pathway by converting nicotinamide and 5′-phosphoribosyl-1-pyrophosphate (5′-PRPP) to nicotinamide mononucleotide (NMN), which is the rate-limiting step in this NAD biosynthesis [11] and [12]. The second enzyme, nicotinamide/nicotinic acid mononucleotide adenylyltransferase (NMNAT), completes NAD biosynthesis by transferring adenine from ATP to NMN. There are three distinct isoforms for NMNAT, NMNAT1-3, which are localized in nucleus, cytoplasm, and mitochondria, respectively, suggesting that NAD biosynthesis mediated by NAMPT and NMNAT might be compartmentalized in each subcellular compartment [40] and [41].”  Later. we discuss how one possible strategy for enhancing body level of NAD is supplementation with NMN.  This has worked remarkably well to produce health benefits in small animals, but the substance is now extremely expensive.

PROPERTIES OF THE NAD SALVAGE CYCLE

A diagram for the salvage pathway has already been offered above.  the following diagram shows both the de-novo and the salvage pathway.

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Image source

Important aspects of the salvage cycle are 1.  The cycle is under circadian clock and SIRT1 control, 2.  salvage cycle activities regulate expression of several genes via SIRT1 promoter activities, 3.  ROS stress activating p53 is a regulator of the cycle, and 4.  The NAD World includes extra-cellular actions as well as ones in different cell compartments. We expand on each of these points.

1.  Circadian clock regulation of the NAD salvage cycle

The 2009 publication Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1  reports: “Many metabolic and physiological processes display circadian oscillations. We have shown that the core circadian regulator, CLOCK, is a histone acetyltransferase whose activity is counterbalanced by the nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase SIRT1. Here we show that intracellular NAD+ levels cycle with a 24-hour rhythm, an oscillation driven by the circadian clock. CLOCK:BMAL1 regulates the circadian expression of NAMPT (nicotinamide phosphoribosyltransferase), an enzyme that provides a rate-limiting step in the NAD+ salvage pathway. SIRT1 is recruited to the Nampt promoter and contributes to the circadian synthesis of its own coenzyme. Using the specific inhibitor FK866, we demonstrated that NAMPT is required to modulate circadian gene expression. Our findings in mouse embryo fibroblasts reveal an interlocked transcriptional-enzymatic feedback loop that governs the molecular interplay between cellular metabolism and circadian rhythms.”

The 2010 publication Clocks  in the NAD World: NAD as a metabolic oscillator for the regulation of metabolism and aging relates: ” Most recently, it has been demonstrated that SIRT1 regulates the amplitude and the duration of circadian gene expression through the interaction and the deacetylation of key circadian clock regulators, such as BMAL1 and PER2. More strikingly, we and others have discovered a novel circadian clock feedback loop in which both the rate-limiting enzyme in mammalian NAD biosynthesis, nicotinamide phosphoribosyltransferase (NAMPT), and NAD levels display circadian oscillations and modulate CLOCK:BMAL1-mediated circadian transcriptional regulation through SIRT1, demonstrating a new function of NAD as a “metabolic oscillator.” These findings reveal a novel system dynamics of a recently proposed systemic regulatory network regulated by NAMPT-mediated NAD biosynthesis and SIRT1, namely, the NAD World. In the light of this concept, a new connection between physiological rhythmicity, metabolism, and aging will be discussed.”

Here is a diagram of the situation:

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Image source ” The circadian clock controls the expression of nicotinamide phosphoribosyltransferase (NAMPT), which encodes the rate-limiting enzyme in mammalian NAD+ biosynthesis from nicotinamide. NAMPT catalyses the transfer of a phosphoribosyl residue from 5-phosphoribosyl-1-pyrophosphate (PRPP) to nicotinamide to produce nicotinamide mononucleotide (NMN), which is then converted to NAD+ by nicotinamide mononucleotide adenylyltransferases (NMNATs; there are three NMNAT genes). Oscillations in NAMPT levels result in circadian variations in NAD+ levels, which determines the activity of sirtuin 1 (SIRT1) and poly(ADP-ribose) polymerases (PARPs). Therefore, SIRT1 determines the oscillatory levels of its own coenzyme, NAD+ (Ref. 23). SIRT1 can also deacetylate and regulate proteins involved in metabolism and cell proliferation. Orange indicates circadian oscillation. FOXO1, forkhead box O1; LXR, liver X receptor; PPARGC1α, PPARγ co-activator 1α; PPi, pyrophosphate.”

Note that SIRT1 is intrinsic to the operation of the cycle, both regulating the cycle and being regulated by it.

Here is another Depiction of clock regulation of the salvage cycle via BMAL1 and CLOCK:

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Image and legend source  source   ” A circadian oscillatory feedback loop regulated by NAMPT, SIRT1, and CLOCK/BMAL1 and a possible functional interplay between adipose tissue and two frailty tissues in the NAD World, pancreatic β cells and neurons. NAMPT and NAD levels display circadian oscillations that are regulated by CLOCK/BMAL1 in peripheral tissues, such as the liver and WAT. This NAD oscillation periodically activates SIRT1, which represses CLOCK/BMAL1-mediated transcription of clock target genes, including Nampt itself, completing an interlocked transcriptional-enzymatic feedback loop involving NAMPT-NAD and SIRT1-CLOCK/BMAL1. In pancreatic β cells and central neurons, intracellular NAMPT (iNAMPT) levels are so low [45] that they may not be able to drive the NAMPT–NAD–SIRT1-dependent circadian feedback loop. However, their NAD oscillation might be generated by incorporating NMN that is likely synthesized from nicotinamide (Nic) by extracellular NAMPT (eNAMPT) that could be periodically secreted from adipose tissue.”

2.  The salvage pathway and SIRT1 regulation of gene expression. 

NAD+ regulates gene expression via histone deacetylase activity of SIRT1 at gene promoter sites.

The 2009 publication Enzymes in the NAD+ salvage pathway regulate SIRT1 activity at target gene promoters reports: “In mammals, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 1 (NMNAT-1) constitute a nuclear NAD(+) salvage pathway which regulates the functions of NAD(+)-dependent enzymes such as the protein deacetylase SIRT1. One of the major functions of SIRT1 is to regulate target gene transcription through modification of chromatin-associated proteins. However, little is known about the molecular mechanisms by which NAD(+) biosynthetic enzymes regulate SIRT1 activity to control gene transcription in the nucleus. In this study we show that stable short hairpin RNA-mediated knockdown of NAMPT or NMNAT-1 in MCF-7 breast cancer cells reduces total cellular NAD(+) levels and alters global patterns of gene expression. Furthermore, we show that SIRT1 plays a key role in mediating the gene regulatory effects of NAMPT and NMNAT-1. Specifically, we found that SIRT1 binds to the promoters of genes commonly regulated by NAMPT, NMNAT-1, and SIRT1 and that SIRT1 histone deacetylase activity is regulated by NAMPT and NMNAT-1 at these promoters. Most significantly, NMNAT-1 interacts with, and is recruited to target gene promoters by SIRT1. Collectively, our results reveal a mechanism for the direct control of SIRT1 deacetylase activity at a set of target gene promoters by NMNAT-1. This mechanism, in collaboration with NAMPT-dependent regulation of nuclear NAD(+) production, establishes an important pathway for transcription regulation by NAD(+).”

3.  NAD+ synthesis in the salvage cycle is in part mediated by ROS stress and p53 activation.

The 2014 publication The NAD+ synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) is a p53 downstream target  reports: “NAD(+) metabolism plays key roles not only in energy production but also in diverse cellular physiology. Aberrant NAD(+) metabolism is considered a hallmark of cancer. Recently, the tumor suppressor p53, a major player in cancer signaling pathways, has been implicated as an important regulator of cellular metabolism. This notion led us to examine whether p53 can regulate NAD(+) biosynthesis in the cell. Our search resulted in the identification of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2), a NAD(+) synthetase, as a novel downstream target gene of p53. We show that NMNAT-2 expression is induced upon DNA damage in a p53-dependent manner. Two putative p53 binding sites were identified within the human NMNAT-2 gene, and both were found to be functional in a p53-dependent manner. Furthermore, knockdown of NMNAT-2 significantly reduces cellular NAD(+) levels and protects cells from p53-dependent cell death upon DNA damage, suggesting an important functional role of NMNAT-2 in p53-mediated signaling. Our demonstration that p53 modulates cellular NAD(+) synthesis is congruent with p53’s emerging role as a key regulator of metabolism and related cell fate.

4.  Action in the NAD World takes place in multiple cell compartments: the cytoplasm, the mitochondria and in the nucleus – and also in the plasma.

This point is illustrated in this diagram:

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Image and legend source   “The subcellular distribution of NAD+ biosynthetic enzymes in mammalian cells is shown on the left-hand side of the figure. All known biosynthetic reactions can take place in the cytosol, although conversion of nicotinamide (Nam) to NAD+ also takes place in the nucleus. However, NAD+ and its biosynthetic intermediates are likely to be freely exchangeable between the cytosol and the nucleus. Mitochondria contain nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT) activity and can therefore generate NAD+ from NMN. An extracellular form of Nam phosphoribosyltransferase (NamPRT), known as eNamPRT (also known as visfatin), is secreted from adipose tissue. Pyridine bases (Nam and nicotinic acid (NA)) and nucleosides (such as Nam riboside (NR)) enter cells by different transport mechanisms. The compartments of predominant NAD+ generation, the nucleus and mitochondria, also harbour the majority of intracellular NAD+-dependent signalling processes (right-hand side of the figure). Nuclear poly(ADP ribose) polymerase 1 (PARP1) generates polyADP ribose and probably accounts for the majority of nuclear NAD+ degradation. Direct interaction with NMNAT1 facilitates NAD+ supply to activated PARP1. Members of the sirtuin family are found in the nucleus, the cytosol and the mitochondria. SIRT1 is a key regulator of gene expression by deacetylating histone and non-histone targets such as p53. SIRT6 is also a nuclear protein. Among others, it activates PARP1, thereby facilitating DNA repair. SIRT7 is mostly localized to the nucleolus. SIRT2 localizes to the cytosol. SIRT3, SIRT4 and SIRT5 are located in mitochondria. SIRT3 is the major deacetylase in these organelles and a key regulator of metabolic pathways, including β-oxidation of fatty acids and the tricarboxylic acid cycle (TCA). Moreover, it deacetylates and activates superoxide dismutase 2 (SOD2), thereby suppressing reactive oxygen species (ROS) production. CD38, the main mammalian NAD+ glycohydrolase, and the majority of monoADP ribosyltransferases (mARTs) are found on the cell surface. All signalling reactions generate Nam as a common product, which can be recycled into the NAD+biosynthesis pathway by NamPRT.”

THE AVAILABILITY OF NAD+ IS CRITICAL FOR MITOCHONDRIAL FUNCTIONALITY

This has been known for a decade now.   The 2004 publication Poly(ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition  relates “—. In astrocytes, extracellular NAD(+) can raise intracellular NAD(+) concentrations. To determine whether NAD(+) depletion is necessary for PARP-1-induced MPT, NAD(+) was restored to near-normal levels after PARP-1 activation. Restoration of NAD(+) enabled the recovery of mitochondrial membrane potential and blocked both MPT and cell death. Furthermore, both cyclosporin A and NAD(+) blocked translocation of the apoptosis-inducing factor from mitochondria to nuclei, a step previously shown necessary for PARP-1-induced cell death. These results suggest that NAD(+) depletion and MPT are necessary intermediary steps linking PARP-1 activation to AIF translocation and cell death.”

The 2011 publication Pharmacological effects of exogenous NAD on mitochondrial bioenergetics, DNA repair, and apoptosis  reported: “During the last several years, evidence that various enzymes hydrolyze NAD into bioactive products prompted scientists to revisit or design strategies able to increase intracellular availability of the dinucleotide. However, plasma membrane permeability to NAD and the mitochondrial origin of the dinucleotide still wait to be clearly defined. Here, we report that intracellular NAD contents increased upon exposure of cell lines or primary cultures to exogenous NAD (eNAD). NAD precursors could not reproduce the effects of eNAD, and they were not found in the incubating medium containing eNAD, thereby suggesting direct cellular eNAD uptake. We found that in mitochondria of cells exposed to eNAD, NAD and NADH as well as oxygen consumption and ATP production were increased. Conversely, DNA repair, a well known NAD-dependent process, was unaltered upon eNAD exposure. We also report that eNAD conferred significant cytoprotection from apoptosis triggered by staurosporine, C2-ceramide, or N-methyl-N’-nitro-N-nitrosoguanidine. In particular, eNAD reduced staurosporine-induced loss of mitochondrial membrane potential and ensuing caspase activation. Of importance, pharmacological inhibition or silencing of the NAD-dependent enzyme SIRT1 abrogated the ability of eNAD to provide protection from staurosporine, having no effect on eNAD-dependent protection from C2-ceramide or N-methyl-N’-nitro-N-nitrosoguanidine. Taken together, our findings, on the one hand, strengthen the hypothesis that eNAD crosses the plasma membrane intact and, on the other hand, provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.”

Declining NAD+ can lead to the collapse of mitochondrially-mediated energy metabolism,  The process appears to be reversible.

The December 2013 publication that catalyzed the current interest in NAD+ and mentioned at the begenning of this blog entry is Declining NAD+ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging.

“Highlights
• A specific decline in mitochondrially encoded genes occurs during aging in muscle
• Nuclear NAD+ levels regulate mitochondrial homeostasis independently of PGC-1α/β
• Declining NAD+ during aging causes pseudohypoxia, which disrupts OXPHOS function
• Raising nuclear NAD+ in old mice reverses pseudohypoxia and metabolic dysfunction

“Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD+ and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.”

” In oncology, the Warburg effect is the observation that most cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria as in most normal cells.[4][5][6] The latter process is aerobic (uses oxygen). Malignant, rapidly growing tumor cells typically have glycolytic rates up to 200 times higher than those of their normal tissues of origin; this occurs even if oxygen is plentiful(ref).”

The following diagrams illustrate aspects of the NAD+ – mitochondrial connection:

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Image source

Image and legend source ” We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded oxphos subunits. We trace the cause to an alternate pgc-1a/b-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD+ and the accumulation of hif-1a under normoxic conditions, with parallels to warburg reprogramming. Deleting sirt1 accelerates this process, whereas raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse in a sirt1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1a/b-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.”

The following two diagrams illustrates matters discussed above that go on in key cell compartments and outside the cell:

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Images and legend source ” Maintenance of the mitochondrial NAD pool. Although separate, the mitochondrial and nuclear/cytoplasmic NAD pools are intricately connected through the NAD/NADH-redox shuttles (most commonly the malate/aspartate and the glycerol-3-phosphate shuttles) and NAD biosynthetic pathways in each subcellular compartment. Multiple cellular processes play an important role in maintaining an optimal NAD/NADH ratio between mitochondria and the cytoplasm, including glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation by the electron transport chain (ETC). Mitochondrial and nuclear/cytoplasmic NAD biosynthetic pathways are balanced in response to nutritional and environmental stimuli.”

Why this is all important for longevity is illustrated by these diagrams for mice:

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Image and legend source 

“Activity of brain mitochondrial enzymes that are markers of aging in relation to mice survival. Complex I, complex IV, and mitochondrial nitric oxide (NO) synthase (mtNOS) activities in male (A) and female (B) mice.”

NAD Part 2

Our perception is that a big new area of longevity science may be opening up connected wih NAD, metabolisn and Sirtuins, one that may well offer new practical interventions that could lead to longer healthier lives. There are strong theoretical as well as experimental reasons for believing that enhancing body levels of NAD+ may have a chance of enhancing health and longevity while more conventional approaches do a limited job or fail.  We identify those reasons here.

  1. There are several alternative approaches now being investigated to achieve such enhancement, including supplementation with NMN, or with nicotinamide riboside (NR) and direct IV infusion of NAD. We don’t know which one will be best. or even the extent to which any one really works in humans to enhance health and longevity.   Nonetheless, we probably are seeing a commercial rush to bring NMN supplements to the market, and NR is already being sold as a supplement.
  2. There are additional theoretical reasons for suspecting that such benefits may not emerge or that they may require additional interventions for their realization. One approach for enhancing NAD+ levels may be far superior to the others.  For example many of the benefits of NAD plus supplementation can be attributed to higher activity of the Surtuin SIRT1.  However, what actually happens with Sirtuin expression is the result of a complex network of feedback-inhibition loops and many known factors can work to limit or eliminate Sirtuin levels or activity.  We have very poor understanding of how these feedback inhibition loops ultimately net out in live mice under various real-life conditions, let alone in us.
  3. We offer many predictions below about probable health benefits of NAD+ supplementation. We need to acknowledge that some or all of these could be wrong.
  4. For this last reasons, it is very important to develop a panel of practically measurable bottom-line biomarkers that tell us what a form of NAD+ enhancement is actually doing in our body to enhance health and possible longevity. And, to tell us other things we don’t understand well, such as how best to synchronize NAD+ enhancement with our circadian rhythms and additional interventions such as fasting and exercise. Otherwise, we will be flying blind with respect to the efficacy of NAD+ enhancement, much as we have been traditionally flying blind with respect to the efficacy of consuming dietary health supplements. We identify a number of such possible biomarkers in the course of this blog entry, although we leave discussion of practical means to measure these biomarkers to a subsequent blog entry in this series.

The Discovery of NAD+ and NAD+ Metabolism

How NAD+ was discovered – Yeast fermentation of sugar

Four Nobel prize winners contributed to the discovery of NAD+, starting with Sir Arthur Harden who studied yeast fermentation of sugar back in 1904 and isolated a low molecular weight fraction and a high molecular weight fraction, both of which were necessary for the fermentation to occur.  He did not know that the low molecular weight fraction was NAD+, but called this low molecular weight fraction “cozymaze”.  Later on Hans von Euhler-Chelpin  showed that cozymaze was made two mononucleotides, AMP and NMN. Then in 1936 Otto Warburg, showed that cozymaze was involved in hydrogen transfer (now called redox reactions).  In 1948, Arthur Kornberg showed that NAD+ was synthesized enzymatically within cells by an enzyme that consumed ATP and linked NMN with AMP to form NAD+. All of these scientists won Nobel prizes during their career. However, it would be another 55 years before the primary structure of the NAD biosynthetic enzyme (NMNAT) was determined. Thus the entire discovery process of NAD+ and NAD+ biosynthesis took over 100 years to get to where we are today.

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Images source

Popular but questionably effective downstream attempts to increase longevity and calorie restriction that actually does this

Next, again as background we review a) why antioxidants, vitamins, and hormones fail to reverse aging, b) the molecular pathways of calorie restriction (CR) which is the best known intervention for life extension, and c) how Sirtuins are the key molecular actors for health and longevity resulting from both calorie restriction and NAD+ enhancement.

Why Antioxidants, Vitamins, and Hormones failed to reverse aging – They treat the symptoms and not the causes

Although many drivers of human aging can be slowed or delayed, many of these factors are thought to be non-reversible reversible. (Ex: DNA gene mutations are not reversible). Most “anti-aging” supplements like anti-oxidants cannot reverse such aspects of aging.  Instead, they merely “control damage,” and many have a mixed track record of efficacy in clinical trials.  Likewise, hormone replacement therapy (HRT) does not reverse aging, despite the claims of some “anti-aging” clinics. In fact, emerging scientific studies have shown that exogenous anti-oxidant supplements and HRTs have a paradoxical effect. For instance, exercise has been shown to increase the expression of anti-oxidant genes and increase the expression of endogenous hormones (hGH, etc.). However, when exogenous anti-oxidants are used, this reduces the expression of anti-oxidant genes induced by the exercise producing a negative effect. Likewise, exogenous HRT use suppresses endogenous hormone production and thereby accelerates the decline in hormone gene expression that occurs with aging (via an epigenetic feedback-this is so good inhibition mechanism). This is why many testosterone users have testicular atrophy. Thus antioxidants and HRTs treat the symptoms (i.e. “downstream effects) of aging, rather than the cause (“upstream events”) of aging.

Current initiatives to restore NAD+ levels in individuals are attempts to affect what we believe are “upstream events” in aging to produce positive downstream events, such as already has been shown to be possible in various studies – like reversal of muscle aging in rodents.

Because nuclear NAD+ deficiency has so many impacts that mimic those of caloric restriction (CR), we look next at how it affects upstream events in molecular aging.  We are also looking at CR for ideas for the best biomarkers for objectively evaluating the “upstream effects” of restoring NAD+ levels in the nucleus.

Why Calorie Restriction (CR) increases life span  – CR affects the “upstream events” in molecular aging

Caloric restriction (CR) is the most scientifically-validated method of increasing life span and health span.  This involves reducing caloric intake by 10-40% (below RDA) without inducing protein-calorie malnutrition. The molecular mechanisms of CR are complex, but simply put, they impede and slow “pro-aging pathways” and amplify “longevity pathways”.  The two major “pro-aging pathways” are the Insulin/IGF-1 pathway and the mTOR pathway. CR inhibits both of these.  The “longevity pathways” involve AMPK,  Sirtuins, Glucagon, and Adiponectin. CR activates all of these. The diagram below shows the molecular mechanism of each of these factors in CR.

NAD+00

The Role of Sirtuins in Caloric Restriction (CR) – Sirtuins are integrally involved with all of the CR pathways and are the main actors for both calorie restriction and NAD+ health and longevity interventions.

In the diagram above, there are three red ovals that comprise a major molecular pathway of CR involving the family of enzymes called Sirtuins. One red ovals contain the symbols for the one of the 7 Sirtuin enzymes called Sirtuin 1 (SIRT1).  Another red oval contains symbol for Nicotinamide adenine dinucleotide (NAD+), which is the substrate that activates all of the Sirtuin enzymes. Sirtuin enzymes consume NAD+, producing a by product called Nicotinamide (Nam), which inhibits SIRT enzymes.  The last red oval represents the enzyme called Nampt, which is the rate-limiting enzyme in the pathway that converts Nam back into NAD+, thereby renewing the supply of NAD+ to activate SIRT enzymes.  If you have read the Part 1 blog entry, you will recognize this pathway of regenerating NAD+ from Nam which is called the “NAD+ salvage pathway.” Activating these “red ovals” and restoring normal Sirtuin function is the goal of interventions intended to elevate or restore nulcear levels of NAD+.

The diagram above only shows one Sirtuin enzyme, SIRT1.  In reality, there are 7 NAD+-dependent Sirtuins (SIRT1-7) which all play a role in CR molecular mechanisms. Sirtuins affect almost every oval in the diagram below because they activate or inhibit other CR proteins by removing acetyl groups from these CR proteins.  In the diagram above, the small “Ac” stands for an “acetyl” group. SIRT1 directly affects all of the CR proteins that have an “Ac” on the diagram. It deacetylates and therefore tends to activate them.

For discussion of many of the benefits of Sirtuins, you can review the blogs on the series Slaying Two Dragons With One Stone (the Sound of Silence)Part 1Part 2 and Part 3.

In addition to the major molecular pathway of CR on aging shown above, there are more important but lesser- known factors that change with CR, such as Klotho, p66shc, and DNA repair pathways. Although none of these pathways alone can account for the beneficial effects of CR, all of them probably work together to increase healthspan in all organisms studied to date.  It is the involvement of NAD+ as a required substrate for the Sirtuin enzymes that is the focus of initiatives to restore the nuclear levels of NAD+. 

Although the diagram does not illustrate this, NAD+ drives the Sirtuin-dependent deacetylation of many of the enzymes shown above, including eNOS, NF-kB, PGC-1a, Foxos, LKB1, and IRS1/IRS2. By NAD+-dependent deacetylation of these enzymes, Sirtuins thereby affect every major molecular pathway that involves aging. This is why many researchers believe restoring NAD+ levels in the cell is so important for affecting the “cause” of aging, rather than the “effects” of aging.

A 2012 review article, Cantó et. al.  Targeting SIRT1 to improve metabolism: all you need is NAD+? anallyzes ” the pros and cons of the current strategies used to activate SIRT1 and explore the emerging evidence indicating that modulation of NAD+ levels could provide an effective way to achieve such goals.”

The Two Roles of NAD+ – Contrasting the Cofactor vs Signaling role of NAD+

(Note that the Part 1 blog entry introduces the main actors in the cast of NAD World, molecular entities mentioned below, like NAD+, NADH, NADPH. ATP. etc.)

NAD+ does two things: a) it serves as a cofactor in redox and important metabolic reactions in which case it is shuttled back and forth between the NAD+ and NADH forms but is not consumed, and b) it serves as a signaling molecule in which case it is consumed as a substrate.

When NAD+ and NADP were discovered, they were thought to function only as a cofactor in redox reactions. Here they functioned as an “energy currency”, transferring high energy electrons from fuel oxidation (carbohydrates, fats, protein or alcohol) to the mitochondria (NADH) or the plasma membrane (NADPH) to generate ATP.  In these reactions, NAD(P)  “accepted” hydride groups (2 electrons and one proton) and NAD(P)H “donated” hydride groups, transferring the high energy electrons to another substrate.  Thus NAD/NADH was the recycled cofactor pair in the catabolic oxidation of carbohydrates, fatty acids, proteins, and alcohol.  On the other hand, NADP/NADPH was the recycled cofactor pair in the anabolic synthesis of fatty acids and cholesterol.  In these redox reactions, no NAD or NADP molecules are consumed.  Over 200 cellular enzymes utilize either NAD+/NADH or NADP/NADPH as a cofactor this way.

It was not until recently, however, that a second role for NAD+ as a signaling molecule was discovered. In these reactions, NAD+ does not accept hydride groups and the molecule is not recycled.  Instead, the NAD+ molecule is consumed as a substrate and the glycosidic bond between the ADP-ribose moiety and the nicotinamide moiety (NAM) of the molecule is broken, releasing NADM and ADP-ribose as byproducts of the reaction. Sirtuins are just one of many enzymes that “consume” NAD+ as a substrate.  The two contrasting roles for NAD+ as a cofactor vs a substrate are illustrated below:

The Redox/Co-Factor Role of NAD+ and NADPH  and The Signaling Compound/Substrate Role of NAD+

NAD+3
NAD+2

Image source

What makes and what eats-up NAD+?  Causes of NAD+ deficiency with aging.

Although NAD+ can be made from the amino acid tryptophan by an 8-step pathway called the de novo pathway,  this pathway does not work very well in most cells.  For this reason, most of the consumed NAD+ is converted to nicotinamide and then “salvaged” by a two step process that converts it back into NAD+.  This pathway is called the salvage pathway and is rate-limited by the first enzyme in the two-step pathway called NAMPT.  NAMPT gene expression can be activated by caloric restriction (CR) or exercise and this may be a major reason why both CR and exercise have long term health benefits.  However even with exercise and caloric restriction, nuclear NAD+ levels decline with aging.

It is unclear now if the cause of age-related nuclear NAD+ deficiency is due to Sirtuins, PARPs, CD38, or a decline in the expression of NAMPT.  Most studies suggest that it is a combination of all four factors and that “genotoxic stress” is a major driving force for nuclear NAD+ deficiency.  The synergistic effects of genotoxic stress inducing single stranded DNA breaks and double-stranded DNA breaks and the combined effect of Sirtuins and PARPs is illustrated below:

NAD+4

Image source

Also, NAD+ can be provided by an exogenous source such as an IV, and its level can possibly enhanced such as by NMN or NR supplementation as discussed below. And also as outlined below numerous factors can affect SIRT activation levels.

NAD+ and aging

Nuclear NAD+ decline and Aging – One cause of aging is thought to be the effect of NAD+-consuming enzymes in the cell nucleus

One of the “upstream events” in aging appears to be the decline in NAD+ within the nucleus of the cell. This appears to occur within all 37 trillion cells within the human body.  The decline is due to several classes of NAD+-consuming enzymes called Sirtuins, PARPs, and ADP-ribose glycohydrolases.  These enzymes perform absolutely essential functions required for cell health and each consumes NAD+ as a substrate.  Aging appears to be associated with increasing insufficiency of enough NAD+ to support the growing demands of these.  Sirtuins are a class of NAD+-consuming enzymes needed for double stranded DNA repair, for epigenetic gene silencing, and for chromatin remodeling.  Poly-ADP-ribose Polymerases (PARPs) is another class of NAD+-consuming enzymes involved with repair of single stranded break DNA damage.  PARPs also initiate cell death (apoptosis) in cancer cells and cells whose DNA is beyond repair.  More recently, a third type of NAD+-consuming enzyme has been found in the nucleus called CD38, once thought to only exist outside the cell.  CD38 is a part of the family of enzymes called ADP-ribose glycohydrolases.  Thus there are at least three families of enzymes all competing for the same pool of NAD+ within the nucleus of the cell.

Aging and NAD+ – Aging in the NAD world-view is due  to a “nuclear NAD+ deficiency” incurred from NAD+-consuming enzymes

For quite some time, caloric restriction (CR) has been regarded as the most scientifically-validated method to retard aging. CR research has lead to the discovery of several major molecular pathways that accelerate or retard aging, including the Insulin/IGF-1 pathway (age accelerator), the mTOR pathway (age accelerator), the AMPK pathway (aging brake), and Sirtuins (aging brake).  It is the role of NAD+ as a required substrate for Sirtuins that lead to the recent discovery that there is a nuclear NAD+ deficiency that affects not only Sirtuins, but another set of important NAD+ consuming enzymes called Poly-ADP-ribose Polymerases (PARPs).  Both the Sirtuins and the PARPs are involved with the DNA damage response to genotoxic stress.  Sirtuins play a major role in double-stranded DNA breaks whereas PARPs play a major role in single-stranded DNA breaks.  Here is a simplified diagram showing how both SIRTs and PARPs use up NAD+ in the nucleus of the cell:

NAD+5

Image source

Restoring nuclear NAD+ levels may reverse aging

Recent work has shown that restoring nuclear NAD+ levels in aging mice actually reversed a common phenotypes of aging – that of mitochondrial dysfunction.  Mitochondrial dysfunction manifests itself as a decline in ATP production, a decline in heat production, excessive cellular free radical production, and increased DNA damage.

Although there are many proposed strategies for restoring nuclear NAD+ levels to normal, the only one that has been successfully accomplished this experimentally so far (with mice) is nicotinamide mononucleotide (NMN).  However, many experts believe that supplementation with nicotinamide riboside (NR) or directly with NAD will also restore nuclear NAD+ levels.  See the 2012 publication Canto et. al.  The NAD+ precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet induced obesity.  “As NAD+ is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38—both NAD+ consumers—increases NAD+ bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD+ precursor with the ability to increase NAD+ levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD+ levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function.”

Others have suggested alternative strategies such as PARP inhibition, CD38 inhibition,  or increasing NAMPT activity.  We are interested in all of the above.  We are not interested in limiting our approach to one particular strategy.  Likewise, we do not believe that any particular NAD precursor, NAD metabolite, or enzyme inhibitor is a replacement for sleep, exercise, and caloric restriction, which have already been scientifically proven to help ameliorate the nuclear NAD+ problem.  Rather, we are interested in doing whatever it takes to restore nuclear NAD+ levels, using sleep, exercise, and CR as fundamental strategies and various NAD precursors, PARP inhibitors, and CD38 inhibitors as adjuncts to a healthy lifestyle.  Further, we strongly suspect that determination of what works best and under what conditions cannot be based on theory and will require monitoring a panel of biomarkers.  My personal  goal is not to increase lifespan but to increase healthspan, although increasing healthspan could increase lifespan.

This diagram projects an increase of lifespan possibly associated with NMN

NAD+6

Image source

How NAD+ activates SIRTs and the competing “NAD+ consumers”

Based on the evidence from CR studies, there is strong scientific evidence that activating Sirtuins has major positive health effects and longevity benefits in most all animal models.  Although there are 7 different Sirtuin enzymes (SIRT1-7), SIRT1 is the most well understood member of this family of enzymes. All 7 Sirtuins require NAD+ as a substrate for the enzymes to work. Sirtuin enzymes consume NAD+, converting it into nicotinamide (NAM). NAM itself inhibits SIRT1 activity, but NAM can be “salvaged” and converted back into NAD+ (see diagram below). This conversion of NAM back into NAD+ is a two-step enzymatic process called the “NAD+ salvage pathway” described in our Part 1 blog entry and  covered more in additional detail here.

NAD+8
NAD+7

Image source  NAD+ is consumed by enzymatic activity of all 7 of the Sirtuin isoforms (SIRT1-7). The consumption of NAD+ produces the byproduct, Nicotinamide (NAM) which can be “recycled” to NAD+  in the 2-step process shown above that is called the salvage cycle, which requires PRPP & ATP to run the cycle.

As discussed in the Part 1 blog entry, there are other enzymes that also require NAD+ as a substrate, such as the PARP family of enzymes as well as CD38 (see diagram below).  These other NAD+-consuming enzymes “compete” with SIRT enzymes in the nucleus and this competition may be the primary reason why nuclear NAD+ levels decline with aging.  Again, this is why restoring nuclear NAD+ levels is thought to be a good idea.

The above two diagrams illustrate the key “suspects” in the etiology of SIRT deficiency-induced aging.  As you can see, this “SIRT1 deficiency” is not a deficiency in the protein, but a deficiency in the activity of the protein.  It has been clearly shown that in most scenarios, there is no decline in SIRT1 protein availability with aging.  This may be because the SIRT1 protein can regulate its own gene expression.  This auto-regulation may be why the expression of SIRT1 protein does not decline with aging.

What is also a mystery is that aging also is associated with a decline in the expression of SIRT3.  It appears that the reason for this decline in SIRT3 expression may be due to the fact that SIRT1 regulates the gene expression of SIRT3. This is why many experts believe that the decline in SIRT1 activity is an “upstream event”, whereas the decline in SIRT3 is a “down stream events”.  The following possible causes for the decline in SIRT1 activity are illustrated in the diagrams above and listed in the table below:

Possible Causes of the Decline in SIRT1 activity with Aging1.

Decline in iNAMPT activity – The NAD+ salvage pathway has a rate-limiting enzyme called Nicotinamide phosphoribosyltransferase (NAMPT). Because there is both an intracellular and an extracellular localization of NAMPT, it is sometimes abbreviated as iNAMPT (intracellular NAMPT) and eNAMPT (extracellular form, which has also been called eNAMPT, PBEF, or Visfatin). eNAMP circulates in the blood and will be discussed elsewhere.  With iNAMPT, several independent studies have shown that iNAMPT gene expression and iNAMPT protein activity declines with aging and that this decline in iNAMPT activity precedes replicative senescence of cells, such as human vascular smooth muscle cells (VSMCs) (Eric van der Veer, Extension of Human Cell Lifespan by Nicotinamide Phosphoribosyltransferase 2007).  Even more interestingly, before the VSMCs became senescent, the decrease in NAMPT enzymatic activity was even greater than the decline in NAMPT gene expression with NAMPT enzyme activity dropping to 14% of baseline prior to VSMC senescence.   This is a very powerful argument that NAMPT is a “longevity protein” that extends the lifespan of VSMCs by activating SIRT1 and restraining the accumulation of p53 (SIRT1 mediates p53 degradation).

The “good news” is that there are 4 ways to increase NAMPT activity: caloric restriction (CR), fasting, exercise, and ATR1 blockade. The mechanism by which CR and fasting increase NAMPT is probably due to the fact that the SIRT1 protein regulates the NAMPT gene (Zhang, Enzymes in the NAD+ Salvage Pathway Regulate SIRT1 Activity at Target Gene Promoters  2009). The mechanism by which exercise induces an increase in NAMPT expression is via AMPK-mediated PGC-1α activity.  Athletes have a 2-fold higher levels of NAMPT in their skeletal muscle compared with sedentary adults (obese, non-obese, and T2DM). Three weeks of exercise training increases NAMPT protein in skeletal muscles by 127%.

AICAR, a potent “exercise mimetic”, increases skeletal muscle NAMPT mRNA by 3.4-fold. (Costford et.al., Skeletal muscle NAMPT is induced by exercise in humans, 2010). Recently, a fourth method to increase iNAMPT gene expression was found: angiotensin receptor 1 (ATR1) blockade. ATR1 blockers such as candesartan and     telmisartan increase expression of NAMPT and SIRT3 genes, increased NAD+ levels, increased mitochondrial biogenesis, and reduced atherosclersosis in in vivo studies.  Even more surprising was that in ATR1 gene knock-out mice, there was a 26% longer lifespan. (Benigni et.al., Disruption of the Ang II type 1 receptor promotes longevity in mice 2009).  This effect was not mediated by the usual CR  pathways, but appeared to be a CR independent mechanism, mediated by an increase in iNAMPT and SIRT3.

Conclusion:  CR, fasting, and regular exercise decelerates the deleterious effects of aging via SIRT1-dependent pathways by stimulating of NAD+ biosynthesis via increase in NAMPT gene expression.  ATR1 blockade increases NAMPT gene expression via an unknown, CR-independent mechanism.

  1. Increase in PARP activity – There is recent evidence that a decline in NAD+ levels occurs in the nucleus of the cell with aging. As we have already indicted, the exact cause of this “nuclear NAD+ decline” is unknown and is the focus of research initiatives for restoration of nuclear NAD+. We have already discusssed the role of the PARPs as NAD+ consumers above and in the Part 1 blog entry.

SIRT1 – The Most Important Sirtuin in Aging and Age-related Diseases

Here, we review some of the most compelling reasons why SIRT1, the queen of the Sirtuins, is so important for health and longevity.

Depletion of SIRT1 by siRNA knockdown significantly alters the expression of about 200 genes (Zhang, Enzymes in the NAD+ Salvage Pathway Regulate SIRT1 Activity at Target Gene Promoters  2009). Here are some of the effects of SIRT1 on various organs in the body that have been scientifically proven in animal models. We believe that restoring nuclear NAD+ levels to normal will probably have all of these effects on the body, as well as many other positive effects, since NAD+ activates all 7 of the Sirtuins, not just the SIRT1 isoform.

NAD+9

Image source: Sirtuins — novel therapeutic targets to treat age-associated diseases

As you can see from the diagram above, there are beneficial effects of SIRT1 activation on almost every organ system in the body. Many of these have to do with ameliorating the signs or symptoms of metabolic syndrome and type II diabetes mellitus. For those that are not familiar with Sirtuin proteins, it is important to understand that SIRT1 does not circulate as a hormone, but instead, is a protein largely confined to the nucleus of the individual cell.  In the cell nucleus, SIRT1 deacetylates many proteins that activate or inhibit gene expression.  These effects are thought to be largely but not exclusively  due to epigenetic mechanisms involving the deacetylation of histone proteins.  SIRT1 deacetylates many other proteins found in the nucleus other than histone proteins. The following sections will cover both the histone and the “non-histone” effects of SRIT1 in the nucleus of the cell. All of these benefits of SIRT1 are easier to understand if one familiarizes themselves with the fundamental aspects of epigenetics. This article does not go into detail about epigenetics, but it would be wise for the unfamiliar reader to delve into this topic first before proceeding to read the following sections.  Past blog entries related to epigenetics are listed here.  And the many prevous blog entries related to Sirtuins are listed here.  For now, we will focus on the specific effects of SIRT1 within the nucleus of a cell, as described below.  Then we’ll cover the other nuclear-localized members of the Sirtuin family, SIRT6 and SIRT7.

Major SIRT1 Functions #1: Preventing cellular senescence  –SIRT1 prevents cell cycle arrest via six mechanisms.  We first list these mechanisms and then review what cellular senescence is and further discuss why it is important, and what these SIRT1 impacts are.

1) SIRT1 helps repair double stranded DNA breaks, which is a major “upstream cause” of cellular senescence

2) SIRT1 epigenetically prevents expression of the p16INK4a/ARF promoter by maintaining promoter hypermethylation

3) SIRT1 epigenetically prevents gene expression of p16INK4a/ARF decetylation of H3K9 histone proteins

4) SIRT1 genetically activates Akt/p70S6K1 signaling, thereby inducing p16INK4a/ARF repression

5) SIRT1 prevents p53-induced cell cycle arrest by deacetylating p53

6) SIRT1 prevents the formation of heterochromatin by deacetylating histone linker proteins (H1K24)

About cellular senescence

Cellular senescence is defined as “cell cycle arrest” and is a major hallmark of aging in all species. In old age, approximately 15-20% of cells in non-human primates show markers for cellular senescence (Herbig, Cellular Senescence in Aging Primates 2006). In human bone marrow stromal cells cultured in vitro, the percentage of senescent cells increases 4% per population doubling in old bone marrow cells vs only 0.4% per population doubling in young bone marrow cells (Stenderup, Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells  2003).

Some past blog entries discussing cell senescence and the roles of p16INK4a/ARF are listed here.

NAD+10

Image and legend source: Cellular Repair and Reversal of Aging: the Role of NAD  “Schematic illustration of NAD+-mediated sirtuins actions on NF-κB. During inflammation reduced levels of NAD+ do not impaired the hyperacetylation of NF-κB which in turn through promoter region of target genes activate pro-inflammatory pathway and senescence. Conversely, increased cellular levels of NAD+ activate SIRT1, 2 and 6 which deacetylate NF-κB inhibiting its transcriptional role and then inflammation.”

Although cell senescence has many beneficial, vital roles in normal life (embryogenesis, wound healing, cancer prevention, red blood cell aging, etc.) senescent cells are rare in young organisms.  In old age, all species start to accumulate old cells that will not divide (i.e. are senescent). Normally old cells undergo apoptosis, but these senescent cells are resistant to apoptosis, which means they hang around for longer than cells should. Moreover, they secrete a toxic mixture of secretions that are pro-inflammatory and actually help cancers grow (called the senescence associated secretory phenotype, aka SASP).  Other than genetic modifications of cells, no “cure” for the accumulation of senescent cells has been found to date.  For this reason, many researchers have believed the best way to prevent aging would be to prevent cellular senescence without increasing the risk of cancer.  Caloric restriction (CR) is the most effective method to date that has been discovered for preventing cellular senescence.  SIRT1 appears to be an important reason why CR prevents cellular senescence.

To understand cellular senescence on a molecular level, it is important to understand the major causes of cellular senescence, which includes DNA damage and telomere shortening. When either of these events occur, three key proteins trigger cell cycle arrest. They are p16INK4a protein, the ARF protein, and the p53 protein.  Since p16INK4a and ARF are both transcribed from the same gene, I will discuss them together here. However, I will briefly discuss DNA damage and telomere shortening first.

The initial discovery of the phenomena of cellular senescence was by Leonard Hayflick when he noted that diploid fibroblasts in culture would only keep dividing for a finite number of “population doublings”, and then would stop dividing. The number of cell divisions that would trigger this phenomena was referred to as the “Hayflick limit”, but the cause was unknown until researchers discovered that telomeres shortened with each cell division due to the DNA “end replication problem”. Later on, an enzyme called telomerease was discovered that could counteract the telomere shortening that occurred with cell divisions.  The researchers who discovered telomerase won the Nobel prize in 2009, but since then, many other factors have been discovered that regulate telomere length (Ex: oxidative stress, the lncRNA called TERRA, Shelterin protein caps on the telomeres that protect the ends, Rap1, Tankyrases, SIRT6, histone protein silencing of telomeric DNA, etc.).

Ten years ago, many of us concerned with aging thought that since telomeres usually get shorter with age and shorter telomeres can lead to cell senescence, health and longevity could be enhanced by interventions that make telomeres longer.  That was a very exciting idea, but it turned out to be wrong.  We now know that telomere lengths are mostly downstream responses to upstream events, just like grey hair and wrinkles are downstream consequences of aging.   See the blog entries Nuclear Aging: The View from the Telomere end of the Chromosome –  Part 1 – context, history, and about telomere lengths and Part 2 – Telomere Molecular Biology and many other blog entries we have writtem about telomeres and telomerase for details.  There is just a possibility that we could be equally wrong about our new exciting idea – that of enhancing NAD+ as a health and longevity intervention.  Time will tell.

When telomeres shorten to a critical point, they trigger the DNA damage response (DDR), which activates p53 and can induce cellular senescence.  Later on, it was discovered that even non-dividing cells and cells with longer telomeres could undergo cellular senescence.  For instance, X-ray or gamma ray radiation can induce cellular senescence in cells with longer telomeres.  Likewise, oxidative stress from intrinsically-produced ROS or exposure to oxidative stress by external ROS (Ex: exogenous H2O2) can also trigger cellular senescence.   In these cases, the cause for the cellular senescence appears to be DNA damage.  Double stranded DNA breaks (DSBs) appear to be the most closely associated with cellular senescence, regardless of whether the DSBs were induced by ionizing radiation (X-rays, UV light, etc.), DNA damaging drugs (chemotherapy), or oxidative stress (endogenously generated or exogenously applied).

A key factor in cellular senescence is the increased expression of two proteins that trigger cellular senescence, p16INK4a and ARF. These two proteins are actually part of the same gene (i.e. they share exons) but because they are encoded in different reading frames, they have different amino acid sequences and different functions. As a result of this “shared gene”,  they are often referred to as the “INK4a/ARF locus”. p16INK4a is an inhibitor of the cyclin-dependent kinases CDK4 and CDK6.  ARF regulates p53 stability through inactivation of the p53-degrading ubiquitin ligase, MDM2.  In young cells and young organisms, the INK4a/ARF locus is epigenetically repressed by Polycomb proteins, but in old age, the INK4a/ARF locus is “derepressed”, the gene is transcribed, and production of these two proteins increases by as much as 42-fold in different organs in older mice (Krishnamurthy, Ink4a/Arf expression is a biomarker of aging 2004). In this context, the activities of SIRT1 listed above  (Numbers 2, 3, and 4) mitigate against cell senescence by repressing p16INK4a/ARF or its effects. 

Interestingly, calorie restriction retards the increase in INK4a/ARF protein accumulation in all organs, reducing the age-associated increase in INK4a/ARF proteins by 2 and 16-fold. (Also Krishnamurthy, Ink4a/Arf expression is a biomarker of aging 2004).   Although the loss of Polycomb protein repression is a major reason for the increase in INK4a/ARF proteins, decline of SIRT1 expression plays a major role here as well.   For instance, glucose restriction (GR) has been found to increase SIRT1 mRNA levels and SIRT1 protein levels within cells and to inhibit p16INK4a/ARF expression. As a result, cellular lifespan was found to be dramatically increased (by 4 weeks) in normal cells with GR, whereas the same glucose restriction increased apoptosis (18%) in immortalized cells causing them to die (Li, Tollefsbol, Glucose restriction can extend normal cell lifespan and impair precancerous cell growth through epigenetic control ofhTERT and p16 expression 2010)(Li, Tollefsbol, p16INK4a Suppression by Glucose Restriction Contributes to Human Cellular Lifespan Extension through SIRT1-Mediated Epigenetic and Genetic Mechanisms 2011).  When the molecular mechanisms were studied on the role of GR in repressing p16INK4a/ARF expression, several mechanisms were found to play a role as follows:

1. Promoter site DNA (CpG) methylation

Glucose restriction induced hypermethylation of the p16INK4a/ARF promoter, which then prevents binding of the E2F-1 transcription factor.  This results in down-regulation of the p16INK4a/ARF locus, preventing cell apoptosis and cellular senescence, which leads to increased cell lifespan. (Li, et.al. as above, 2010).  Recently, other researchers have shown that SIRT1 can deacetylate DNA methyltransferase 1(DNMT1) and that compared to other HDACs, SIRT1 is the most robust deacetylator of DNMT1 (Peng, SIRT1 Deacetylates the DNA Methyltransferase 1 (DNMT1) Protein and Alters Its Activities  2011). “In summary, we show that DNMT1 is acetylated at multiple lysines and that SIRT1 deacetylates DNMT1 in vitro and in vivo. Deacetylation of DNMT1 at specific lysines enhanced its methyltransferase activity, changed its transcription repression activity and cell cycle regulatory function, and impaired its capacity to silence TSGs. In contrast to class I HDACs, which boost the silencing effect of DNMT1 by chromatin modification or stabilization of DNMT1 (5981), SIRT1 directly modifies DNMT1 activity.”

The same authors showed that nicotinamide, an inhibitor of SIRT1, reduced DNA promotor methylation by DNMT1. Since the main role of DNMT1 is to maintain CpG methylation (as opposed to de novo methylation),  Thus it is likely that SIRT1 plays a role in maintaining DNA promoter site (CpG) methylation of the p16 promoter, although this mechanism not yet been specifically proven for the INK4a/ARF locus.  Again, the key point of interest here is that supporting methylation and therefore inactivation of ther p16 promoter site is a likely action of SIRT1 for countering age-related cell senescence.

  1. Promoter site Histone deacetylation

The best way to understand the role of SIRT1 in cellular senescence is to understand the molecular mechanisms of how  Sirtuin enzymes work. Although Sirtuins do have other molecular functions, their primary job is to remove acetyl groups from lysine amino acids on proteins. In the nucleus, SIRT1 removes acetyl groups (i.e. deacetylation) from many proteins, but the major target of SIRT1 are histone proteins (H1, H3, and H4). Histone proteins make up the “spools” (called nucleosomes) which DNA is wound around.  When the spools are tightly compacted together, genes cannot be transcribed from the DNA wound around the nucleosomes. This chromatin compacted state is called “heterochromatin”. When the nucleosomes are spread out and not compacted, genes can be transcribed from the DNA and this chromatin state is called “euchromatin”. SIRT1 plays a major role in determining if chromatin is in the heterochromatin vs the euchromatin state. Specifically, SIRT1 rmoves an acetyl group from histone subunit 1 (abbreviated as H1K24). When this occurs, the chromatin remains in the euchromatin state and can be transcribed.  When there is a nuclear deficiency of NAD+, H1K24 becomes acetylated and chromatin compaction (heterochromatin) occurs.  The formation of heterochromatin is a hallmark of cellular senescence and aging. This is illustrated in the diagram below.  On a molecular level, there is no clearer “upstream explanation for aging” than this diagramt, illustrating what happens in young cells when NAD+ levels are high (SIRT1 activation) and what happens in old age when nicotinamide levels are high (SIRT1 inhibition). The 4 main SIRT1 anti-aging effects involve protein deacetylation in the nucleus:

1) deacetylation of the histone linker protein, H1;

2) deacetylation of the  tumor suppressor, p53;

3) deacetylation of histone H4 at lysine 16 and

4) deacetylation of histone H3 at lysine 9.

When H1 is deacetylated, chromatin is relaxed and gene expression occurs (euchromatin). If p53 is deacetylated, it cell survival occurs and cell cycle arrest does not occur (i.e. senescence).  When H4K16 is deacetylated, genes can be silenced.  With low levels of nuclear NAD+,

NAD+11

Image and legend source: Sirtuins: critical regulators at the crossroads between cancer and aging “SIRT1 expression and activity decrease during aging. High levels of SIRT1 protein in young cells in combination with high levels of nicotinamide adenine dinucleotide lead to deacetylation of p53 and histone proteins to promote longevity (left panel). SIRT1 levels are lower in aged cells, and higher levels of nicotinamide inhibit SIRT1 activity (right panel). The resulting hyperacetylation of p53 can induce replicative senescence. Deacetylation of histone H1 leads to its degradation that promotes formation of senescence-associated heterochromatic foci (SAHFs). Failure to downregulate SIRT1 during aging may promote cell survival after oxidative damage, leading to the accumulation of mutations, and an increased risk of cancer development.”

When there is inadequate SIRT1, H1K24 gets acetylated and the chromatin becomes compacted, forming “senescence associated heterochromatin” (SAH). SAH is a major cellular marker of aging. Thus the loss of SIRT1 H1K24 deacetylation directly causes this aspect of aging. However, if SIRT1 H2K24 deacetylation activity is maintained in old age, then SIRT1 becomes a “longevity protein”.  Unfortunately, with aging, there is a nuclear deficiency of NAD+.  As a result, SIRT1 cannot deacetylate H1K24 and senescent cells accumulate.

Note a point of clarification here on the different contexts for and meanings of deacetylation. The specific histone H1 relates to links between spindles of DNA,  Keeping it deacetylated via SIRT1 results in uncompacted chromatin which facilitates ready gene expression.  When it comes to histones H3 and H4, acetylation has the opposite effect; it locally uncompacts the chromatin and allows ready gene expression.  And deacetylation inhibits gene expression in these cases,  So, H3 and H4 deacetylation is very useful for silencing pro-aging genes such as senescence-associated ones.

Major SIRT1 Functions #2: Gene silencing  – H3K9 and H4K16 deacetylation in the nucleus

Two histone proteins that are deacetylated by SIRT1 are histone subunit 3 (H3) and subunit 4 (H4) . SIRT1 removes an acetyl group from a specific lysine (K9) on H3 and a specific lysine (K16) on H4.  H3K9 is a “site-specific” deacetylase target of SIRT1 and SIRT6, whereas  H4K16 is a “site-specific” deacetylase target of only SIRT1.  Both H3K9 and H4K16 deacetylation of histones have the effect of silencing genes as mentioned above.  See the discussion and the diagrams in the blog entry  Slaying Two Dragons with the Sound of Silence: – How to Keep Your Repetitive DNA Turned Off with “3 Songs”: Sirtuins, Polycomb Proteins, and DNMT3.

The major (non-histone) “longevity mechanism” is the effect of SIRT1 on p53, also shown below.

NAD+12

In the diagram above, SIRT1 deacetyates the p53 protein thereby reducing its transcriptional activity.  p53 is the master “guardian of the genome”, and serves as a tumor suppressor to prevent cancer formation by shutting off the cell cycle in damaged cells. So, turning it off in cancer cells is not a good idea.  p53 also increases cell survival in non-cancerous cells and also activates apoptosis when the cells are old or too damaged to repair.

From the 2012 publication Current advances in the synthesis and antitumoral activity of SIRT1-2 inhibitors by modulation of p53 and pro-apoptotic proteins” — As sirtuins are involved in many physiological and pathological processes, their activity has been associated with different human diseases, including cancer. Especially two sirtuin members, SIRT1 and SIRT2, have been found to antagonize p53-dependent transcriptional activation and apoptosis in response to DNA damage by catalyzing p53 deacetylation. The findings that SIRT1 levels are increased in a number of tumors highlight the oncogenic role of sirtuins, in particular, in the down-modulation of p53 oncosuppressor activity. Along this lane, cancers carrying wild-type (wt) p53 protein are known to deregulate its activity by other mechanisms. Therefore, inhibition of SIRT1 and SIRT2, aimed at restoring wt-p53 transcriptional activity in tumors that retain the ability to express normal p53, might represent a valid therapeutic cancer approach specially when combined with standard therapies.”

In addition, SIRT1 deacetylates a specific site on the histone 4 subunit of the nucleosomes, which are the spools on which DNA is wound.  When SIRT1 deacetylates lysine at position 16 on the histone 4 protein (H4K16), this silences the gene on the DNA wound around this histone.  Thus SIRT1 has multiple effects all due to its ability to remove acetyl groups from proteins.  These are all dependent on the nuclear levels of NAD+ within the cell.

Image and legend source: Sirtuins: critical regulators at the crossroads between cancer and aging “ SIRT1 expression and activity decrease during aging. High levels of SIRT1 protein in young cells in combination with high levels of nicotinamide adenine dinucleotide lead to deacetylation of p53 and histone proteins to promote longevity (left panel). SIRT1 levels are lower in aged cells, and higher levels of nicotinamide inhibit SIRT1 activity (right panel). The resulting hyperacetylation of p53 can induce replicative senescence. Deacetylation of histone H1 leads to its degradation that promotes formation of senescence-associated heterochromatic foci (SAHFs). Failure to downregulate SIRT1 during aging may promote cell survival after oxidative damage, leading to the accumulation of mutations, and an increased risk of cancer development.”

Major SIRT1 Functions – #3: Circadian Clock Control – keeping the clocks in every cell regulated

We discussed the role of SIRT1 in controlling circadian clocks in the Part 1 blog entry,  Also, you can see the blog entries Circadian Regulation,NMN, Preventing Diabetes, and Longevity  and Shedding new light on circadian rhythms. Pathways involved are illustrated in this diagram:

NAD+13

Image source: Circadian integration of metabolism and energetics

Major SIRT1 Functions – #4 : Reducing Inflammation – inhibition of both glucose and fatty acid-induced inflammation.

Inflammation and aging seem to go hand-in-hand, a process that has been referred to as “inflammaging”.  Caloric restriction can reverse a significant amount of this inflammaging as a result of both the reduction in insulin/IGF-1 pathway signaling and also via a reduction in free fatty acid-induced inflammation.  Sirtuin activation associated with CR has been shown to have a major role in CR as a molecular mediator of this effect on inflammaging.  Here are some of the ways this occurs:

SIRT1 and Glucose-mediated inflammaging – Insulin resistance is the hallmark of this pathway of inflammaging.

SIRT1 improves insulin sensitivity in insulin resistant states by inhibiting protein tyrosine phosphatase 1B (PTP1B), which is a direct intracellular inhibitor of insulin action (This effect is only seen in insulin resistant states, however, and not in insulin sensitive states).  In addition, SIRT1 increases the circulating plasma hormone that is made in fat, called adiponectin, which increases insulin sensitivity.  Adiponectin also directly inhibits TNF-α and the conversion  of macrophages to foam cells.  This reduces adhesion molecules and the number of macrophages attached to endothelial cells.  These SIRT1-induced effects reduce the atherosclerotic effects of insulin, high sugar diet, and high fat diets.

Probably the greatest positive effect of SIRT1 on inflammaging is by its inhibition of NF-kβ signaling. Normally, insulin signaling increases inflammation via Akt-mediated activation of the “master inflammatory switch”, NF-kβ, a transcription factor that turns on all of the major inflammatory genes. However, when SIRT1 activity is increased, SIRT1 deacetylates NF-kβ and this inactivates the transcription factor.  As a result, all of the NF-kβ controlled inflammatory genes show less expression, including TNF-α, IL-6, C-reactive protein, and the cJun N-terminal Kinase (JNK) transcription factor. The diagram below illustrates this well:

NAD+14

Image and legend source:  Sirtuins in neurodegenerative diseases: an update on potential mechanisms” Anti-inflammatory mechanisms of SIRT1. SIRT1 deacetylates p65 and blocks the transactivation of NF-κB-dependent gene expression. SIRT1 suppresses the activity of PARP-1, a coactivator of NF-κB-dependent transcription, by deacetylation and by inhibiting its expression. PARP-1 activation could deplete NAD+, resulting in inhibition of SIRT1 and NF-κB activation. On the epigenetic level, SIRT1 represses NF-κB-dependent inflammatory gene expression by deacetylating H4K16 and also by recruiting more components of repressor complexes. SIRT1 deacetylates and activates histone methyltransferase SUV39H1, which suppresses expression of inducible inflammatory genes. DNA methylation is associated with suppressed expression.”

In summary, SIRT1 has a net anti-inflammatory effect in glucose-mediated inflammaging and increases insulin sensitivity in insulin-resistant conditions, but not in baseline conditions of insulin-sensitive states.

SIRT1 and Fatty acid-mediated inflammaging – High fat diets have also been shown to induce inflammaging. Here the pro-inflammatory effects are independent from insulin and are mediated by intracellular free fatty acid overload within cells.  These high free fatty acid levels inhibit the forkhead transcription factor, FOXO1. As a result of FOXO1 inhibition, there is an increase in ROS, IL-6, PAI-1, and MCP-1 gene expression.  SIRT1 increases adiponectin levels which in turn, activates FOXO1 activity and increases the interaction between FOXO1 and C/EBPβ transcription factors.  In summary, SIRT1 has a net anti-inflammatory effect in fatty-acid mediated inflammaging and this effect is mediated primarily by adiponectin and the FOXO1 transcription factor.

Conclusion: Restoring NAD+ levels to normal in the nucleus of cells should increase insulin sensitivity, increase adiponectin levels, and reduce inflammation induced by both a high glucose and high fat diet.  We predict that biomarkers for insulin sensitivity (fasting blood sugar, 2-hour glucose tolerance testing, fasting insulin, 2-hour insulin tolerance testing, HOMA2, HOMA-IR, and Glycomark tests will improve with increases of nuclear NAD+, but only in insulin resistant states.  We also predict that biomarkers for inflammation will decline with such nuclear NAD+ increases, especially adiponectin (increase), TNF-α (decrease), IL-1β (decrease), IL-6 (decrease), IL-8 (decrease), andC-reactive protein (decrease).  However, if these values are normal in baseline conditions in a person undertaking to increase their nuclear NAD+, no change in these numbers will be evident.

Major SIRT1 functions: #5 – DNA repair – It is a key enzyme in repairing double stranded DNA breaks (DSBs)

Homologous recombination is the highest-fidelity mechanism for repairing DSCs.  See UBI et al.  Role of SIRT1 in homologous recombination (2010) ” — Recent reports revealed that SIRT1 also deacetylates several DNA double-strand break (DSB) repair proteins. — Using nuclear foci analysis and fluorescence-based, chromosomal DSBrepair reporter, we find that SIRT1 activity promotes homologous recombination (HR) in human cells. Importantly, this effect is unrelated to functions of poly(ADP-ribose) polymerase 1 (PARP1), another NAD(+)-catabolic protein, and does not correlate with cell cycle changes or apoptosis.”   That is, the effect is independent of the PARP-related mechanism for repairing single-stranded breaks.

Major SIRT1 functions: #6 – deacetylation nonhistone proteins – transcription factors, co-activators, co-repressors, methyl binding proteins, etc.

In addition to the effects of SIRT1 on histones, p53, and DNA repair, SIRT1 deacetylates many other proteins within the nucleus.  Most of these are transcription factors or co-activators which increase gene expression. They include the ones listed in this diagram:

NAD+15

Image and legend source: Mammalian Sirtuins and Energy Metabolism  “ The diverse functions of SIRT1 in central nutrient sensing and peripheral energy metabolism. The activity of SIRT1 is regulated by the cellular metabolic status, small molecule activators, interacting proteins, as well as post-translational modifications. After activation, SIRT1 modulates a variety of metabolic activities systemically and locally through either direct protein deacetylation or indirect chromatin remodeling.”

Following is a list of the non-histone protein targets of SIRT1 shown above, along with several other CR proteins not shown in the diagram.  SIRT1 deacetylates over a dozen non-histone proteins, including p53, HSF1, eNOS, STAT3, FOXOs, PGC-1a, PPARγ, LXR, NF-kB, PER2, CLOCK, UCP2, MyoD.   Other effects of SIRT1 are paradoxic are the effects of SIRT1 on FOXA2, LKB1, and Nrf2 (E2F1).

p53Ac – The most important non-histone target of SIRT1 is the protein called p53 already mentioned here. Both SIRT1 and SIRT2 deacetylate p53 and thereby increase cell survival.  SIRT1 deacetylates lysine 382 (K382) on the SIRT1 protein. On the other hand, inhibiting both SIRT1 and SIRT2 (but not one of these two SIRTs) induces cell death in both cancer cells and non-cancer cells. Thus p53 has often been referred to as the “master tumor suppressor” or the “guardian of the genome”.  Regardless of the terminology describing p53, it is clear that both SIRT1 and SIRT2 deacetylate p53 and promote cellular survival.  This is one of the most important functions of SIRT1/2 and is a fundamental reason why Sirtuins probably promote longevity (Peck, SIRT Inhibitors Induce Cell Death and p53 Acetylation through Targeting Both SIRT1 and SIRT2  2010).

HSF1Ac – Heat shock factor 1 is a thermally sensitive protein that trimerizes migrates into the nucleus in response to heat stress or oxidative stress.

LXRAc – Caloric restriction has a positive effect on lowering cholesterol.  This is due to the fact that SIRT1 deacetylates the nuclear           receptor liver X receptor (LXR) protein

FXRAc – The farnesoid X receptor regulates the expression of a number of its target genes(ref).

eNOSAc – Reduced caloric intake decreases arterial blood pressures in healthy individuals and improves endothelium-dependent vasodilation in obese individuals.  Endothelial nitric oxide synthetase (eNOS) is an enzyme that makes nitric oxide from L-arginine, relaxes blood vessels and thereby lowers blood pressure with CR. L-arginine is a popular “anti-aging” supplement that is marketed as a “CR mimetic”, but has failed to lengthen life span or increase health span.  The likely reason for the “arginine failure” is that SIRT1 is needed to remove the “acetyl group” from lysines 496 (K496) and 506 (K506) from eNOS.   This can only be accomplished by activating SIRT1 with CR or fasting.  However, with old age, there is inadequate NAD+ to drive SIRT1 deacetylation.  It is for this reason that we predict that NAD+ or NAD+ precursors like NMN will lower blood pressure in healthy individuals and improve flow-mediated dilation (FMD) in both healthy and obese individuals.

FMD (flow-mediated dilation) can easily be measured with clinically validated testing devices such as the EndoPAT or the VENDYS systems, which use a brachial blood pressure cuff and finger sensors which measure reactive hyperemia index (EndoPAT) or finger temperature warming (VENDYS).  Both systems are clinically validated as non-invasive measurements of eNOS activity.

FOXAAc – The FOXA symbol in the diagram above is an upstream event that activates Glucagon signaling and inhibits Insulin signaling.  Reduced caloric intake reduces insulin levels yet increases insulin sensitivity, increases glucagon production, and improves pancreatic β-cell function via the forkhead transcription factor called FOXA2.  FOXA transcription factors play a key role in gene regulation of genes involved with glucose metabolism by binding to promoter regions on these genes with SIRT1. When nutrients are scare or when cells are starved, FOXOA2 can be acetylated in at least 7 different lysine residues. This increases FOXOA2 protein stability.  SIRT1deacetylates these lysine residues in times of CR or starvation.  NAD+ depletion disrupts this SIRT1-mediated stress response and results in excessive accumulation of FOXOA2 proteins. Restoring nuclear NAD+ levels should restore the normal interaction between FOXOA2 and SIRT1.  This could be measured with oral glucose tolerance testing (OGTT), insulin tolerance testing (ITT), fasting blood glucose and fasting insulin, as well as HbA1c and Glycomark testing.

FOXOAc – Caloric restriction, fasting, and reducing Insulin/IGF-1 pathway signaling all promote the nuclear localization of the Forkhead homeobox type O transcription factors (FOXOs).  FOXOs play a major role in cellular stress resistance by turning on genes that code for anti-oxidant enzymes, such as MnSOD (SOD2) and catalase.  FOXOs also control genes for apoptosis. Unfortunately, none of these benefits occur unless blood sugar levels are low and insulin/IGF-1 signaling is low.  In these conditions, the FOXOs can all migrate into the cell nucleus and turn on many genes responsible for cellular stress resistance.  Both SIRT1 and SIRT2 deacetylate FOXO transcription factors and thereby activate the FOXOs. For instance, SIRT1             deacetylates FOXO1 which then increases gluconeogenesis in the liver. SIRT2 deacetylates FOXO3a and also promotes hepatic gluconeogenesis.

We predict that restoring nuclear NAD+ levels with NAD+ precursor therapy will increase cellular stress resistance and this could be measured by stem cell survival with stem cell harvesting, stem cell transplantation, and stem cell banking (protection from freeze/thawing induced apoptosis).  This could be easily measured in the laboratory by culturing stem cells under conditions of cellular stress. 

LKB1Ac – Liver kinase B1 (LKB1) is an enzyme that mediates much of the effect of mitochondrial biogenesis with CR or fasting. This occurs due to the fact that LKB1 is a direct activator of AMPK, the master regulator of metabolism and mitochondrial biogenesis.  Recently, it has been disclosed that LKB1 also functions as a tumor suppressor by maintaining epithelial integrity. In tumors where LKB1 is mutated, cells loose an orderly epithelial configuration and the cancers start to grow rapidly  LKB1 must be deacetylated by SIRT1 for proper tumor suppression and for AMPK activation. SIRT1 deacetylates lysine 48 (K48) on LKB1 and causes the LKB1 to leave the nucleus and go to the cytoplasm where it can associated with the adaptor protein STE20, activating itself and AMPK.

Thus, SIRT1 deacetylation of LKB1 induces LKB1 and AMPK activity in the cytoplasm. (Ruderman, SIRT1 Modulation of the Acetylation Status, Cytosolic Localization, and Activity of LKB1 2008). Both of these effects promote longevity and increase energy. For this reason, we predict that restoring nuclear NAD+ levels will have a direct effect on reducing age-induced fatigue.  This could be measured with a VO2 max measuring device and endurance testing.  We also predict that restoring nuclear NAD+ levels will re-organize skin cells that have lost their epithelial polarity, restoring better looking skin and improving skin histologic architecture on light microscopy.

MyoDAc – MyoD is a key transcription factor in skeletal muscle differentiation.

STAT3Ac – Skeletal muscle insulin resistance is a key component of the underlying cause of type II diabetes. Fasting and CR both can increase whole body insulin sensitivity, even when practice for brief periods of time (4-20 days). Although there are multiple ways that CR changes Insulin/IGF-1 signaling, the primary method by which CR promotes insulin sensitivity appears to be via the (inhibition of )  STAT3.  SIRT1 deacetylates STAT3, which inactivates the transcription factor. This reduces gene expression for two subunits of the enzyme PI3K (p55a/p50a). This results in more efficient PI3K signaling with insulin stimulation.  In summary, SIRT1 inhibits STAT3 which inhibits gene expression for PI3K subunits, thereby increasing energy expenditure.

IRS1/IRS2Ac – CR has a paradoxic effect on insulin receptor substrate 1 and 2 (IRS1/IRS2) gene expression. IRS1 and IRS2 are the first proteins in the molecular cascade of events that occurs when Insulin binds to the Insulin/IGF receptor on the cell.  With CR, however, there is an increase in protein expression of IRS1/IRS2 in muscle, which in turn increases intracellular signaling for the Insulin/IGF-1 pathway.  SIRT1 deacetylates IRS1/IRS2, thereby “turning off” this pro-aging pathway.

Nrf2Ac (E2F1) – Sirtuins have a paradoxic effect on the transcription factor called Nuclear factor Erythroid 2-related factor 2 (Nrf2 or E2F1). Normally, cellular stress activates the nuclear localization of Nrf2 which then in turn activates gene transcription by binding to the antioxidant response element (ARE) found in the promoter sites of these genes. This can occur due to direct effect of ROS on Nrf2 binding to its binding partner, Keap-1. It can also occur by the acetylation of Nrf2 by p300/CBP (aka CREB-binding protein). Paradoxically, both SIRT1 and SIRT2 can deacetylate Nrf2, SIRT1 deacetylates at two specific  lysine residues (K588 and K591) which promotes cytoplasmic localization of the Nrf2 protein and prevents nuclear localization of Nrf2. As a result, SIRT1 actually is an inhibitor of Nrf2  antioxidant gene transcription. This is one of the paradoxic effects of SIRT1 and CR on Nrf2 (Kawai, Acetylation-Deacetylation of the Transcription Factor Nrf2 (Nuclear Factor Erythroid 2-related Factor 2) Regulates Its Transcriptional Activity and Nucleocytoplasmic Localization 2010).

Another paradoxic effect is the effect of SIRT2 on Nrf2.  SIRT2 also deacetylates Nrf2 and therefore can decrease Nrf2 gene expression.  One of these genes is the major cellular iron exporters called FPN1.  Nrf2 activates FPN1 gene expression and SIRT2 inhibits FPN1 gene expression (Yang, Sirtuin 2 mediated-deacetylation regulates cellular iron homeostasis

2014).  Thus SIRT2 controls regulates iron levels within the cell, decreasing iron export by FPN1, by deacetylating Nrf2.

p66shc – The adaptor protein p66shc is a major “pro-aging” factor within the cell.  When growth factor signaling or ROS activates p66shc, the protein is phosphorylated on Serine (S).  This promotes mitochondrial migration of p66shc into the mitochondrial matrix where it increases ROS production from mitochondria. SIRT1 decreases p66shc activity by decreasing both p66shc mRNA and p66shc protein levels.  The molecular mechanism responsible for SIRT1 repression of p66shc gene expression is thought to be mediated by the epigenetic silencing of the p66shc gene by SIRT1-mediated histone deaetylation. Thus SIRT1 is a negative          regulator of ROS production by epigenetically repressing p66shc gene expression (Xu, Salvianolic acid A preconditioning confers protection against concanavalin A-induced liver injury through SIRT1-mediated repression of p66shc in mice 2013) (Paneni, Molecular pathways of arterial aging 2015).

PGC-1αAc – Peroxisome proliferator activated receptor PPAR-γ co-activator-1α (PGC-1 α) is a master gene co-activator that works with over a dozen different transcription factors to turn on hundreds of genes involving peroxisome and mitochondrial biogenesis.  Thus, PGC-1α turns on all of the genes involving making and burning of fat, producing of ketones, making ATP, and generating energy.  SIRT1 also promotes gluconeogenesis in the liver by deacetylating PGC-1α (along with deacetylating FOXO1).  Earlier blog entries discussing PGC-1 α can be found in this list,

PPARγ – Caloric restriction also activates the master transcription factor for the production of peroxisomes, called peroxisome proliferator activator receptor gamma (PPARγ). PPARγ induces long chain fatty acid oxidation, which is the first step required before fats can be oxidized in the mitochondria by beta-fatty acid oxidation.  SIRT1 deacetylates PPARγ and then recruits a coactivator for PPARγ called Prdm16.  Then the PPARγ/Prdm16 complex can activate the genes required to make brown fat.  You can see the blog entry Getting skinny from brown fat,

NF-kβ – Nuclear factor kappa-β is a transcription factor that is the “master switch” for inflammation. SIRT1 deacetylates NF- kβ at the p65 subunit of NF-kB at lysine 310.  This “turns off” gene expression for all of the genes involving inflammation, such as CRP, TNF- α, IL-1β, etc.  Since arthritis is a universal feature of aging and all of these inflammatory biomarkers are elevated in joints with OA, we predict that restoring nuclear NAD+ with NAD+ precursor therapy will reduce the signs and symptoms of OA.  This could be measured with various validated clinical scoring systems, such as the K-L scoring system based on plain, X-rays with weight bearing, the WOMAC scale, the IKDC scale, etc.  All of these should improve with NMN or other NAD+ enhancing therapy.

Fine Tuning of SIRT1 Protein Activity

Endogeneous Activators and Inhibitors of Sirtuins – NAD+, NAM, AROS, lamin A, Tenovins, DBC1/CCAR2, CD38,  HIC1, Cathepsin D

Alas, in biology for any generalization there is too often one or more “An the other hands, —.”  We have discussed SIRT1expression here as if it were mainly driven by NAD+ level.   Actually, it is affected both positively and negatively by many interacting variables. SIRT1 activity is increased by increasing nuclear NAD+, by fasting, and by caloric restriction. Most of these positive effects on SIRT1 are mediated by increases in nuclear NAD+ levels. SIRT1 activity is also inhibited by nicotinamide. This may be why niacin supplementation has not been shown to have a longevity effect, since it is converted into nicotinamide within the cell.  In addition to activating SIRT1 with NAD+, there are naturally derived exogenous compounds such as reseveratrol and other plant-based compounds that bind to a different site on SIRT1, increasing it’s activity independently from NAD+. There are also several exogenous small molecule inhibitors of SIRT1, such as Tenovin-1 and Tenovin-6 which prevent the deacetylase activity of SIRT1. There are also endogenous proteins that bind directly to SIRT1 and increase or decrease the activity of SIRT1, such as AROS, lamin A, and DBC1.  AROS is a protein that binds to SIRT1 and increases its activity.  DBC1 (aka CCAR2) is a protein that binds to SIRT1 and inhibits SIRT1.  We discussed these two in the Part 1 blog entry.  HIC1 is a protein that binds to the SIRT1 promoter and increase SIRT1 gene expression. Another recently discovered protein that binds to SIRT1 is lamin A, a nuclear cytoskeletal protein that binds to SIRT1 and activates the protein.  Another endogenous inhibitor of SIRT1 is the enzyme CD38. Although initial reports labeled CD38 as an “ectoenzyme”, several reports have shown that the CD38 protein is found on the inner membrane of the nucleus of the cell. Aksoy and colleagues have  shown that CD38 is an SIRT1 inhibitor (Aksoy, Regulation of SIRT 1 mediated NAD dependent deacetylation: A novel role for the multifunctional enzyme CD38  2006).

In inflammatory joint diseases such as osteoarthritis and rheumatoid arthritis, there is another endogenous disruptor of SIRT1 – Cathepsin B.  Normally SIRT1 has a positive effect within the joint, inhibiting chondrocyte apoptosis and promoting extracellular matrix synthesis of proteins such as alpha2(I) collagen. However, when there are high levels of TNF-a within the joint, TNF-a mediates a Cathepsin-B induced cleavage of the SIRT protein at amino acid 533, creating a 75-kd SIRT1 fragment that is no longer functional.  As a result, there are low levels of SIRT1 activity within the chondrocytes and synovial cells of arthritic joints.

Some of these activators and inhibitors of SIRT1 activity are illustrated below:

NAD+16

Image source: How does SIRT1 affect metabolism, senescence and cancer?

DBC1 (aka CCAR2) is one of the primary inhibitors of  SIRT1 within the nucleus and it is activated by DNA damage (genotoxic stress).  Fasting also inhibits the interaction of DBC1 and SIRT1.  This may be a NAD+-independent molecular mechanism for how fasting increases SIRT1 activity.  Likewise, a high fat diet activates SIRT1 binding to DBC1. This may be an NAD+-independent effect of a high-fat diet inhibiting Sirtuins.

NAD+17

SIRT1 Phosphorylation – SIRT1 phosphorylation can increase or decrease SIRT1 activity and SIRT1 half life

In addition to the above factors, SIRT1 is modified  by at least 7 enzymes that phosphorylate SIRT1 at 13 “site-specific” locations on the SIRT1 protein. These enzymes phosphorylate different sites, and thereby either inactivate SIRT1 (AMPK),  activate SIRT1 (cAMP/PKA, Cyclin B/Cdk1, CK2, JNK1, DYRKs) or increase the half life of the SIRT1 protein (JNK2). For instance AMPK phosphorylates SIRT1 at Thr344, which inactivates the p53 deacetylase function of SIRT1.  On the other hand, DYRKs (Dual specificity tyrosine phosphorylation-regulated kinases) phosphorylate SIRT1 at Thr522 in response to environmental stress. This DYRK-mediated phosphorylation prevents SIRT1 from forming oligomeric aggregates of SIRT1 proteins. As a result, the Thr522 phosphorylated SIRT1 forms only monomers and thereby becomes a more active deacetylator of p53. Thus SIRT1 phosphorylation by AMPK and DYRKs have opposite effects on SIRT1 deacetylation of p53. Two phosphorylation sites on SIRT1 help regulate the cell cycle. Specifically, the CyclinB/Cdk1 complex phosphorylates SIRT1 at Thr530 and Ser540.  Thus CyclinB/Cdk1 phosphorylation of SIRT1 increases cell proliferation.  This is why SIRT1 is so important for cell growth and is present in high levels in mitotically active cells. Even the two cJun N-terminal kinases, JNK1 and JNK2, have different phosphorylation sites on SIRT1 and different effects. For instance, JNK1-mediated phosphorylation of the Ser47 residue on SIRT1 increases the histone 3 (H3) deacetylase activity of SIRT1, whereas JNK2-mediated phosphorylation of the Ser27 residue of SIRT1 increase the half life of the SIRT1 protein from 2 hours to 9 hours. Thus each of these different “SIRT1 phosphorylators” add a phosphate group at specific sites on SIRT1. This increase specific activities of SIRT1 and these effects are independent of NAD+ levels within the cell. Last of all, UV light and free radicals such as hydrogen peroxide (H2O2) inactivates SIRT1 by another protein called SENP. SENP inactivates SIRT1 by removing a sumoyl group from the SIRT1 protein.  Here are some diagrams that illustrates how “SIRT1 phosphorylators” activate SIRT1 activity and how de-sumoylation of SIRT1 inhibits SIRT1.

De-SUMOylating SIRT1

UV light or H2O2 inactivate SIRT1 via a UV/ROS sensitive protein called SENP.  SENP removes a SUMO group from SIRT1, thereby inactivating the protein.

NAD+21

ROS activation of SIRT1

Free radicals such as H2O2 or superoxide can activate the SIRT1 protein via a protein kinase called c-Jun Kinase (JNK), increasing the ability of SIRT1 to deacetylate histone proteins such as H3.

NAD+19

SIRT1 and Cell Survival

DYRKs are enzymes that are activated by  ROS  or (environmental stress) or DNA damage. DYRKs activate SIRT1-mediated deacetylation of p53, thus increasing cell survival in stressful times.

NAD+20

SIRT1 and the Cell Cycle

Two proteins involved with cell cycle regulation form a complex with SIRT1 and phosphorylate the SIRT1 protein.  This increases cell proliferation and is a key factor in allowing mitotic cells to keep dividing.

NAD+18

In summary, SIRT1 activity is dependent on NAD+ levels, nicotinamide levels, phosphorylation status, DBC-1 status, AROS status, ROS or other stressors, and the presence of exogenous SIRT1 activators (resveratrol) or inhibitors (Tenovins).

The over-all importance of Sirtuins, however, is their ability to affect gene expression in the nucleus. Changing gene expression is is considered an “upstream event” in aging.  In this aspect, SIRT1 is not the only Sirtuin in the nucleus of the cell.  There are two others that we will discuss now – SIRT6 and SIRT7.

SIRT6 – The Master DNA Repair and Genomic Stability Sirtuin

The effects of SIRT6 on Genome Stability

Another Sirtuin family member that directly affects gene expression is SIRT6.  SIRT6 is also found in the nucleus and is also an NAD+-dependent enzyme.  Like SIRT1, SIRT6 plays a vital role in both single strand DNA breaks (SSBs) and double stranded DNA breaks (DSBs). With DSBs, SIRT6 is one of the earliest factors that is recruited to the site of the DSB. This occurs within 5 seconds in a healthy cell. SIRT6 recruits the chromatin remodeler, SNF2H, to the site of the DSB and deacetylates histone 3 at lysine 56 (H3K56). This allows SNF2H to open up the chromatin at the DSB site, which then allows the DNA DSB repair proteins to be recruited (BRCA1, 53BP1, and RPA).  This is shown in the diagram below.

SIRT6 – The Early Responder to DSBs

Double stranded DNA breaks are the most lethal DNA damage type and are primarily responsible for cancer and aging.  Of the seven SIRTs, the SIRT6 isoform probably plays the greatest role in DNA repair.  In the diagram that follows, a double stranded DNA break occurs and within 5 sec, SIRT6 shows up to deacetylate H3K56. This recruits the chromatin remodeler, SNF2H, which then “opens up” the chromatin, allowing the key DNA repair proteins to bind to the site of injury. The key DNA repair proteins include 53BP1, RPA, BRCA1, and CtIP (not shown). Collectively, these proteins repair double-strand DNA breaks by the more accurate repair program called “homologous recombination”, or HR.  The other DNA DSB break repair pathway, NHEJ, is not as accurate a repair pathway as the HR pathway, which is the pathway shown in the diagram.

NAD+22

Image and highlights source SIRT6 Recruits SNF2H to DNA Break Sites, Preventing Genomic Instability through Chromatin Remodeling  “Highlights: •SIRT6 arrives to DNA break sites within 5 s, •Lack of SIRT6 and SNF2H increases sensitivity to genotoxic damage, •SIRT6 directly recruits SNF2H, which in turn opens chromatin at break sites. •SIRT6 and SNF2H are necessary to recruit 53BP1, RPA, and BRCA1 to the damage sites”

With DSBs, SIRT6 plays crucial roles in both the nonhomologous end joining (NHEJ) pathway and the homologous recombination (HR) pathway.  With HR, SIRT6 deacetylates a key protein called CtIP which works with the BRCA1 protein to generate single stranded DNA (aka “DNA end resection”) at the site of the DSB. Unless CtIP and BRCA1 can form a single strand of DNA at the break site by “DNA end resection”, then the non-homologous end joining (NHEJ) method of DNA repair takes over, which does not repair DSBs nearly as accurate as HR.  In addition to the effects of SIRT6 on HR and NHER repair of DSBs,  SIRT6 activates PARP1 directly as well.  This increases DNA repair for single stranded DNA breaks (SSBs) by increasing base-excision repair (BER) pathway for SSBs and also increases double stranded DNA repair by both HR and NHEJ pathways.  All of these effects have a net effect of increasing genome stability within the cell.

NAD+23

Image and legend source: 2011 Repairing split ends: SIRT6, mono-ADP ribosylation and DNA repair  “SIRT6 regulates genomic stability. SIRT6 promotes genome stability by regulating DNA single-strand and double strand break repair pathways and by facilitating telomere maintenance. The deacetylase and the mono-ADP ribosyltransferase activities of SIRT6 both contribute to this function.”

Based on the three mechanisms illustrated above, SIRT6 plays a key role in both SSB and DSB repair.  As a result, when SIRT6 knock-out mice are created, they have an accelerated aging-like phenotype.

The Other Effects of SIRT6 besides Genome Stability

Other than genomic stability, the other primary effect of SIRT6 are mediated by histone protein modifications of specific lysines found on Histone 3 (H3K9 and H3K56).  Although SIRT1 has some histone protein deacetylase function, it has a much greater mono-ADP-ribosyltransferase activity and an even greater “deacylation” function. “Deacylation” refers to the removal of long chain fatty acids (palmitic acid, myristic acid, oleic acid, and linoleic acid) from lysine amino groups on proteins.  One of the SIRT6 targets for ADP-ribosylation is PARP1 (see above).  PARP1 is mono-ADP-ribosylated by SIRT6 which activates PARP1.  The “deacylation” function of SIRT6 was just recently discovered. Here is a diagram that shows the effects of SIRT6 deacetylase function and the effects of SIRT6 on CtIP which also occurs via its effects on gene expression in the nucleus of every cell.  (The mono-ADP-ribosyltransferase and deacylation function of SIRT6 are not shown in the diagram).

NAD+24

Illustration  and legend reference “SIRT6 is a critical regulator in genome stability, metabolism, and inflammatory response. By deacetylation of H3, SIRT6 regulates metabolic homeostasis and inflammatory response in peripheral tissues, while functioning as a central regulator of somatic growth.”

As you can see, most of the effects of SIRT6 are mediated by histone protein deacetylation of two specific lysine residues found on the histone 3 (H3) subunit of the histone “spools” that wind up DNA and compact it into the nucleus.  The H3 subunit has two specific lysine residues found on the tail of “H3” protein called H3K9 and H3K56.  When these two sites are deacetylated by SIRT6, this inhibits the type of metabolism seen in cancer cells called “Warburg metabolism” where the cells are totally dependent on sugar for generating energy and cannot burn fats.  Also, SIRT6 inhibits the inflammation caused by the major inflammatory “on switch” called NF-kB.  The other mechanisms of action of SIRT6 have not been fully elucidated, such as the effects of SIRT6 on growth hormone, IGF-1 and telomere stability.  What appears clear, however, is that all of the effects of SIRT1 and SIRT6 occur in the nucleus of the cell and all of the activity of both SIRT1 and SIRT6 are dependent on the availability of NAD+ in the nucleus of the cell.

The effects of SIRT7 within the cell, SIRT7 activators and SIRT7 inhibitors

NAD+25
NAD+26

Image source: A Big Step for SIRT7, One Giant Leap for Sirtuins… in Cancer

Outlier Research

Unfortunately, there are research results that challenge some of the basic assumptions of those who are looking for health creation through enhancing body NAD+ levels. And, they chellenge some of our predictions listed above.   For example there seems widespread agreement that:

  1. If you want to activate SIRT1, you must have adequate levels of nuclear NAD+.

2,  Having adequate levels of nuclear NAD+ is the best way to keep SIRT1 active.

  1. The impact of SIRT1 is universally health-producing.

Unfortunately, there appears to be research that suggest that these broad assertions ain’t always necessarily so.  We have above discussed how SIRT1 phosphorylation can increase or decrease SIRT1 activity and a number of proteins that bind to SIRT1 and increase or decrease its activity.  Cold is another example.

Cold can activate SIRT1 in a way that is completely independent of NAD+

You can use cold to activate SIRT1 and PGC1alpha regardless of your nuclear NAD+ status. The December 2011 article The cAMP/PKA Pathway Rapidly Activates SIRT1 to Promote Fatty Acid Oxidation Independently of Changes in NAD+ reports: “Highlights

  • Stimulation of the cAMP/PKA pathway results in phosphorylation of SIRT1 serine 434
  • SIRT1 S434 phosphorylation increases intrinsic deacetylase activity
  • SIRT1 activation by S434 phosphorylation is rapid and independent of changes in NAD+
  • S434 phosphorylation induces PGC-1α deacetylation and increased fatty acid oxidation”

“The NAD+-dependent deacetylase SIRT1 is an evolutionarily conserved metabolic sensor of the Sirtuin family that mediates homeostatic responses to certain physiological stresses such as nutrient restriction. Previous reports have implicated fluctuations in intracellular NAD+concentrations as the principal regulator of SIRT1 activity. However, here we have identified a cAMP-induced phosphorylation of a highly conserved serine (S434) located in the SIRT1 catalytic domain that rapidly enhanced intrinsic deacetylase activity independently of changes in NAD+ levels. Attenuation of SIRT1 expression or the use of a nonphosphorylatable SIRT1 mutant prevented cAMP-mediated stimulation of fatty acid oxidation and gene expression linked to this pathway. Overexpression of SIRT1 in mice significantly potentiated the increases in fatty acid oxidation and energy expenditure caused by either pharmacological β-adrenergic agonism or cold exposure. These studies support a mechanism of Sirtuin enzymatic control through the cAMP/PKA pathway with important implications for stress responses and maintenance of energy homeostasis.”

We have discussed cold as a simple and practical hormetic healh intervention in previous blog entries(ref)(ref)(ref)(ref).

SIRT1 turns off Nrf2 and its health-producing affects

We mentioned this above.  We have published several blog entries on the numerous impacts of activating Nrf2 which turns on hundreds of positive “antioxidant response genes.” See this list.  One of the last things we would expect to find is that SIRT1 which also produces numerous health benefits actually turns Nrf2 off. but that appears to be the case. It tends to keep Nrf2 in the cytoplasm rather than allowing it to migrate to the nucleus where it can do good.  Even worse, resveratrol, our favorite SIRT1 activator actually inhibits expression of Nrf2.

SIRT1 “turns off” Nrf2-mediated antioxidant gene expression?

The 2010 publication: Acetylation-Deacetylation of the Transcription Factor Nrf2 (Nuclear Factor Erythroid 2-related Factor 2) Regulates Its Transcriptional Activity and Nucleocytoplasmic Localization reports: “Activation of Nrf2 by covalent modifications that release it from its inhibitor protein Keap1 has been extensively documented. In contrast, covalent modifications that may regulate its action after its release from Keap1 have received little attention. Here we show that CREB-binding protein induced acetylation of Nrf2, increased binding of Nrf2 to its cognate response element in a target gene promoter, and increased Nrf2-dependent transcription from target gene promoters. Heterologous sirtuin 1 (SIRT1) decreased acetylation of Nrf2 as well as Nrf2-dependent gene transcription, and its effects were overridden by dominant negative SIRT1 (SIRT1-H355A). The SIRT1-selective inhibitors EX-527 and nicotinamide stimulated Nrf2-dependent gene transcription, whereas resveratrol, a putative activator of SIRT1, was inhibitory, mimicking the effect of SIRT1. Mutating lysine to alanine or to arginine at Lys588 and Lys591 of Nrf2 resulted in decreased Nrf2-dependent gene transcription and abrogated the transcription-activating effect of CREB-binding protein. Furthermore, SIRT1 had no effect on transcription induced by these mutants, indicating that these sites are acetylation sites. Microscope imaging of GFP-Nrf2 in HepG2 cells as well as immunoblotting for Nrf2 showed that acetylation conditions resulted in increased nuclear localization of Nrf2, whereas deacetylation conditions enhanced its cytoplasmic rather than its nuclear localization. We posit that Nrf2 in the nucleus undergoes acetylation, resulting in binding, with basic-region leucine zipper protein(s), to the antioxidant response element and consequently in gene transcription, whereas deacetylation disengages it from the antioxidant response element, thereby resulting in transcriptional termination and subsequently in its nuclear export.”

A brand new 2015-dated publication seems to say the direct opposite to the just-cited publication Polydatin promotes Nrf2-ARE anti-oxidative pathway through activating Sirt1 to resist AGEs-induced upregulation of fibronetin and transforming growth factor-β1 in rat glomerular messangial cells.  “Highlights:

  • PD increased Sirt1 levels, promoted Nrf2-ARE pathway activation, and reduced ROS levels in AGEs-treated GMCs. (PD is a resveratrol glycoside)
  • PD resisted AGEs-induced upregulation of FN and TGF-β1 by activating Sirt1-Nrf2-ARE pathway.
  • PD ameliorated DN in a STZ-induced diabetic rat model.”

“Sirt1 and nuclear factor-E2 related factor 2 (Nrf2)-anti-oxidant response element (ARE) anti-oxidative pathway play important regulatory roles in the pathological progression of diabetic nephropathy (DN) induced by advanced glycation-end products (AGEs). Polydatin (PD), a glucoside of resveratrol, has been shown to possess strong anti-oxidative bioactivity. Our previous study demonstrated that PD markedly resists the progression of diabetic renal fibrosis and thus, inhibits the development of DN. Whereas, whether PD could resist DN through regulating Sirt1 and consequently promoting Nrf2-ARE pathway needs further investigation. Here, we found that concomitant with decreasing RAGE (the specific receptor for AGEs) expression, PD significantly reversed the downregulation of Sirt1 in terms of protein expression and deacetylase activity and attenuated FN and TGF-β1 expression in GMCs exposed to AGEs. Under AGEs-treatment condition, PD could decrease Keap1 expression and promote the nuclear content, ARE-binding ability, and transcriptional activity of Nrf2. In addition, PD increased the protein levels of heme oxygenase 1 (HO-1) and superoxide dismutase 1 (SOD1), two target genes of Nrf2. The activation of Nrf2-ARE pathway by PD eventually led to the quenching of ROS overproduction sharply boosted by AGEs. Depletion of Sirt1 blocked Nrf2-ARE pathway activation and reversed FN and TGF-β1 downregulation induced by PD in GMCs challenged with AGEs. Along with reducing HO-1 and SOD1 expression, silencing of Nrf2 increased FN and TGF-β1 levels. PD treatment elevated Sirt1 and Nrf2 levels in the kidney tissues of diabetic rats, then improved the anti-oxidative capacity and renal dysfunction of diabetic models, and finally reversed the upregulation of FN and TGF-β1. Taken together, the resistance of PD on upregulated FN and TGF-β1 induced by AGEs via oxidative stress in GMCs is closely associated with its activation of Sirt1-Nrf2-ARE pathway.”

How do these and possibly many other contrarian or contradictory findings play out in-vivo? We simply don’t know.  We don’t know how all the regulatory feedback inhibition loops will affect each other when we make a major intervention like enhancing body levels of NAD+.  And we don’t know what the effect of many other variables will be, such as initial oxidative state, circadian state, relationship to stressors, disease states, hormonal states, age, sex, etc. Our theories which look at one pathway at a time under standardized conditions cannot tell us that. We will only know the effect of NAD+ enhancement therapy through observing net health effects and key health surrogate biomarkers. And we have to do that on and highly individualized basis That is why we regard the issue of individual biomarkers to have such great importance.

NAD Part 3

Ever since David Sinclair’s seminal 2013 publication relating insufficiency of NAD+ to mitochondrial dysfunctionality(ref, and and for some time before that, many research scientists have thought that insufficient nuclear levels of NAD+ are a major cause of diseases and aging processes including mitochondrial dysfunctionality,  associated deleterious metabolic consequences, and insufficient DNA repair.  Based on much research, this point seems to be solid and incontrovertible.  Having higher levels of NAD+ would doubtlesly be a good thing, especially for older people whose levels decline with age.  The major though not the only avenues of benefit are realized via sirtuins, particularly SIRT1. As is well-recognized and pointed out in many of out earlier blog entries, the sirtuins are essential for multiple key biological processes such as DNA repair and healthy mitochondrial metabolism.  Further, with aging and disease they tend to be in declining supply.  When there is not enough SIRT1. serious consequences can ensue .

In the growing excitement about NAD science during last two years, some researchers and entrepreneurs have further thought that human NAD+ levels can likely be enhanced by ongoing supplementation with NAD precursors such as nicotinamide riboside (NR) or  with  nicotinamide mononucleotide (NMN), The thinking has been that such supplementation might make a significant difference in human health and longevity.  However direct evidence  that NAD levels can be non-transiently enhanced in humans, either intracellular or extra-cellular, is thin to nonexistent.  No direct research shows this.  Likewise, evidence that human health or longevity benefits will result from continuous NAD precursor supplementats  is equally thin,  Enhancement of SIRT1 levels in mice via NMN supplementation and associated health benefits has been observed in only short term trials with mice, ones that lasted only 7 or 10 days (ref)(ref). There appears to be no clear evidence however, either human or animal, that continuing to take a NAD precursor supplement over a long term can lead to higher continuing levels of NAD+ or the many health benefits hypothesized to ensue.

This has led Jim Watson to investigate just what determines human NAD levels and what determines SIRT1 levels.  He found at least 50 such factors  In this current blog entry reports on what he believes are the most important 30 of these. 

This is another Chapter in the extended NAD story, but not the final one.  We expect soon to publish a Part 4 blog entry in this NAD World series.  That blog entry will look with more detail into several additional areas including NAD/Sirtuins and inflammation, and NAD and the much-discussed Warburg effect.  Despite a now-popular perception that scarcity of NAD+ is the main cause of the Warburg effect, Jim Watson points out that the effect  has multiple other causes including three long non-coding RNAs that promote it happening as a key effect in aging and cancer processes. Related to these causes,Jim discusses some possible intervention strategies that go beyond those normally discussed in the longevity literature.

1.  Subcellular compartment levels of NAD regulate aging 

The content of NAD in the nucleus as well as total cellular NAD levels declines with aging.

A.  Animal studies that showed how total cellular NAD decline with aging – DNA damage seems to be the most significant biomarker that correlates with the change in cellular NAD levels

Several papers in the 1990s showed that PARP activity increased with aging in both animal and human cells, but the evidence that NAD declined with aging was not discovered until the past 10 years.  In fact, a 1983 paper by Chapman, Zaun, and Gracy suggested the opposite effect – that NAD levels increased with aging.   The first paper to show that NAD depletion occurred was actually a heart failure study published in 2005.  Pillai and colleagues showed that PARP1 activation resulted in NAD depletion, reduced SIRT2 activity, and myocyte cell death. The next report was in 2008 by Parihar and colleagues, and was astudy of rat hippocampal neurons.  In this in vitro study, they showed that a 50% decline in NAD(H) levels occurred in the aged neurons. (see reference below).  The 3rd report was a study in Wistar Rats, which showed a 4-7 fold decline in NAD+ levels with aging.  ThIs paper by Braidy and colleagues at the U of New South Wales in Sidney, Australia was the first study that specifically was designed to look at declining levels of NAD with aging. (2011).  What was remarkable in this study was that the DNA damage biomarker, pH2AX, seemed to correlate with the decline in NAD levels, even more than other biomarkers for oxidative stress.

B,  Human Studies showing that total cellular NAD levels decline with aging– DNA damage seems to be the most significant biomarker that correlates with the change in cellular NAD levels

Until recently, it was not known if human cells also underwent a decline in NAD content with aging.  The first paper to show this effect in humans was also from Sidney, Australia.  In 2012, Massudi and colleagues showed a precipitous drop in NAD levels within human skin cells, harvested from areas of skin with no sun exposure from infancy to old age.   Although measures of oxidative stress changed with aging (TBARS, MDA, F2-isoprostanes, etc.), the most significant change that correlated with aging was the DNA damage marker, pH2AX, which showed a significant increase with age in both males and females (p < 0.003). This suggests that the most important feature of cellular aging is DNA damage, not lipid oxidation or oxidative stress, per se.   Along with this increase in DNA damage was a dramatic increase in PARP activity (10-fold) in males.  PARP enzymes are NAD-consuming enzymes that sense and help repair single stranded DNA breaks via the base-excision repair (BER) pathway.  Paradoxically, the increase in PARP activity  was not statistically significant in the female cohort.  Even more surprising was the fact that SIRT1 activity did now show a statistically significant change with aging in either males or females, however.  Again, this is puzzling, since NAD levels declined so much.

References: 

C,  Studies that showed how nuclear levels of NAD decline with aging – Studies of Wallerian degeneration in the brain and mitochondrial biogenesis in muscles linked NAD deficiency in the nuclear subcellular compartment to the occurrence of Wallerian degeneration, and the “mitochondrial failure” seen with aging. 

Long ago, there was circumstantial evidence that NAD levels may be regulated on a subcellular compartmental level, the significance of this was unknown, since no one had measured NAD levels in various sub cellular compartments.  For instance, it was well-known that three isoforms of NMNAT existed [NMNAT-1 in nuclear compartment, NMNAT-2   in the Golgi complex, and NMNAT-3 in the mitochondria], the significance of this was not understood.  NMNAT is a key enzyme since it is the only enzyme shared by both pathways for NAD synthesis, the NAD biosynthesis de novo pathway  and the NAD Salvage cycle pathway.

One clue to this significance was the discovery of a mutant mouse model of  Wallerian degeneration called the “Wlds mouse“,  which was protected from developing Wallerian degeneration following axonal injury.  It was discovered that the Wlds mouse  had an 3 extra copies of a 85 kb tandem triplication on the end of chromosome 4 that contained two protein-coding genes for UCH-L1 (UPS gene) and NMNAT-1 (NAD synthesis  gene for the nuclear isoform of NMNAT).  These extra copies of the NMNAT-1 gene in the Wlds mouse resulted in a 3-fold higher NMNAT activity in the brain, but whole brain NAD+ levels were not increased.  This Wlds mouse study was published by Wang and He from Boston in 2009, but they did not show that nuclear NAD levels were actually reduced with aging or Wallerian degeneration.

More recently, in a December 2013 paper Gomes and colleagues from Sinclair’s lab in Boston showed that in mice, declining NAD levels in the nucleus of  muscle cells induced a pseudohypoxic state, disrupting nuclear-to-mitochondrial communication with aging.  In 3 separate experiments, he “knocked down” NMNAT-1, NMNAT-2   and NMNAT-3.  Only NMNAT-1 knock-down mimicked aging and the mitochondrial dysfunction seen with aging.  Although not published, Sinclair has measured NAD levels in the nuclear subcellular compartment and has showed that nuclear NAD levels decline with aging in mice.  In his key 2013 paper, he did publish evidence that IP supplementation with 400 mg/kg of NMN for one week reversed this “pseudohypoxic state” by promoting Tfam-dependent transcription of mitochondrially-encoded OXPHOS genes.

In summary, the indirect evidence about nuclear NAD levels from Wang and He’s work with the Wlds mice with 3 extra copies of the nuclear-specific isoform of NMNAT (NMNAT-1) as well as the more direct work on NMNAT-1 knockdown by Gomes and colleagues in Sinclair’s laboratory have provided tantalizing clues to a major cause of a “Universal phenotype of Aging:”   that of the decline in mitochondrial-encoded OXOPHOS genes,which includes increased ROS generation, Warbug-type metabolism, oxidative stress, NAD depletion, and eventually   ATP depletion in cells.  By turning on the Tfam gene via a SIRT1-mediated mechanism, induced by intraperitoneal high-dose NMN supplementation, Gomes and colleagues   increased expression of mitochondrial DNA encoded genes, thereby increasing the expression of mitochondrially-encoded OXPHOS genes.

Caveat:  The above three paragraphs make a strong case that one major cause of aging is a decrease in the nuclear levels of NAD co-enzyme, required for so many nuclear proteins involving DNA repair, epigenetic gene regulation, and apoptosis.  Unfortunately, no one has yet pin-pointed the cause of this “nuclear NAD decline” or demonstrated  that it can be reversed for more than one week.  The rest of this blog entry goes over 29 possible reasons why nuclear levels of NAD decline with aging.  Until someone demonstrates that they can permanently restore nuclear NAD levels, the exact cause of this aging phenomena must still be considered speculative.  Koch’s postulates must be proven.

2.  NAD/NADH ratio regulates aging independently of NAD content

The longevity gene, NQO1, regulates aging by altering the NAD/NADH ratio in cells.   NQO1 does this by oxidizing NADH to NAD.   Beta-lapachone increases NQO1 enzyme activity and quercetin increases Nrf2-mediated gene expression of NQO1. 

Not only is the NAD content in the nucleus important for delaying/preventing aging, the redox ratio of NAD/NADH is also very important for delaying/preventing aging independently of the total NAD found in the cell.  Many genetic studies in model organisms have searched for “longevity genes” that regulate lifespan.  One of the curious findings from these studies is the gene that codes for the protein “NADH-quinone oxidoreductase 1″, or NQO1.  NQO1 oxidizes NADH to NAD, thereby increasing the NAD/NADH ratio.   Interestingly, Lee and colleagues from Korea recently showed that feeding animals beta-lapachone (aka Beta-L), an exogenous NQO1 co-substrate, prevented the age-dependent decline of motor and cognitive function in aged mice.  Beta-lapchone is a compound originally obtained from the Lapcho tree and has been used for medical purposes for many years.  Beta-L fed mice did not alter their food intake or locomotor activity, but did increase their energy expenditure as measured by VO2max and by heat generation.  The Beta-L fed mice developed changes in gene expression that mimicked 30% caloric restricted diets.  Another molecular effect of beta-Lapachone is that it induces apoptosis in breast and prostate cancer cells.

Gene polymorphisms in the NQO1 gene are strong prognostic indicators for breast cancer.  For instance, the NQO1 2 genotype (P187S) predicts poor survival from breast cancer(ref).  The relative risk for breast cancer in this with the P187S genotype is 6.15, when compared to control groups.  The P187S genotype does not affect local recurrence, but affects survival.

Interestingly, benzene poisoning is associated with the mutation of the NQO1 gene at codon 187, which creates the 609C-T mutation of the NQO1 gene.  This results in complete loss of the enzymatic activity of NQO1 protein.  By this mechanism, benzene produces a NOA1 “loss-of-function” mutation and induces hematological malignancies. This appears to be a major mechanism for chemotherapy-induced secondary malignancies, which are called “therapy-related malignancies”.  Two diseases that benzene induces are “therapy-related  leukemia” and “therapy-related myelodysplastic syndrome“.  The NQO1 gene can also be transcriptionally unregulated by a polyphenol called quercetin.  Specifically, quercetin increases gene expression of the NQO1 gene via an Nrf2 transcription factor mediated pathway(ref)(ref)(ref).  Specfically, quercetin enhances the binding of Nrf2 to the NRF-ARE binding site on the NQO1 gene promoter.   Quercetin also increases Nrf2-mediated transcriptional activity by up regulation gut e expression of Nrf2 mRNA and Nrf2 protein.  Quercetin also reduces the level of Keap1 protein, the binding partner of Nrf2, which prevents Nrf2 nuclear translocation.  Quercetin reduces Keap-1in a post-translational mechanism, thereby reducing Nrf2 ubiquitination and proteasomal degradation.  Another unusual up regulator of NQO1 is the toxin, dioxin.

The NAD World Part 4 blog enry contains additional discussion related to NQ01 and how it is egulated, beta lapachone and other topics mentioned in this item

References:

3.  Clock/BMAL1

CLOCK and BMAL1 regulate the circadian expression of the SIRT1 gene.  Day/night cycles are thus the #1 factor that determines SIRT1 expression.SIRT1 regulates circadian gene expression by deacetylating PER2

With long lists like those in this blog entry, it is easy to throw up your hands and say “its too complicated!”  Well it isn’t!  If you want to skip the list and just ask “What is the most important regulator of SIRT1 expression,” I do not think anyone would argue with the statement that CLOCK/BMAL1 bind to the gene promoter for SIRT1 and regulate the diurnal change in SIRT1 gene expression. (see references).

For example, it has been well-documented that liver insulin sensitivity correlates with the two circadian transcription factors CLOCK and BMAL1.  BMAL1, CLOCK, and SIRT1 all must work together to “turn on” and “turn off” 15% of the genome in human cells every day.  Unless the expression of these three proteins is coordinated, hepatic insulin resistance develops.   Constant darkness “dysregulates” the coordinated timing of BMAL1 and SIRT1.  As a result, BMAL1 and SIRT1 expression decreases with constant darkness and hepatic insulin resistance is induced.  Interestingly resveratrol can dramatically reverse the “dysregulation” of SIRT1-dependent circadian genes by increasing SIRT1 activity.  SIRT1 regulates circadian gene expression by PER2 deacetylation.

References:

4.  Long non coding RNAs 

The long non-coding RNA transcribed from the anti-sense strand of the SIRT1 gene regulates SIRT1 gene expression via a “trans-regulatory” mechaism.   Long non-coding RNAs also appear to regulate cellular senescence, metabolism and many other cellular functions.  

Introduction/Background

Recently, there has been dramatic shifts in the thinking of geneticists about the role of “junk DNA” in human disease, aging, and normal development.  Specifically, over 56,000 unique RNA transcripts have been identified via next generation sequencing (also called “deep sequencing” or RNAseq).  Of these, over 9,000 long non-coding RNA “genes” have been identified that make many more than 9,000 different RNA transcripts.  Although there are many other types of non coding RNA besides long non-coding RNA, these RNA sequences that are > 200 base pairs in length have garnered the most attention since they seem to have such powerful regulatory functions over and above even epigenetic gene regulatory mechanisms.

Some recent papers have suggested that of the 6,000+ gene polymorphisms (SNPs) that have been linked to disease by GWAS studies, as many as 93% of these SNPs are not due to protein-coding regions of the human genome, but instead are in regulatory areas.  Another surprising finding was that at least 5,000 (of the more than 9,000 DNA sequences that encode for long non-coding RNA) are not evolutionarily conserved.  What does this mean?  It means that these long non-coding RNA may be the “youngest” forms of gene regulation and may account for the unique characteristics of homo sapiens, such as our ability to make tools, form language, develop written communication, music, art, religion, socialization, etc.  In short, long non-coding RNA may be why we are “human”, rather than looking and acting more like our nearest cousins, the chimpanzees and bonabos.

SIRT1 gene-specific lncRNA

It is not surprising then that a long non-coding RNA has been discovered that regulates SIRT1 gene expression.  (see reference below).  This long non-coding RNA is transcribed from the anti-sense strand of the SIRT1 gene.  This lncRNA is called “SIRT1 antisense long non coding RNA” or SIRT1AS lncRNA for short.  Wang and colleagues from Shanghai, China discovered this and published their finding in April, 2014.  They were able to show that SIRT1 AS lncRNA expression results in an increase in expression of the SIRT1 gene.  They isolated SIRT1 AS lncRNA from differentiating myotubes from developing skeletal muscle, as well as from the spleen.  It appears that the SIRT1 AS lncRNA expression is both temporal and tissue-specific.  For instance, higher levels of SIRT1 AS lncRNA were expressed in undifferentiated, younger tissues,   Likewise, SIRT1 AS lncRNA was expressed in a tissue-specific fashion.  In the spleen, SIRT1 AS lncRNA levels were higher than in skeletal muscle.

SIRT1 AS lncRNA may counteract microRNA that inhibit SIRT1

miRNA that down regulate SIRT1 gene expression appear to be blocked or their effect attenuated by the SIRT1 AS lncRNA.  Thus, the function of SIRT1 AS lncRNA may be to counteract the effects of miR-34a, miR-217, miR-181a, and the other microRNA that increase SIRT1 mRNA degradation and thereby reduce SIRT1 protein expression.

Long non coding RNAs and cellular senescence

Gene-specific lncRNAs can regulated only one specific gene, like the SIRT1 AS lncRNA described above.  However, many more lncRNAs regulate many genes rather than one specific gene.  Gene-specific lncRNAs typically regulate nearby genes, which is a method called “regulation in-cis”.   Since SIRT1 plays a role in cellular senescence, it is likely that the “Anti-sense” senescence-associated lncRNAs play an role in regulating genes that SIRT1also regulates.  A list of the top 12 up-regulated Antisense lncRNAs with cellular senescence along with a list of the top 15 Anti-sense  lncRNAs that are down regulated with cellular senescence are listed below.

In addition to the Anti-sense lncRNAs described above, a number of lncRNAs have been found that originate in pseudogenes, genes that no longer code for proteins, but are still transcribed from their sense or anti-sense strand.   lncRNAs can regulate a single gene or regulate hundreds of genes as well. lncRNAs that regulate many genes typically do so via a method called “regulation in-trans”.  These lncRNAs are not located near by the genes that they regulate, but instead, are typically located long distances away, even on other chromosomes.  Many of these long non-coding RNAs are found in areas with no nearby protein-coding genes.  For this reason, they are called “Long intergenic non coding RNAs” or lincRNAs.

Several lincRNAs have been discovered that regulate cellular senescence via “trans regulatory” methods.  These lncRNAs appear to be “novel” and not within or associated with a particular protein-coding gene or a pseudogene.

Long non coding RNAs and metabolism

Since lncRNAs have been discovered that regulate cellular senescence, it is no surprise to find out that lncRNAs regulate metabolism as well.  There is strong evidence that insulin resistance may be mediated in part by lncRNAs.  This includes tissue-specifc lncRNAs and generalized lncRNAs.  For instance, the long non coding RNA called “H19″may be involved in the intergenerational transmission of diabetes mellitus.  In a large next-generation RNA sequencing study of pancreatic RNA transcripts, over 1,000 lncRNAs were discovered in pancreatic islet beta cells.  Of these, 40% were long intergenic non coding RNAs (lincRNAs) and 55% were Anti-sense strand long non-coding RNAs (AS lncRNAs).  Interestingly, the non-coding RNA sequences found via this next generation sequencing study were more “tissue-specific” than the mRNA for protein-coding genes in the pancreatic tissue examined.  In other words, non-protein coding RNAs had a more “tissue specific signature” than the protein-coding mRNAs that were sequenced.  This is quite remarkable.

References:

Antisence Long Non-coding RNAs that are Differentially  Regulated with the induction of Cellular Senescence

NAD3-15n

Summary:  It is likely that much of the regulation of protein-coding genes and the unique aspects of humans, human aging, and human disease may be due to the “invisible human genome”, which is the large number of non-coding RNAs.  This field is exploding, with new RNA sequences being discovered every day.  Already, an Antisense strand long noncoding RNA has been discovered at the SIRT1 gene, which increases SIRT1 gene expression by a yet unknown mechanism.  One possibility is that this lncRNA prevents or inhibits the functions of miRNAs which increase SIRT! mRNA degradation.   Other lncRNAs have been found that regulate cellular senescence, metabolism, etc. via “trans regulatory” or “cis regulatory” methods.

5.  Nicotinamide (Nam)

Nicotinamide is a direct inhibitor of both the SIRT enzymes and the PARP enzymes.

The accumulation of excess nicotinamide in cells is probably a major cause of aging.  Whereas we typically associate “NAD deficiency” with aging, “Nam excess” may have a similar effect.  To no one’s surprise, the levels of the two compounds are inversely related in aging.

Nam plays a role in both aging and in disease.   In hypertension and in aging individuals with normal blood pressure, Nam inhibits the methylation-mediated degradation of catecholamines.  Thus Nam excess plays a role in hypertension (see references below).

Nicotinamide also has an epigenetic effect. When SIRT1 is inhibited, cells age and cancer oncogenes are re-activated.  SIRT1 silences these genes  by histone deacetylation of H3K9 and H4K16 residues on the histones of these oncogenes.

A recent article showed that in rats, nicotinamide supplementation during pregnancy causes global DNA hypomethylation in rat fetuses.  Nicotinamide has detrimental effects in development, detrimental metabolic effects, and detrimental epigenetic effects when given to young rats.  Low dose nicotinamide increased weight gain in developing rats.  High dose nicotinamide did not, however.  The livers of nicotinamide-fed young rats had more DNA damage (8oxoG), impaired glucose tolerance, and increased insulin resistance.  Nicotinamide increased the levels of N-methylnicotinamide in the blood and decreased betaine levels in the blood.  This resulted in a global hypomethylation of DNA in the rat genome.  Nicotinamide also had “gene-specific effects” on CpG islands within the promoters of the following genes:

  1. NNMT gene – this was down-regulated
  2. DNMT genes – these were down-regulated
  3. Homocysteine metabolism genes – these were down-regulated
  4. Antioxidant genes and oxidative stress protection genes – these were down-regulated

Since niacin is converted into nicotinamide in human tissues, high dose niacin probably produces all of the above effects.  A recent paper called niacin and nicotinic acid “methyl consumers” and strongly suggested that high niacin/nicotinic acid intake is bad.

Excess nicotinamide has also been shown to increase plasma serotonin and histamine levels in humans, due to disrupting the metabolism of these neurotransmitters.  This is probably due to the fact that methyl donors and methylation enzymes are needed for serotonin/histamine metabolism.   Most importantly, nicotinamide is a direct inhibitor of the Sirtuin enzymes (SIRT1-7) and the PARP enzymes (all 17 of the PARPs).

The molecular mechanism by which Nam works is very straightforward. Nam acts as a direct inhibitor of the SIRT1 enzyme pocket where NAD binds.  Thus Nam is a “competitive inhibitor” of NAD and is “bad” when it comes to most cancers, aging, and most diseases.

On the other hand, inhibition of SIRT1 by Nicotinamide may be a “good thing” in the brain, where it may prevent NAD+ depletion and thereby protect neurons against excitotoxicity and neuronal cell death induced by PARP1.

As the cell consumes NAD (by SIRT1-7, PARP1, PARP2, Tankyrases, CD38, CD157, ARTs, and other enzymes), the NAD is consumed, leaving the by-product, Nam.   There are two primary ways  of “disposing of Nam”.  They are methylation/excretion or recycling of Nam into NMN (and subsequently NAD) via the “NAD salvage cycle”.  

Here are the problems with both of these methods of reducing Nam levels in the cell.  (see #2 and #3 below).

References:

Conclusion:  It is now clear that high concentrations nicotinamide are harmful to health.  HIgh doses of dietary niacin probably produce the same effects, despite the many benefits of high dose niacin.  With aging, nicotinamide levels already go up.  Adding more nicotinamide is probably not going to “cure” aging.  Adding a methyl donor to eliminate nicotinamide (such as betaine) may be a good thing.

6.  NAMPT

-NAMPT is the rate-limiting step in the NAD Salvage Cycle, and is regulated in a circadian fashion.

The enzyme that converts Nam back into NMN in the “NAD salvage cycle” is Nicotinamide phosphoribosyl transferase, or NAMPT.  Unfortunately, NAMPT is regulated by circadian rhythms and is primarily up-regulated at night.  It is inhibited by not sleeping, however.  It is also down-regulated by eating and sedentary lifestyle.  Fasting and exercise dramatically up-regulate NAMPT.   I really do not see how NMN or NR will really change the gene expression of NAMPT.  If anything, since NMN is the “product” of the NAMPT enzyme, high levels of NMN may actually have a “feedback inhibition effect” on NAMPT, just like Nictoinamide has a “feedback inhibition effect” on SIRTs and PARPs.

Paradoxically, in a recent study in zebrafish, resveratrol actually DECREASES the expression of NAMPT.  This may be via a “feedback inhibition effect”, since SIRT1 “auto regulates” its own gene expession (see references).   Another interesting “twist” is that Angiotensin II receptor blockers (ATR type 1 blockers) actually increase NAMPT gene expression.  This may be the molecular mechanism behind the longevity effects of ATR1 blocker medications like Telmisartan.

References:

 7.  NNMT – Methylation of Nicotinamide – Nam)

Methylnicotinamide is a “mitohormetic compound” that regulates longevity

Of all of the unusual aspects of Sirtuins and NAD metabolism, N-methylnicotinamide is probably the hardest one to understand.  Recent evidence has shown that for the longevity effects of NAD metabolism to occur in nematodes (C. elegans), NAM must be methylated and then used by the gene, GAD-3, to produce low levels of hydrogen peroxide, thereby acting as a “mitohormetic compound”.  This low level of hydrogen peroxide induces mitochondrial biogenesis and is necessary for nematode lifespan extension.

Reference:  2013 Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide

This article came out last year and one of the co-authors is David Sinclair.

However, that is not the entire story.  There is much more to the story of the methylation of nicotinamide.  Most of this has to do with stopping the toxic effects of nicotinamide from occurring, due to the inhibitory effects of Nam on both the Sirtuin enzymes (SIRT1-7) and the PARPs (1-17).

Over 85% of transmethylation reactions occur in the liver, including the methylation of nicotinamide.  With the oral intake of nicotinamide, the liver could either methylate the nicotinamide or convert it to NAD.  From many studies, it is clear that the liver preferentially methylates nicotinamide, rather than converting it to NAD.   The reason why the liver methylates nicotinamide is that high concentrations of nicotinamide cause cell injury and cell death, since nicotinamide inhibits all 7 of the Sirtuins and also inhibits the PARP enzymes (PARP1,PARP2, etc).

For this reason, with oral intake of Nam, serum levels of N-methylnicotinamide increase.  Only 15% of nicotinamide methylation occurs outside of the liver.  Nicotinamide methylation by NNMT also requires a methyl donor, such as SAMe or trimethylglycine, which is also called betaine.  When nicotinamide is methylated, it is then excreted, thereby reducing Nam levels in the body and preventing SIRT/PARP inhibition.

GWAS studies of polymorphisms in the NNMT gene have revealed an amazing association with Non-alcoholic steatohepatitis (NASH).  The NNMT gene SNP, rs694539, is a SNP found in the regulatory portion of NNMT gene.  The “GG” genotype protects against NASH, with an OR of 0.58.  The “AA” genotype increases the risk of NASH with an OR of 7.3.   This suggests that methylation of nicotinamide is an important factor in preventing NASH (see references below).   This same polymorphism (rs694539) has been linked to bipolar disorder recently.  The association was “female gender-specific” and did not influence male bipolar risk.

A recent novel theory discussing how “too much NNMT” and “too little NNMT” may both play a role in aging and disease has been proposed. This theory suggests that excess dietary nicotinic acid consumption results in molecular/cellular  “methyl consumption” and plays a role in disease. This new theory has been published in Nature and is being taken seriously.  The main hypothesis is the increase in “methyl consuming compounds” in our diet contributes to metabolic syndrome and many other “man-made diseases”.  Another aspect of this new theory is that NNMT is a “fat accumulation gene”  (see references below), since the expression of NNMT is unregulated with increased dietary intake of food, especially foods that are rich in niacin and nicotinic acid.   Specifically, the expression of the NNMT gene correlates with the percentage of fat in 20 different mouse strains.  The main cause of the up regulation of the NNMT gene is overeating/overfeeding.   In fact, Kraus and colleagues recently showed that the administration of methylnicotinamide inhibited NNMT and this increased NAD levels and SAM-dependent gene expression.

References:

This theory has been proposed because of recent studies which show a paradoxical effect occurs when NNMT is over-expressed.  When this occurs, the NNMT over-expression results in diet-induced obesity.  This has been shown in humans and in animal models.  For instance, the NNMT gene is unregulated in fat cells with obesity and T2DM. When the NNMT gene is “knocked down”, it protects against diet-induced obesity.

Other references for nicotinamide methylation:

Here is the recent (2014)article on NNMT knock-down and the protection of diet-induced obesity: Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity

8.   PARP-1 and PARP-2

PARPs are the #1 Intracellular “NAD consumers” .  PARP-2 is also a direct inhibitor of SIRT1.

  • The Poly-ADP-ribose polymerases (PARPs) are a large family of enzymes involved with DNA damage detection, DNA damage repair, and also cellular apoptosis.  They are voracious “NAD consumers” and use as much as 100-150 molecules of NAD when activated by one DNA break.   As a result, cells can become NAD depleted in the nucleus, where the PARPs reside.  They are also voracious “ATP consumers” and use up as much as 100-150 molecules of ATP when activated by one DNA break.  As a result, cells can become ATP depleted, which then induces cellular death.  Thus PARPs may be an important way of killing a cell by “suicide,” if there is too much DNA damage.
  • PARP-1 is not a direct inhibitor of SIRT1, but because it consumes so much more NAD than Sirtuins do, PARP1 inhibitors or PARP-1 “knock down” have been shown to increase SIRT1activity in cells.
  • PARP-2, on the other hand, is a direct negative regulator of SIRT1, independent of NAD activity. When PARP-2 activity increases, which it does with aging (by 10-fold), SIRT1 is inhibited, regardless of NAD levels.  However, the deletion of the PARP-2 gene results in hepatic cholesterol accumulation and decreased HDL lipoproteins.

References:

Conclusion: There is probably an “antagonistic cross talk” between SIRT1, PARP-1, and PARP-2 due to their mutual demand/need for NAD.  With aging, there is a “sterile inflammation” that occurs, often referred to as “inflammaging”.  Inflammaging appears to be directly under the control of the NF-kB transcription factor with antagonistic crosstalk between SIRT1, PARP1, and PARP2 signaling pathways.  There may be a role for a PARP inhibitor for health.  Pharmacological inhibition of PARPs has already been shown to improve skeletal muscle fitness and mitochondrial function in rodent models. In Part 4 of this NAD World series, we will discuss inflammationaging and the roles of NF-kB, NAD and SIRT1 in much further detail

9.  CD38

CD38 is probably the #1 Extracellular “NAD consumer” (whereas PARPs are the #1 intracellular consumer)

CD38 is one of several “ectoenzymes” found outside the cell that are “NAD consumers”.  (The others are CD157, ART1, ART2, ART3, and ART4).  CD38 is a multifunctional membrane-bound extracellular enzyme that plays a key role in immunity, autoimmunity, and calcium signaling.  CD38 consumes NAD and makes cyclic-ADP ribose (cADPR).  Unfortunately, CD38 is a very inefficient enzyme and consumes as many as 100 NADs for every one cyclic-ADP-ribose that it makes.  For this reason, some experts on CD38 feel that the #1 function of CD38 is to regulate cellular NAD levels.  A strong argument for this theory is the recent discovery that CD38 is found inside the cell as well, bound to membranes on the inner portion of the cell nucleus.  Here it could deplete nuclear NAD.  Interestingly, the apple skin-derived flavanoid, apigenin, is a powerful inhibitor of CD38.  Treatment of cell cultures with apigenin increased NAD levels in the cells, reduced global acetylation of proteins, and reduced the acetylation of p53 and RelA-p65 subunits of NF-kB.

Reference: 2012 Flavonoid apigenin is an inhibitor of the NAD+ ase CD38: implications for cellular NAD+ metabolism, protein acetylation, and treatment of metabolic syndrome.

The classic description of CD38 has not been that of an “intracellular NAD level regulator”, but part of a signaling system involving cyclic ADP-ribose and calcium. Cyclic-ADP-ribose (cADPR) functions as a second messenger in the cell to trigger calcium release from the sarcoplasmic reticulum. CD38 is a very important membrane-bound enzyme found on the surface of many cells, but the highest density of CD38 are on the surface of immune cells such as monocytes/macrophages.

In the brain, CD38 is very important for the secretion of oxytocin from oxytocin-producing cells in the hypothalamus. Oxytocin has traditionally been thought of as the “maternal milk/nurturing hormone”, but in the brain it functions as a neurotransmitter.  In the brain, there are many locations for oxytocin type 1 (OTR1) and oxytocin type 2 (OTR2) receptors, located mostly in the telencephalon.  Oxytocin appears to be the “Peptide that binds our hearts in love”.  It has clearly been linked to maternal love, brotherly love, spousal/opposite sex affection, and community spirit.  It may be the peptide that is secreted in response to religious experiences of love for God as well.

References:

Here is a diagram that illustrates the possible way that CD induces NAD depletion and therefore causes metabolic syndrome:

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Ref for diagram:  CD38 as a regulator of cellular NAD: a novel potential pharmacological target for metabolic conditions

” Possible mechanism of regulation of SIRT1 and AMPK pathway by CD38 inhibition.

10.  MicroRNAs

16 microRNA have been found to regulate SIRT1 expression.  These miRNA are mostly negative regulators of SIRT1 expression by their binding to the 3′ UTR of SIRT1 mRNA, increasing SIRT1 mRNA degradation before they can be transcribed.  Different microRNA are expressed by different triggers, such as EtOH, p53, and Diabetes type II.

16 different micrRNAs have been found that regulate SIRT1 expression (see table below).   Different triggers induce the expression of each miRNA.   These microRNA all down-regulate SIRT1 expression.  This occurs because microRNA bind to a region in the 3′ untranslated region (3′ UTR) in the SIRT1 mRNA,  thereby increasing the degradation of SIRT1 mRNA.  Thus microRNA are a “post-transcriptional regulators” of SIRT1 gene expression.  This miR-mediated effect is likely the mechanism by which cancer increases the expression of SIRT1 and also the mechanism by which aging decreases the expression of SIRT1.

miR-217 – an miR that is activated by drinking EtOH

Ethanol also activates miR-217 gene expression and is one of the primary mechanisms responsible for alcoholic fatty liver disease and NASH (which is the clinically symptomatic form of alcoholic fatty liver disease).  NASH can lead to alcoholic cirrhosis, liver failure, ascites, and death.  miR-217 is also the mechanism by which HIV infections shorten lifespan, since tat-activated LTR expression induces miR–217.

miR-34a – an miR that is activated by p53

miR-34a has been the most studied microRNA when it comes to SIRT1 expression. It is involved in pancreatic cancer, colorectal cancer, prostate cancer, brain cancer, liver cancer, normal neural differentiation, liver metabolism, endothelial cell senescence, and endothelial progenitor cell senescence.

Ectopic miR-34a reduces SIRT1 expression. The gene for miR-34a is “turned on” by p53 protein.   miR-34a has been shown to induce cancer cell apoptosis in colon cancer cells.  miR-34a also promotes endothelial cell senescence in atherosclerosis.

miR-181a – an miR that is increased in diabetes type II

miR-181a binds to a region in the 3′ untranslated region (3′ UTR) in the SIRT1 mRNA, thereby increasing the degradation of SIRT1 mRNA.  Thus miR-181a is a “post-transcriptional regulator” of SIRT1 gene expression.   Over-expression of miR-181a results in insulin resistance.  Studies of the serum (plasma) of diabetics has shown that miR-181a is increased in the serum.  Studies of diabetics has also shown that miR-181a is increased in hepatocytes.  There is hope that inhibiting miR-181a may be a strategy for treating diabetes type II.

There are many other miRNAs that regulate SIRT1, listed below.

NAD3-15o

Table reference

References:

11.  p53

There are two binding sites for p53 on the promoter of SIRT1. They are located at positions 168 and 178 upstream from the TSS.  p53 binding to the SIRT1 promoter prevents gene expression of SIRT1, except with starvation. With starvation stress, p53 dissociates from the SIRT1 promoter and FoxO3a can “knock p53 off these two binding sites (168 and 178), thereby removing the repressive effects of p53 from the SIRT1 promoter, thereby resulting in SIRT1 gene expression.

References:

12.  FoxO3a and Eating (especially sugar):

Glucose induced Insulin signaling activates the Insulin/IR/IRS-1/PIP3K/Akt pathway which prevents Foxo3a from migrating into the cell nucleus and activating the SIRT1 gene. Fasting does the opposite. FoxO3a also enters the nucleus with fasting and “bumps” the two p53s off the two binding sites on the SIRT1 promoter, thereby abolishing the repression of SIRT1 by p53.

Reference:2004 Stress-Dependent Regulation of FOXO Transcription Factors by the SIRT1 Deacetylase

13.  E2F1

The cell cycle and apoptosis regulator.

In actively growing cells, E2F1 is the transcription factor which controls cell-cycle fluctuations in SIRT1 levels. E2F1 induces SIRT1 gene expression in response to other factors also, such as cellular oxidative stress from exercise, low dose ETOH, chemo, XRT, and maybe your “oxidative stress water.” If this water would work as a “controllable timeable ROS dose,” this is the molecular mechanism by which it would work. OBTW, SIRT1 also has a negative feedback loop, inhibiting E2F1.

References:

14.  HIC1:CtBP co-repressor complex

The SIRT1 promoter has a binding site for a co-repressor complex called “HIC:CtBP”, which decreases SIRT1 gene expression. With CR and fasting or “fasting Mimetics like 2-DG”, the affinity of CtBP for HIC goes down, thereby increasing SIRT1 gene expression several fold. This is one of the primary molecular mechanisms of fasting.

References:

15.  ChREBP

 The carbohydrate response element binding protein.

ChREBP, is up-regulated with the dietary intake of carbohydrates. ChREBP is the “molecular link” between carbohydrate ingestion and high triglycerides in the blood.  (i.e. the glucose-induction of triglyceride synthesis.)   How does this occur?  Well, ChREBP represses the transcription of SIRT1.

ChREBP may play a major role in alcoholism and alcohol-induced fatty liver disease.  In a mouse model of binge drinking, ChREBP acetylation was increased dramatically and was recruited to gene promoters in mice.  The acetylation of ChREBP was dependent on alcohol metabolism rate.  In mice with mutant forms of ChREBP that could not be acetylated, the ChREBP-dependent genes could not be “turned on”.  ChREBP silencing in mice that were fed alcohol prevented the increase in triglycerides that normally occurs with binge drinking.  In addition, SIRT1 was down-regulated in these mouse models of EtOH binge drinking, due to the direct inhibitory effect of ChREBP on SIRT1 gene expression.

References:

Conclusions:  ChREBP is a “glucose sensor”.   ChREBP is the direct molecular link between high fructose or high glucose intake and the hepatic formation of triglycerides.  ChREBP is also a major inhibitor of SIRT1 gene transcription.  More importantly, ChREBP is the “binge drinking gene.”

16.  CREB

The cyclic-AMP response element binding protein,

CREB, activates SIRT1 gene expression. This is activated by low glucose levels, which happens with fasting.  CREB may also be the transcription factor that explains some of the paradoxical effects of SIRT1 over-expression.  In mice that over-express SIRT1, an atherogenic diet does not worsen glucose metabolism.  Instead, SIRT1 over-expression protects these mice fed an atherogenic diet from glucose dysregulation (i.e. insulin resistance).

However, in these SIRT1 over-expressed mice on an atherogenic diet, their atherosclerotic lesions actually get much worse than controls.   The reason for this is that SIRT1 deacetylates CREB, preventing its cyclic-AMP phosphorylation.  Thus SIRT1 inhibits CREB from activating gluconeogenic genes and inhibits CREB from activating hepatic lipid metabolism and excretion. In summary, CREB activates SIRT1 gene expression, and SIRT1 has a negative feedback effect on CREB function.  This explains how SIRT1-mediated CREB deacetylation regulates the balance between glucose and lipid metabolism.

ReferenceS:

Conclusion:  CREB is a “glucose and lipid sensor” with reciprocal interactions with SIRT1.  CREB activates SIRT1, which is the opposite of ChREBP.  Fasting activates CREB, whereas fasting inhibits ChREBP.  Moreover, when there is too much fat, but SIRT1 is over-expressed, CREB cannot prevent atherosclerosis and atherosclerosis worsens.

17.  TLX

TLX is one of the “orphan nuclear receptors”.

TLX binds to a TLX-response element in the SIRT1 promoter.  TLX is short for “Tailess.”

TLX is a very important transcriptional repressor in the brain, especially in neural stem cells and is vital to normal growth and development. As of now, no endogenous ligand for these ligand-dependent transcription factors has been identified.  It is a “druggable” target, however.

So far, only three compounds have been found out of a 20,000 compound high-throughput screen that bind to TLX (ccrp1, ccrp2, and ccrp3).  Although many functions for TLX have been discovered, the main cellular function of TLX appears to be keeping neural stem cells in their undifferentiated, proliferative state.  TLX regulates the expression of another nuclear receptor, the “retinoic acid receptor” or RAR.  Thus TLX is an important receptor. TLX is an oncogene-induced senescence suppressor inside and outside of the brain.  It has been shown to be effective in the prosate.

TLX  co-regulates the cyclin-D kinase inhibitor, CDKN1A (aka p21WAF/CIP1) with SIRT.  Very little is known about this orphan nuclear receptor other than the fact that it activates SIRT1 gene expression.

References:

18.  C/EBP-alpha and beta

C/EBP-alpha and C/EBP-beta – the two hepatic CCAAT/enhancer binding proteins with opposite effects on SIRT1/

In the liver, there are two opposing transcription factors that play a major role in liver biology, glucose metabolism, and fat metabolism: CCAAT/enhancer binding proteins alpha and beta.  These two transcription factors are also expressed elsewhere, outside of the liver, but the general role of the two appear to be similar – they have opposing roles on gene expression.

  •  The CCAAT/enhancer-binding protein alpha (C/EBP-alpha) is a transcription factor that represses many genes and activates many genes.  The most important 3 are SIRT1, p53, and PGC-1a.
  • There is also a C/EBP-beta that has the opposite effects on these genes.

For instance C/EBP-alpha represses the hTERT gene, thereby preventing cancer induction of telomerase.   C/EBP-beta, on the other hand increases the expression of the hTERT gene, thereby increasing telomerase enzymes.

C/EBP-alpha activates SIRT1 gene expression via binding to the promoter region of SIRT1.  The opposite is true about C/EBP-beta.  It represses SIRT1 gene expression.

In old age, SIRT1 cannot be up-regulated very well due to the repressor effects of the C/EBP-beta/HDAC1 complex, which both work together to suppress SIRT1 gene expression.

Interestingly, pomegranate seed oil has 3 ingredients in it (xanthigen, fucoxanthin, and punicic acid) that appear to down regulate C/EBP-beta and thus prevent fat accumulation.  Xanthigen up regulates SIRT1 and AMPK signaling in differentiated fat cells also.

References:

Conclusion: The two CCAAT/enhancer-binding proteins alpha and beta have important opposing effects on tissue regeneration, glucose metabolism and fat metabolism.  In general, C/EBP-alpha prevents fat accumulation and promotes liver regeneration, whereas C/EBP-beta has the opposite effect.  C/EBP-alpha increases SIRT1 gene expression, whereas C/EBP-beta and HDAC-1 combine to repress the promoter of SIRT1.

19.  BRCA1

BRCA1 increases the expression of NAMPT, PARP1 and SIRT1, whereas BRCA1 mutation, promoter methylation, or knockdown decreases NAMPT, PARP1, and SIRT1 gene expression, but paradoxically increases NAD levels, which then increase SIRT1 activity (but not SIRT1 gene expression).  Thus BRCA1 may be a “balancer” between SIRT1 gene expression and SIRT1 protein activity

Two decades ago, the first breast cancer susceptibility gene was discovered and called “BRCA1”.  Now we know the BRCA1 protein (which is involved in double stranded DNA repair), is mutated in hereditary forms of breast and ovarian cancer.  Inherited BRCA1 mutations can lead to cancers of the breast, ovary, and many other organs.  For breast cancer, the risk due to the mutation is increased to 56-80%.  For ovarian cancer, the risk is increased to 15-60%.   BRCA1 regulates the expression of 7% of the mRNA in cancer cells.  This is probably why it is so important and should therefore be called a “transcriptional regulator”.

Although a lot of research was being done about BRCA1 and cancer, no one had linked BRCA1 to Sirtuins or NAD until very recently.  Recently, the BRCA1 protein was found to control NAMPT-mediated NAD synthesis, which was a surprise.  Another surprise was that NAD levels could have a “feedback activation” of BRCA1 expression.  Moreover, it has been shown that BRCA1 is a positive regulator of PARP1 levels and NAD-dependent PARP1 activity.  Most recently, BRCA1 was found to also positively regulate SIRT1 expresión. BRCA1 binds to the SIRT1 promoter and activates SIRT1 gene expression. SIRT1 then inhibits the expression of Survin by deacetylating the H3 histones of the Survivin gene. BRCA1 also inhibits Survivin. Without SIRT1 or BRCA1, Survivin levels increase and Cancer develops.

BRCA1 may also play a role in preventing hypertension.  One of the major pathways in spontaneous hypertension is the Angiotensin II pathway and the activation of the ATR1 and ATR2 receptor by Angiotensin II.  ATR1 receptor activation induces NAD(P)H oxidase-induced free radical formation and the vasoconstriction of blood vessels due to reduced NO production.  A recent article showed that BRCA1 limits Angiotensin II ATR1-mediated redox signaling, thereby improving vascular reactivity and reduces blood pressure in spontaneously hypertensive mice.

References:

NAD3-15p

Illustration Reference:  2015 Linking BRCA1 to NAD World  “Figure 1. Proposed model of crosstalk among BRCA1, SIRT1 and PARP1. A, BRCA1 inactivation may regulate SIRT1 and PARP1 levels, and induce an increase in NAD-mediated SIRT1 and PARP1 activity. B, the model shows a significant effect of BRCA1 in the maintenance of SIRT1-related biological processes. C, a proposed model to maintain stable BRCA1 and PARP1-related DNA repair ability.”

20.  SNPs in the SIRT1 promoter

Evolution and the “Feast or Famine” SNP in the SIRT1 promoter region in Northern India. rs12778366 is a single nucleotide polymorphism (SNP) found 1.46 kb upstream from the TSS in the SIRT1 promoter that predisposes N Indians to type II diabetes. > 80% of N. Indians have this SNP and have a 6-9 fold higher risk of T2DM. This SNP was probably “selected by famines” that often occurred in N India for 1,000s of years. Now it is a liability, since famines no longer occur

21.  EGR1 

Mechanical stretching of muscles:

The SIRT1 gene is upregulated by the stretching of muscle fibers via the transcription factor “Early Growth Response Factor 1”, or EGR1. Mechanical stretching of muscles increases mRNA for SIRT1 by 2.2 fold and SIRT1 protein by 100%! This is why stretching before and after exercise is so important, since SIRT1 expression causes FoxO3a deacetylation (and thereby induction of the mitochondrial SOD gene) as well as Nrf2 Deacetylation (and thereby induction of many antioxidant genes).

22.  DBC1 (aka CCAR2)

“Deleted in Breast Cancer 1” is probably a bad name for this protein, since it may not even be deleted in most breast cancers.

For this reason, the new name for DBC1 is “Cell cycle activator and apoptosis regulator 2”, or CCAR2. DBC1 directly interacts with SIRT1 by forming a stable complex with DBC1, thereby preventing the activity of SIRT1 in vitro and in vivo.  Interestingly, SIRT1 and DBC1 protein levels are higher in breast cancer tissues, compared to age-matched controls, but not at the transcriptional level.  At the transcriptional level, there does not appear to be up regulation of SIRT1 and DBC1.  This up regulation of SIRT1 and DBC1 is therefore at the “postranscriptional level” (i.e. via microRNA or microRNA sinks).  In gastric adenocarcinoma, overexpression of SIRT1 and DBC1 are actually associated with a better prognosis.

Reference:

23.  c-Myc

There is now evidence that the SIRT1 gene expression is “downstream” from the oncogene, c-Myc.  In cancer, there is a “positive feedback loop” that occurs, which results in contributes to the development of cancer.  Specifically, c-Myc activates SIRT1, which in turn promotes SIRT1 function.   Likewise, SIRT1 promotes c-Myc function.  Here is the article on this.  This “mutual positive feedback” between SIRT1 and c-Myc is why there is so much confusion as to whether SIRT1 is a tumor suppressor or a tumor enhancer.

Reference:  2011 The c-MYC oncoprotein, the NAMPT enzyme, the SIRT1-inhibitor DBC1, and the SIRT1 deacetylase form a positive feedback loop

“Silent information regulator 1 (SIRT1) represents an NAD+-dependent deacetylase that inhibits proapoptotic factors including p53. Here we determined whether SIRT1 is downstream of the prototypic c-MYConcogene, which is activated in the majority of tumors. Elevated expression of c-MYC in human colorectal cancer correlated with increased SIRT1 protein levels. Activation of a conditional c-MYC allele induced increased levels of SIRT1 protein, NAD+, and nicotinamide-phosphoribosyltransferase (NAMPT) mRNA in several cell types. This increase in SIRT1 required the induction of the NAMPT gene by c-MYC. NAMPT is the rate-limiting enzyme of the NAD+ salvage pathway and enhances SIRT1 activity by increasing the amount of NAD+. c-MYC also contributed to SIRT1 activation by sequestering the SIRT1 inhibitor deleted in breast cancer 1 (DBC1) from the SIRT1 protein. In primary human fibroblasts previously immortalized by introduction of c-MYC, down-regulation of SIRT1 induced senescence and apoptosis. In various cell lines inactivation of SIRT1 by RNA interference, chemical inhibitors, or ectopic DBC1 enhanced c-MYC-induced apoptosis. Furthermore, SIRT1 directly bound to and deacetylated c-MYC. Enforced SIRT1 expression increased and depletion/inhibition of SIRT1 reduced c-MYC stability. Depletion/inhibition of SIRT1 correlated with reduced lysine 63-linked polyubiquitination of c-Myc, which presumably destabilizes c-MYC by supporting degradative lysine 48-linked polyubiquitination. Moreover, SIRT1 enhanced the transcriptional activity of c-MYC. Taken together, these results show that c-MYC activates SIRT1, which in turn promotes c-MYC function. Furthermore, SIRT1 suppressed cellular senescence in cells with deregulated c-MYC expression and also inhibited c-MYC–induced apoptosis. Constitutive activation of this positive feedback loop may contribute to the development and maintenance of tumors in the context of deregulated c-MYC.

24.  AROS

Active Regulator of SIRT1

AROS is an endogenous activator of SIRT1.  AROS may bind to the site where resveratrol and other STAC activators bind to SIRT1, but this is still unclear.  What is clear is that AROS increases SIRT1 activity and works with SIRT1 to suppress p53 activity.  Specifically AROS works with SIRT1 to deacetylate p53, thereby reducing p53-mediated transcriptional activity (gene expression of genes dependent on p53).  Thus SIRT1 and AROS are negative feedback regulators of p53.

Reference: 2007 Active Regulator of SIRT1 Cooperates with SIRT1 and Facilitates Suppression of p53 Activity

25.  HuR

The Hu protein called HuR is an RNA binding protein that stabilizes the SIRT1 mRNA, preventing its degradation

Many RNA binding proteins have been recently discovered that degrade or stabilize messenger RNA (mRNA).  This includes the mRNA degrading RNA-binding proteins, AUF1, BRF1, TTP, and KSRP. The mRNA-stabilizing RNA binding proteins include the elav/Hu proteins, of which HuR is one.  HuR is probably the most well-known RNA-binding protein that reduces mRNA degradation.  It binds to the SIRT1 mRNA in the cytoplasm to prevent the SIRT1 mRNA from being degraded. Whereas the 16 different miRNA that bind to SIRT1 mRNA all promote its degradation, HuR binds to the same 3′ UTR region on the SIRT1 mRNA, thereby preventing the miRNA-mediated degradation of the SIRT1 mRNA.  The net result of HuR is that there is more SIRT1 protein as a result of the same level of SIRT1 gene transcription.

Reference: 2007 Phosphorylation of HuR by Chk2 Regulates SIRT1 Expression

26.  JNK2

The c-Jun Kinase, JNK2 phosphorylates the SIRT1 protein, thereby stabilizes the SIRT1 protein 

The SIRT1 protein has several phosphorylation sites on Serine amino acid side chains.  Ser27 is one of these sites that gets phosphorylated indirectly by JNK2 activation.  When the Ser27 site on SIRT1 is phosphorylated, the SIRT1 protein becomes much more resistant to proteasome-mediated degradation.  Thus it increases the half life of the SIRT1 protein from < 2 hrs to > 9 hours.  This is a very important part of maintaining SIRT1 protein levels within the cell.

Reference: 2008 JNK2-dependent regulation of SIRT1 protein stability

27.  Resveratrol, SRT1720, SRT2104, EX527

Resveratrol is a natural STAC activator.  Synthetic STAC activators such as SRT1720 have also been synthesize

The initial excitement about Sirtuins was primarily directed towards the natural compound, reseveratrol, found in red grape skins, Japanese knotweed, and many other plants.   Resveratrol and other STAC activators only activate SIRT1 and not SIRT2-7 or the PARP enzymes.  For this reason, resveratrol may hold certain advantages over NAD therapy or NAD precursor therapy such as NR or NMN.  The STAC-activating site on SIRT1 is near amino acid E320.  This site is not present on the other 6 isoforms of mammalian SIRT (SIRT2-7).

References:

28.  Lamin A

Lamin A is part of the nuclear cytoskeleton and may bind to the C-terminus of SIRT1.   Resveratrol may activate SIRT1 in a Lamin A-dependent manner.

There is a link between accelerated aging and the Laminopathies.  The most well-known laminopathies is Hutchinson-Gilford Progeria Syndrome, or HGPS.  In HGPS, a mutation in the Lamin A gene produces a mutant protein called “progerin”, which results in a breakdown of the cytoskeleton of the nuclear matrix.  This results in premature aging and usually death due to an MI or stroke during teenage years.  Recently, Gosh and colleagues from China have linked Lamin A and SIRT1.  According to their work, the C-terminal tail of SIRT1 binds to Lamin-A.  Thus lamina A may serve as a “SIRT1 anchor” in the cell nucleus.

Gosh and colleagues also showed that reseveratrol activates SIRT1 in a Lamin A-dependent manner.  Specifically, they showed that resveratrol increased the binding of SIRT1 to Lamin A and also down-regulated FoxO3 acetylation (SIRT1 is a known FoxO3 deacetylator).  Not all experts agree with this mechanism of action.  It has not been verified by a 2nd research laboratory.   However, this is very intriguing that reseveratrol may be a compound that could help treat HGPS.

Reference: 2013 Resveratrol activates SIRT1 in a Lamin A-dependent manner

Aging 

Aging lowers SIRT1 activity by multiple mechanisms.   The primary mechanism was once thought to be due to decreased gene expression of the SIRT1 gene, but this may not be true in the majority of cases.  Instead, age-induced reduction in SIRT1 activity is probably due to declining levels of NAD, increasing levels of Nicotinamide, and increasing levels of DBC1.

Reference: 2011 Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats

NAD Part 4

  1. The ratio NAD+/NADH in cells and the body, which may be as or more important than absolute levels of NAD+ for driving health.
  2. The NQ01 gene which drives the NAD+/NADH ratio, and factors related to its activation and expression: BET proteins, the 20S proteasome, BET inhibitors, NQ01 regulation of PGV-1alpha, Nrf2 regulation, etc
  3. The Warburg effect, changes in cell metabolism characteristic of cancers and aging, its causes, effects and how NAD+ level is only one of several factors affecting it. Why the view that “The Warburg effect is caused by a nuclear state of pseudohypoxia which is caused by insufficient NAD+” is incomplete.
  4. Reversing Warburg metabolism – known approaches and the possible use of phytosubstances; possible limits of reversal.
  5. SIRT1 and inflammation, and why control of inflammation has such paramount importance
  6. In the course of these discussions, a review of possible intervention approaches. ones not commonly discussed in the longevity litterture.

NAD+ kinase Image source

  I.  The NAD+/NADH ratio and what affects it. The NQ01 gene

 What is of prime importance for health and longevity may not be the actual concentration of NAD+ in cells or cell nucli, but rather the NAD+/NAD ratio which may not be affected by NAD precursor supplements and rather be driven by other matters such as expression of the NQ01 gene,

In the Part 1 blog entry, we characterized the NAD Salvage Cycle by which NAD+ and NADH (the oxidized and reduced forms of NAD) are cycled back into each other and the de-novo pathway  via which new NAD is introduced into the body and cells.  And. in Parts 1,2 and 3 we discussed how various factors affect the cycling process such as circadian clock gene control.  In Part 3 we introduced and initially discussed the NQO1 gene which is a point of departure for the current discussion. “The longevity gene, NQO1, regulates aging by altering the NAD/NADH ratio in cells.   NQO1 does this by oxidizing NADH to NAD.   Beta-lapachone increases NQO1 enzyme activity and quercetin increases Nrf2-mediated gene expression of NQO1.”

I am now confidence that supplementation with a NAD+ precursor like NMN or NR transiently increased the ratio of NAD+  to NADH (NAD/NADH), but the ratio returns to normal in the course of continued supplementation.  This is because there is an enzyme that regulates the NAD/NADH ratio.  The ratio is NOT determined by dietary intake or IV intake of any compound.  It is enzymatically controlled. That enzyme is called  “NAD(P)H dehydrogenase, quinone 1″, or NQO1 for short.  NQO1 is an unusual gene in that it requires NADH as a co-factor but does not convert the NADH into nicotinamide, like the Sirtuins or PARPs.  Instead, it converts NADH to NAD+.  (i.e. it only oxidizes NADH to NAD+).

The oxidation of NADH increases NAD+ levels  within the cell and at the same time, decreases NADH levels within the cell.  As a result, the NAD+/NADH ratio  increases (or the NADH/NAD+ ratio decreases).

Why is NQO1 such an important gene?  Well here are the “Top 10 Reasons”.

1,  NQO1 regulates the intracellular redox state of the cell and thus, the ratio of NAD/NADH in the cell.

This is the main reason why I now think that NQO1 is so important.  Mice that are homozygous negative (both genes knocked out) for NQO1 have an increase in the NADH/NAD ratio (and an increase in the NADPH/NADP ratio).  NQO1 knockout mice strangely enough have lower blood glucose levels, less abdominal fat.  However, they have higher levels of triglycerides, beta-hydroxybutyrate, pyruvate, and lactate.   They also have higher levels of glucagon.   More importantly, the NQO1 “knock out” mice have lowered rates of pyridine nucleotide synthesis,  reduced glucose metabolism, and reduced fatty acid metabolism.  This is not surprising since NQO1 is the controller of the 20S PC mediated degradation of PGC-1alpha.

Unfortunately, cancer cells have also discovered this wonderful property of NQO1.  Many cancers unregulate NQO1 either via Nrf2 pathways or by other methods, such as the loss of miR suppression of mRNA for NQO1.  A recent study shows that higher levels of the NQO1 protein predict poor prognosis in non-small cell lung cancer.  This is a sobering thought – cancer cells up-regulate antioxidant genes!  This does not mean that we should avoid phytosubstances that up-regulate NQO1, it just means that “cancer cells are smart”.

Introducing:  BET proteins

The opposite problem occurs in aging cells.  Aging cells have lower levels of expression of the gene NQO1.  This is not just due to a “lack of broccoli” or a “lack of exercise”.   Instead, the gene NQO1 is down-regulated by proteins called “epigenetic readers”.  The two “epigenetic readers” that suppress the Nrf2-induction of the NQO1 gene are called “Bromodomain and Extraterminal Proteins” (or BET proteins).  Specifically, Brd2 and Brd4 proteins “sit on top” of the histone protein acetylated lysines at the promoters of the Nrf2-dependent genes.  As a consequence, Nrf2 and the other transcription factors that “turn on” NQO1 gene cannot turn the gene on.  (This is why BET inhibitors like JQ1 are so exciting).

Summary:  NQO1 regulates the ratio of NAD/NADH and the ratio of NADP/NADPH by oxidizing NADH to NAD+.   Warburg-type metabolism ensures that most of the NAD(H) within the cell is in the reduced form (NADH).  NQO1 is one of the few genes that oxidizes NADH to NAD+.  When both genes for NQO1 are  “knocked out”, there is even more NADH in the reduced state.  This results in a lower NAD/NADH ratio (or higher NADH/NAD ratio).  Thus, NQO1 is the “anti-Warburg gene”.   This is why NQO1 is so important.

References:

2001 In Vivo Role of NAD(P)H:Quinone Oxidoreductase 1 (NQO1) in the Regulation of Intracellular Redox State and Accumulation of Abdominal Adipose Tissue

2015 NQO1 protein expression predicts poor prognosis of non-small cell lung cancers

2013 Bromodomain and extra-terminal (BET) proteins regulate antioxidant gene expression

2.  NQO1 regulates the level of the co-activator, PGC-1a,within the cell by controlling degradation rate via the 20S proteasome.

PGC-1a is my “favorite co-activator” because it is so important in mitochondrial biogenesis.  I always thought that exercise activated PGC-1a gene via the “exercise kinase” called AMPK. (See he blog enry PGC-1-alpha and exercise). This is why I was shocked to find out that NQO1 actually regulates PGC-1a, not by the increase in expression of the NQO1 gene, but the the rate that PGC-1a is degraded.  Expression of NQ01 keeps PGC-1a from being degraded.  What I found out is that the level of PGC-1a protein in a cell is primarily determined by its degradation rate, not its synthesis rate.

Like many regulatory factors, PGC-1a has an extremely short half life.  All of these extremely short-lived proteins are regulated by degradation rates, not synthesis rates.  In the past, it was thought that PGC-1a degradation was only regulated by the ubiquitin-proteasome system (UPS).  The UPS method involves a “protein tagger” that goes around putting a ubiquitin “tag” on the protein to be degraded.

Introduce: the 20S proteasome

However, recently a new process of proteasomal degradation has been discovered that does NOT involve any ubiquitination.  Specifically, this proteasome does NOT require ubiquitination of the protein and this proteasome system is called the “20S proteasome catalytic particle”  (aka 20S PC).  Unlike the ubiquitin-dependent, 26S proteasome system (UPS), the 20S proteasome does not require protein unfolding to degrade the protein.  (i.e. it can degrade proteins even without unfolding them).  Moreover, the 20S proteasome can handle oxidized proteins much better than the UPS 26S proteasome.  As a consequence, the 20S proteasome is the “oxidized protein degrader in stressed cells”.  For instance, it takes 4 times as much hydrogen peroxide to inhibit the 20S proteasome as it does to inhibit the 26S proteasome of the UPS.

Introducing: intrinsically disordered proteins (IDPs)

Not all proteins are degraded by the 20S proteasome, however. The main type of proteins degraded by the 20S PC system are called “intrinsically disordered proteins” (or IDPs).  Interestingly, the 20S proteasome system seems to be regulated by oxidative stress, via the glutathionylation of cysteine residues in the alph-rings of the 20S proteasome.

In conclusion, PGC-1a is a “intrinsically disordered protein” (IDP) that is regulated by its degradation rate.  When PGC-1a is damaged by oxidation or when the cell is under oxidative stress (like with aging), the 20S proteasome controls its degradation rate and thus the levels of PGC-1a within the cell.   Other IDPs besides PGC-1a include p53, c-fos, C/EBPa, p63, p33, p73a, and ornithine decarboxylase (ODC).

Interestingly, the 20S PC system has a “gate keeper” that inhibits the IDPs from being degraded.  Guess who the “gatekeeper” is for 20S PC? Yes, it is NQO1. That is how NQO1 expression keeps PGC-1a around.

Summary:

There is strong evidence now that the levels of PGC-1a in cells is regulated primarily by the degradation rate of PGC-1a, and only secondarily by the gene expression of the PGC-1a gene.  There are two degradation pathways for PGC-1a.  The two pathways are the Ubiquitin Proteasome system (UPS) and the ubiquitin-independent proteasome system called 20S PC..

Under conditions of no oxidative stress, the UPS system may regulate PGC-1a levels within the cell.  However when the cell is under cellular stress and the PGC-1a protein is damaged by ROS-induced oxidation, the 20S proteasome controls the degradation rate of PGC-1a. NQO1 is the “gate-keeper” for this 20S PC system that prevents PGC-1a from being degraded during periods of cellular oxidative stress.  Thus with aging, the 20S PC system is more important than the26S proteasome  (i.e. the UPS) and thus the 20S proteasome degrades PGC-1a in the cell, unless NQO1 protects it from degradation. Thus it appears that under conditions of oxidative stress, such as with aging, NQO1 may be a major factor that controls the concentration of PGC-1a in the cell. 

References:

2013 The Protein Level of PGC-1α, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

2001 Degradation of oxidized proteins by the 20S proteasome

2014 Regulating the 20S Proteasome Ubiquitin-Independent Degradation Pathway

1998 Comparative resistance of the 20S and 26S proteasome to oxidative stress

2013 The Protein Level of PGC-1α, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

2006 20S proteasomes and protein degradation “by default”

1996 Degradation of oxidized proteins in K562 human hematopoietic cells by proteasome

2013 Redox regulation of the proteasome via S-glutathionylation

3.  Lower levels of NQO1 leads to increased sensitivity to chemical-induced skin carcinogens.

A very interesting study was done looking at skin-induced cancer from carcinogens.  This study looked at NQO1-null mice and found that their levels of NADH was higher (as expected).

Normally, when skin is exposed to chemical carcinogens, p53 is rapidly unregulated.  In the NQO1-null mice, exposure to chemical carcinogens did not induce p53 and as a result, the cells did not undergo apoptosis.  Instead, they underwent transformation to cancer cells.  This is an amazing and very important finding.  One  feature of p53 induction in the skin is an increase in the appearance of melanin in the skin.  This is normally called a “tan” by most people, but on a molecular level, a “tan” is actually p53 induction.  This is why people with dark skin have a lower incidence of skin cancer (it is not all to do with the sun block effect of melanin.  It is all about p53).

In a study of various mutations in the NQO1 gene in human basal cell carcinomas, 3.1% of 457 cases were found to have “loss-of-function” mutations in the NQO1 gene.  Those “NQO1 loss-of-function” individuals were found to have more skin cancers than those with other mutations, but this was not statistically significant.  The authors concluded that NQO1 mutations were clearly associated with skin cancer risk, but that these mutations only accounted for a minority of skin cancers.

Summary:  NQO1 stabilizes p53 and prevents its degradation. p53 levels in the cell is tightly regulated by two separate degradation pathways – a ubiquitin-dependent pathway that is dependent on the p53 binding partner, Mdm2;  or the ubiquitin-independent pathway that is dependent on NQO1.   It appears that the NQO1-dependent (ubiquitin-independent) pathway is the most important pathway for regulating p53 levels within the cell.

In the experiment above, NQO1-null mice did not induce p53 in response to carcinogens and the damaged skin cells would not undergo apoptosis, as they should to prevent cancer formation.  As a result, the NQO1-null mouse skin cells developed cancer.  These effects were thought to be directly due to the lack of binding of NQO1 to p53, which would preven the 20S PC degradation of p53 in the cell.  This means that p53 induction and cell apoptosis is dependent on NQO1-mediated stabilization of p53, preventing the 20S PC degradation of p53.  This is another reason why HQO1 is so important.   However in humans, loss-of-function mutations in the NQO1 gene only account for 3.1% of human skin cancers.

References:

2005  Lower Induction of p53 and Decreased Apoptosis in NQO1-Null Mice Lead to Increased Sensitivity to Chemical-Induced Skin Carcinogenesis

2003 p53 hot-spot mutants are resistant to ubiquitin-independent degradation by increased binding to NAD(P)H:quinone oxidoreductase 1

1999 Association of NAD(P)H:quinone oxidoreductase (NQO1) null with numbers of basal cell carcinomas: use of a multivariate model to rank the relative importance of this polymorphism and those at other relevant loci

4.  NQO1 regulates blood pressureby eNOS, ACE, and an LKB1/AMPK-mediated preservation in GTPCH-1

Another fascinating study showed that activation of NQO1 ameliorates spontaneous hypertension in a rat model.  As you may know, spontaneous hypertension does not normally occur in rodents.  But in certain strains of inbred rats, bred to develop spontaneous hypertension, high BP does occur and is thought to be mediated by a decline in nitric oxide production by endothelial cells.  In this rat model of spontaneous hypertension, activation of NQO1 by beta-lapachone relieved the hypertension in these rats. The positive effects of beta-lapachone were thought to be mediated by NQO1-induction of endothelial nitric oxide (eNOS).

Another study showed that the effect of NQO1 was to regulate the acetylation of eNOS.  When an eNOS inhibitor was used, the positive effects of beta-lapachone was completely blocked.

In a separate study, beta-lapachone was used to study the effects of the shedding of the enzyme angiotensin converting enzyme (ACE), which converts Angiotensin I to Angiotensin II in the blood stream.  This study showed that beta-lapachone increased NQO1 activity which resulted in reduced cleavage and secretion of ACE into the extracellular space surrounding the cells that synthesized ACE.

In the most recent study, further elucidation of the eNOS mediated mechanism was analyzed and figured out. In this study, they showed that the increase in NAD+ levels in the aortic endothelial cells resulted in an increase in LKBA deacetylation, and AMPK phosphorylation.  This was followed by an increase in GTP-cyclohydrolase-1 preservation and tetrahydrobiopterin/dihydrobiopterin ratio.  This explained the rest of the story on how beta-lapachone reduced blood pressure.

Both beta-lapachone and the polyphenol, epicatechin, have the effect of reducing blood pressure.  Beta-lapachone does this via the direct activation of NQO1, whereas epicatechin does this by activating Nrf2.   Nrf2 is the transcription factor that turns on the NQO1 gene.

Summary:  NQO1 elevated the ratio of NAD/NADH in the endothelial cells and increases eNOS activity via an AMPK-dependent mechanism.  The increase in AMPK phosphorylation resulted in a preservation of the GTP cyclohydrolase-1 (GTPCH-1), which resulted in a lowering of blood pressure. The elevation in the NAD/NADH ratio also results in a reduced cleavage and secretion of ACE into the bloodstream, thereby reducing Angiotensin II formation.  As a result of the eNOS-mediated method and the ACE-reduction mediated molecular mechanism, the hypertension in rats resolved.   As a result of all this research, NQO1 activation has been recently proposed as a strategy for controlling hypertension (see lst reference below).

References:

2011  Activation of NAD(P)H:quinone oxidoreductase ameliorates spontaneous hypertension in an animal model via modulation of eNOS activity

2013 NQO1 Activation Reduces Blood Pressure via Regulation of eNOS Acetylation in Spontaneously Hypertensive Rats

2013 NQO1 Activation Regulates Angiotensin–Converting Enzyme Shedding in Spontaneously Hypertensive Rats

2014 Enhanced activation of NAD(P)H: quinone oxidoreductase 1 attenuates spontaneous hypertension by improvement of endothelial nitric oxide synthase coupling via tumor suppressor kinase liver kinase B1/adenosine 5′-monophosphate-activated protein kinase-mediated guanosine 5′-triphosphate cyclohydrolase 1 preservation

2012 Epicatechin lowers blood pressure, restores endothelial function, and decreases oxidative stress and endothelin-1 and NADPH oxidase activity in DOCA-salt hypertension

2014 NQO1 activation: a novel antihypertensive treatment strategy?

5.  “Loss of function” Polymorphisms in the NQO1 gene are associated with carotid artery atherosclerosis/plaques and stroke risk

This was the most amazing study.  There is a well-known polymorphism in the NQO1 gene called the “C609T variant.”  The C609T polymorphism results in a complete loss of enzymatic activity of NQO1 due to protein instability.  The C609T SNP is very common in Asia and has been well-described in Japanese, Korean, and Chinese ethnic groups.  Individuals with the “C allele” have a lower risk of carotid plaque disease, whereas individuals with the “T allele” have a higher risk of carotid atherosclerotic disease (OR = 1.65).  In Korea, 42% of the population have one “T allele” and 1% of the population have two copies of the “T allele”.  16% of Caucasians have one or two copies of the C609T variant, whereas 49% of Chinese have one or two copies of this SNP.

Another common polymorphism in the NQO1 gene is the C465T mutation.  This SNP results in a reduction in enzyme activity, but not a complete loss of function like the C609T SNP.   The incidence of this SNP is very low in all populations, varying from 0-5% (see reference below).

References:

2009  The C609T variant of NQO1 is associated with carotid artery plaques in patients with type 2 diabetes

2009 An Association between 609 C → T Polymorphism in NAD(P)H: Quinone Oxidoreductase 1 (NQO1) Gene and Blood Glucose Levels in Korean Population

6.   Bromodomain and Extraterminal Proteins (BET) supppress Nrf2-mediated gene expression (including NQO1)

Introducing:  BET protein inhibitors

We mentioned Bromodomain proteins in Item 1 above.  As you may recall, these are “epigenetic readers” that “read” the post-translational modifications of histone proteins.  The BET proteins (Brd2, Brd3, Brd4, and BrdT) bind to acetylated lysine residues on histone and non-histone proteins.  As a result, they either INCREASE or DECREASE the transcription of the genes associated with these acetylated lysine histones.  As it turns out, the Nrf2 genes are regulated by Brd2 and Brd4 proteins.  These complex with the acetylated lysine residues on histones located at the promoters of Nrf2-regulated genes.  As a result, Nrf2 either cannot increase or decrease the expression of these target genes.  When they gave the cells a BET protein inhibitor called JQ1, it increased the expression of Nrf2-genes including HO-1, NQO1, and GCLC, all of which are important in anti-oxidant defense and regulation of intracellular redox state. JQ1 administration resulted in a 10-fold increase in HO-1messenger RNA and a 3-fold increase in HO-1 protein levels.  JQ1 administration resulted in a 3-fold increase in NQO1 mRNA expression and a 3-fold change in NQO1 protein expression.  JQ1 also increased mRNA expression for GCLC by 2-4 fold and GCLC protein expression by 3-4 fold.

Interestingly, JQ1 also inhibited the expression of the ROS-producing protein, Nox.  As a result of this down-regulation of Nox, there was less free radicals in the cell (less H2O2).  This resulted in less oxidative stress in the cell due to both a reduced ROS production and increased anti-oxidant enzymes.

This page lists 14 other BET inhibutors,  And this 2011 paper discusses the discovery and characterization of small-molecule BET inhibitors

Conclusion: Bromodomain “epigenetic readers” can “shut off” the expression of Nrf2 genes and increase the expression of free-radical producing genes (Nox).  Inhibition of BET proteins with JQ1 has the effect of increasing the expression of Nrf2 genes and decreasing the expression of free radical-producing genes (Nox).  This may be a key discovery as one of the major causes of oxidative stress-induced aging may be BET proteins.)  And may also be why eating Broccoli and exercising have failed to lengthen life span.  JQ1, the most well-studied BET inhibitor, suffers from poor pharmacokinetics with a high clearance and low oral biovailabillity in animal studies.  This is why a lot of work is going into developing better BET inhibitors.

References:

2014 Bromodomain and Extra-Terminal (BET) proteins suppress nuclear factor E2-related factor 2 (Nrf2) -mediated antioxidant gene expression

2014  Bromodomain and Extraterminal Proteins Suppress NF-E2–Related Factor 2–Mediated Antioxidant Gene Expression

2014 Bromodomain and Extra-Terminal (BET) proteins suppress nuclear factor E2-related factor 2 (Nrf2) -mediated antioxidant gene expression

2013 Bromodomain and extra-terminal (BET) proteins regulate antioxidant gene expression

2014  Bromodomain and Extraterminal Proteins Suppress NF-E2–Related Factor 2–Mediated Antioxidant Gene Expression

2014 Bumping into BET inhibitors

2014 New benzazepine BET-inhibitors with improved oral bioavailability

7.  The NQO1 gene is regulated by other factors besides Nrf2 – c-Jun, Nrf1, Jun-B, Jun-D, etc.

Quercetin “turns on” the NQO1 gene via Nrf2.   Dioxin “turns on” the NQO1 gene via both AhR, Arnt, and Nrf2.  Luteolin inhibits expression of NQO1 and drug-metabolizing enzymes via AhR and Nrf2 pathways.

As you may know, the levels of Nrf2 and the location of Nrf2 proteins within the cell is primarily regulated by the binding partner of Nrf2, aka Keap1.  There is a new name for Keap1, called INNrf2, but this new name is having trouble getting any attention in the scientific literature  This Important transcription factor and its binding protein havr been often discussed in this blog.  A comprehensive  series of entries on Nrf2 was published in this blog in 2012: Part1Part2 and Part3.

An older article from 2000 showed that the NQO1 gene is regulated by several factors other than Nrf2 binding to the ARE segment of the NQO1 promoter.  They showed the transcription factor, c-Jun, can also bind to the ARE promoter sites on Nrf2-dependent genes.  So do the transcription factors Nrf1, Jun-B, and Jun-D.  Polyphenols can “turn on” NQO1 via Nrf2.  Toxins like dioxin can also ‘turn on” Nrf2, but require the assistance of the aryl hydrocarbon receptor, AhR, and the the aryl hydrocarbon receptor nuclear translocator, Arnt.

References:

2000 Regulation of genes encoding NAD(P)H:quinone oxidoreductases

2001 Induction of human NAD(P)H:quinone oxidoreductase (NQO1) gene expression by the flavonol quercetin

1991 Human NAD(P)H:quinone oxidoreductase (NQO1) gene structure and induction by dioxin

2000 NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms

2015 Constitutive expression of the AHR signaling pathway in a bovine mammary epithelial cell line and modulation by dioxin-like PCB and other AHR ligands

2014 Luteolin modulates expression of drug-metabolizing enzymes through the AhR and Nrf2 pathways in hepatic cells

8.  The Asian vegetable, “pak choi,” reduces colon inflammation and colon cancer even better than broccoli sulforaphanes

In Asia, there is a very popular type of green, leafy vegetable called “pak choi”.  As a child, I ate this frequently as part of our regular diet in Thailand.  It is not a particularly “tasty” vegetable and reminds me of a cross between spinach and cabbage, but when boiled or steamed, it is a common vegetable eaten with rice.

A recent study showed that pak choi and brassica vegetables both activated cytoprotective genes, but the sets of genes that were activated were different.  Specifically, pak choi, broccoli, brussel sprouts, and other brassica vegetables all activated the typical Nrf2-target genes (NQO1, GSTM1, SRXN1, GPX2), whereas pak choi alone activated the AhR target gene,CYP1A1.  The relevance in the current context is that NQO1 belongs to a group of the aryl hydrocarbon receptor (AhR) battery of drug-metabolizing enzymes that are characteristically induced by both AhR agonists and Nrf2 activators(ref).               r

More importantly, in the studied mouse models of colitis and colon cancer, the glucosinolate-rich pak choi drastically reduced colitis and colon tumor number, whereas the broccoli-diet did not reduce colitis or colon tumor number in mice.

Conclusion:  The presence of glucosinolates (sulphoraphanes, etc.) in food does not necessarily reduce colon inflammation,  and colon cancer.  It appears that certain foods may have more stable or different glucosinolates that are more effective than others at preventing cancer and inflammation.  The Asian vegetable, pak choi,  appears to be more effective than broccoli and other brassica vegetables in down-regulating inflammation and preventing colon cancer.  (This study was done in Germany, by the way, and was not sponsored by the pak choi industry).

Reference:  2014  Glucosinolates from pak choi and broccoli induce enzymes and inhibit inflammation and colon cancer differently

9.  Physical methods and many readily available drugs and phytosubstances increase NQO1 expression or increase NQO1 activity

Including hyperthermia, heat shock, photodynamic herapy, sulindac, dimethylfumarate, taxifolin, sulforaphane,  resveratrol, and cisplatin.

A recent discovery that an old, common, generic NSAID that is still available at the drug store also activates the NQO1 gene.  Sulindac, a long neglected compound used to treat arthritis, activates the NQO1 gene.  Also, previous work has shown that several other compounds up-regulate the NQO1 gene or increase the activity of NQO1.   This includes cisplatin, resveratrol, dimethyl fumarate, taxifolin, sulforaphane, and the glucosinolates in pak choi.  Since the cancer-killing effects of beta-lapachone are dependent on the levels of NQO1, all of the above compounds work synergistically to kill cancer with beta-lapachone.

Several physical methods have been shown to increase NQO1 gene or protein activity.  This includes hyperthermia, heat shock, and photodynamic therapy.  This may be how photodynamic therapy works in cancer cells.  Interestingly, beta-lapachone works synergistically with these physical methods as well.

References:

2014 Sulindac Compounds Facilitate the Cytotoxicity of β-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

2015 NQO1 protein expression predicts poor prognosis of non-small cell lung cancers

2014 The Chemotherapeutic Effects of Lapacho Tree Extract: β-Lapachone

2013 Preventive Effects of NSAIDs, NO-NSAIDs, and NSAIDs Plus Difluoromethylornithine in a Chemically Induced Urinary Bladder Cancer Model

2015 Effect of glycosylation patterns of Chinese eggplant anthocyanins and other derivatives on antioxidant effectiveness in human colon cell lines

10.  Beta-lapachone, a compound found in the bark of the South American Lapacho tree, is a potent activator of the NQO1 protein and produces ROS in cancer cells, but reduces ROS in non-cancer cells.  It also inhibits pathological retinal neovascularization, but does not inhibit physiological neovascularization. 

The most exciting thing about NQO1 is that there is a natural, cheap, compound found in the tree bark of a South American tree.  The compound is called beta-Lapachone and is a NQO1 activator.  Specifically, NQO1 is a “two-electron transfer” enzyme that can extinguish free radicals in normal cells, but produces free radicals in cancer cells.  It has been shown to be a very effective compound for treating lung cancer.  Here is how it works:

  • Beta-lapachone undergoes a redox cycle by NQO1, which reduces beta-lapachone to an unstable semiquinone.  The semiquinone then rapidly undergoes a two-step oxidation back to the parent stable compound, beta-lapachone.  This produces what is called a “perpetuating futile redox cycle”.This results in an unbalance of intracellular reactive oxygen species in cancer cells, resulting in the cell death of the cancer cells.  This “perpetual futile redox cycle” is totally dependent on the concentration of NQO1 within cells. Here is a diagram of the reaction:
PT4-a

Illustration reference:   2014 The Chemotherapeutic Effects of Lapacho Tree Extract: β-Lapachone

The downstream effects of perpetual futile redox cycling include 4 apoptotic pathways and one necroptotic pathway:

  1. Mitochondrial-induced apoptosis – The induction of ROS in mitochondria opens the MPTP pores and results in PARP activation and caspace activation.  This induces apoptosis.  
  2. ER-induced apoptosis – The induction of ER stress induces sarcoplasmic release of calcium which induces high levels of cytoplasmic Ca++.  This also induces apoptosis via the ER.
  3. DNA-damage mediated apoptosis – beta-lapachone also induces Topoisomerase I and II.  The activation of topoisomerases  induces DNA breaks, which induces PARPs.  This PARP hyper-activation induces apoptosis independently from mitochondrial ROS or ER stress.
  4. Cell cycle arrest-induced apoptosis – The futile redox cycling of beta-lapachone also induces cycle cycle arrest via the activation of p21, p27, and the phosphorylation of JNK, PI3K, and Akt.  This induces cancer cell apoptosis as well.
  5. Calpain-induced cell necrosis – Unlike the 4 pathways above, futile redox cycling also induces calcium influx into the cells independently of ER stress.  This calcium influx into the cell activates Calpain, which induces cell death by the necrosis pathway, not the apoptosis pathway.

Conclusion:  beta-lapachone induces cancer cell death by five different pathways, all dependent on perpetual futile redox cycling which is dependent on NQO1 expression.   Here is a diagram that illustrates these 5 pathways:

PT4-b

Illustration reference:   2014 The Chemotherapeutic Effects of Lapacho Tree Extract: β-Lapachone

Interestingly, Sulindac, the old generic FDA-approved non-steroidal anti-inflammatory drug, augments the cytotoxicity of beta-lapachone in lung cancer cells.  This appears to be due to its effect of up regulating expression of the NQO1 gene.  Cancer cells with higher levels of NQO1 actually are more sensitive to beta-lapachone than cancer cells with lower expressions

of NQO1.  This may be an “Achilles heel” discovery for cancer cells, allowing us to exploit one of the vulnerable points of cancer cells that have unregulated their anti-oxidant capacity (Many cancers have unregulated Nrf2).

Beta-lapachone shows a lot of promise in treating the most common cause of blindness – neovasculariation of the retina. In the retina, beta-lapachone inhibited pathological retinal neovascularization induced by HIF-1alpha.    Unlike VEGF inhibitors that are currently being used for inhibiting retinal neovascularization, beta-Lapachone does not inhibit physiologic angiogenesis…..only pathologic angiogenesis.

References:

2014 Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

2014 Beta-lapachone inhibits pathological retinal neovascularization in oxygen-induced retinopathyvia regulation of HIF-1α

2014 Sulindac Compounds Facilitate the Cytotoxicity of β-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

2014 The Chemotherapeutic Effects of Lapacho Tree Extract: β-Lapachone

II.  The Warburg effects; its causes and consequences and roles in aging in the NAD World

WAARBURG

Image source video

The Warburg effect is an important phenomenon characteristic of aging.  It is best known for its existence in cancer cells as well as in aging, and for having several negative biological consequences, though there can be a positive side to it too. The essence of the Warburg effect is a metabolic transformation from energy production  from oxidative phosphorylationin the mitochondria to glycolosis.  The effect was originally seen in cancer cells. ” Warburg’s hypothesis was postulated by the Nobel laureate Otto Heinrich Warburg in 1924.[3] He hypothesized that cancer, malignant growth, and tumor growth are caused by the fact that tumor cells mainly generate energy (as e.g. adenosine triphosphate / ATP) by non-oxidative breakdown of glucose (a process called glycolysis). This is in contrast to “healthy” cells which mainly generate energy from oxidative breakdown of pyruvate. Pyruvate is an end-product of glycolysis, and is oxidized within the mitochondria. Hence, according to Warburg, the driver of cancer cells should be interpreted as stemming from a lowering of mitochondrial respiration. Warburg reported a fundamental difference between normal and cancerous cells to be the ratio of glycolysis to respiration; this observation is also known as the Warburg effect(ref).”  Although much of the research literature related to the Warburg effect is cancer-related, in recent years it is being seen to be very important in aging as well.  Specifically, I think there is strong evidence that Warburg metabolism reduces the NAD/NADH ratio in the nucleus, the cytoplasm, and the mitochondria.  And, the Warburg effect may be the main reason why that decline is observed in aging.

The Warburg effect and cancers

I don’t think I understood Warburg metabolism until I read the following articles.

2009 Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation

Wikipedia: Warburg hypothesis

2006  Cancer’s Molecular Sweet Tooth and the Warburg Effect

2008 Cellular life span and the Warburg effect

2004 Glycolytic Enzymes Can Modulate Cellular Life Span

2009 Nutrient transporters in cancer: Relevance to Warburg hypothesis and beyond

2006 Cancer’s Molecular Sweet Tooth and the Warburg Effect

2007 Protection from oxidative stress by enhanced glycolysis; a possible mechanism of cellular immortalization

2007A High Glycolytic Flux Supports the Proliferative Potential of Murine Embryonic Stem Cells

2009 Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation

2013 Oncogene-induced cellular senescence elicits an anti-Warburg effect

Conclusions:

1. Precancerous cells shift to Warburg type metabolism to achieve immortality. The cost of this shift is a decrease In the NAD+/NADH ratio, which inhibits DNA repair by DNA repair enzymes that are dependent on NAD (PARP, SIRT1, SIRT6) and epigenetic histone modifiers that are dependent on NAD (ARTDs, SIRTs).   Also, the previously discussed costs in mitochondrial metabolism which leads to further the Warburg effect.

The poor (slow) DNA repair -allows for DNA mutations to occur at much more rapid rates than what is seen with normal evolution (I.e. Stochastic mutation rates), allowing tumor suppressor genes to be mutated and oncogenes to loose their regulation. The epigenetic dysfunction induced by the change in NAD+/NADH ratios also allow for gene activation or gene silencing to occur by Bromidomain (BET) proteins, histones, DNA methylation, miRNA, and lncRNA over expression. Thus, both DNA mutations and Epigenetic changes occur as a result of the Change in DNA repair that occur with precancerous stages of Cancer.

2. Warburg type metabolism makes precancerous and Cancer cells resistant to oxidative stress because  they upregulate gene expression of antioxidant enzymes via Nrf2, Nrf1, c-Jun, FoxO3a, Jun-B, Jun-C, etc. They accomplish this by activating Nrf2 with endogenously produced ROS, which is a potent activator of Nrf2 nuclear localization, as well as Nrf1, FoxO3a, c-Jun, Jun-B, and Jun-C.

3. Warburg type metabolic shifts activate the  telomerase gene and  cell cycle genes needed for cellular proliferation and survival. This has been well documented in stem cell research. Lowering Oxygen levels in cell culture activates telomerase and cell cycle genes very potently, without the need for TA-65 or any other supplement.

4. An unbiased screen of gene expression in Warburg metabolism cells showed that the “driver” of the Warburg metabolism phenotype is the expression of phosphoglycerate mutase (PGM) and phosphoglycerate isomerase (PGI). SiRNA knockdown of these glycolytic genes induced Cancer cell cycle arrest and cell senescence. Because p53 down-regulates PGM, Cancer cells that survive are the ones that either epigenetically “turn off” p53 or p53 is mutated due to poor DNA repair (see #1 above)

Reference:  2005 Glycolytic Enzymes Can Modulate Cellular Life Span

5.  Eating sugar is NOT the cause of Cancer. This is a common fallacy in layperson circles, such as Mercolas Website, etc. Sugar may help spur cancer growth, but the primary contribution of sugar to Cancer cells is that sugar enables mTOR signaling,

6. The last and most important reason that Cancer cells “actively induce the shutting off of mitochondrial function” is that mitochondria are the subcellular organs that induce apoptosis (I.e. Cellular suicide). When mitochondria are “shut off”, you effectively shut off all of the intrinsic suicide pathways in one “fell swoop”!  The Caspace and Caspace-independent pathways are all inactivated! Cancer cells call this “A genius maneuver”. We humans call this “Cancer chemotherapy and Radiation resistance” (Cancer cells have a different point of view than we humans do!).  Thus the ultimate weapon of the Warburg effect is to inactivate apoptosis!

7. We can possibly counteract the Warburg effect and decline of the NAD/NADH ratio. We can start out to do this by activating NQO1. It will take more than one serving of Broccoli, however.  We must address the epigenetic reader problem (Bromidomain proteins 2 and 4 that prevent Nrf2 activation of NQO1 gene expression). See the above discussion in Section I on Brd2 and Brd4. Here are some ideas: In addition to

  • upregulating the NQO1 gene by inhibiting Brd2 and Brd4 Bromidomain proteins with BET inhibitors,
  • activating perpetual redox cycling with beta-lapachone, and
  • inducing Nrf2 with exercise, broccoli, and Pak choi;

there may be some value to trying metabolic inhibitors of Warburg-type metabolism to restore the NAD/NADH ratio to normal. The best established metabolic inhibitors are DCA, 3-BrOP, and 2-DG. These all restore mitochondrial respiration and mitochondrial-mediated apoptosis, thereby stopping cancer cell proliferation and inducing cancer cell apoptosis.  There might be equally effective phytosubstance inhibitors.  See Section IIIB below, Countering the Warburg Effect with Phytosubstances.

Aging and the Warburg effect

While the Warburg effect has primarily been studied in the context of cancers, it can arise powerfullyin the context of aging.  We note first that the effect is not necessarily all-or-none (mitochondrial vs, glycolic metabolism).  It can arise gradually over time in the process of aging.  Indeed David Sinclair et al’s paper Declining NAD+Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging identifies a mechanism by which age-related decline of NAD+ leads to mitochondrial dysfunction which leads to the Warburg effect.

In a cell affliced by the Warburg effect, the cell converts most of the NAD+ into NADH with aerobic glycolysis.  Two molecules of NAD+ are consumed for every  glucose molecule that is oxidized in aerobic glycolysis.  Thus you loose two moles of NAD+ for every mole of glucose you consume via the Warburg effect! 

The Warburg effect is the use of the glycolytic pathway in the presence of oxygen, whereas anaerobic glycolysis is the glycolytic pathway in the absence of oxygen. Either way, all of the NAD gets converted into NADH with the Warburg effect. Here is a diagram of how/why the Warburg effect lowers NAD+ levels in the cell:

GLpath1
GLpath2

Diagram reference:  Glycolysis, Krebs Cycle, and other Energy-Releasing Pathways

Normally, the NADH produced is converted into ATP in the mitochondria.  However, when mitochondria do not work properly in aging and with age-related diseases, more and more NADH accumulates within the cell.  Some of the NADH is converted to NAD+ when the pyruvate is converted into lactate and then exported to the liver (i.e. the Cori cycle), but the Cori cycle does not keep NAD+ levels high.

Conclusion:  Warburg metabolism associated with aging results in low levels of NAD+ within the cell…….this is the culprit!  It is also the cause of itself in an unvirtuous cycle

Note that there is a positive feedback loop at work here lowering NAD+:  1.  An insufficient level of nulear NAD+ leads to expression of HIF-1alpha. 2.  This leads to a pseudohypoxic state that disrupts nuclear-mitochondrial communication leading to  deficiency in mitochondrially-encoded proteings resulting into dysfunctional mitochondria. 3.  This leads to metabolic reprogramming of the cell to support glycolosis and a Warburg-type metabolism, 4.  The Warburg metabolism converts NAD+ into NADH as illustrated in the diagram above.  Thus, this cycle of events reinforces itself.

Long non-coding RNAs can induce Warburg-type metabolism

Other actors can promote the Warburg effect.  I have now found 3 lncRNAs that induce Warburg-type metabolism as follows:

1. UCA1 (uroepithelial cancer-associated 1) – this is a lncRNA that was discovered in bladder Cancer cells, but appears to be Ubiquitously expressed in other types of cancers and oncancerous cells as well. It upregulates the TOR/STAT3/microRNA-143 pathway, which upregulates the First enzyme in glycolysis, hexokinase 2 (HK2).

Reference:  2014 Long non-coding RNA UCA1 promotes glycolysis by upregulating hexokinase 2 through the mTOR–STAT3/microRNA143 pathway

2. LncRNA p21 – this lncRNA also   Induces Warburg-type metabolism by a completely different molecular mechanism. LncRNA p21 interferes with the binding of VHL to HIF-1a.   As a result, HIF-1a is not degraded and HIF-1a turns on many glycolytic enzymes, even though there is plenty of oxygen present.

Long noncoding RNA may be the missing “dark matter” that explains the unaccounted for risk of developing cancer, disease, and even aging itself.  There appears to be almost 60,000 of these “genes” hidden in the noncoding portion of the human genome, which accounts for 98% of our DNA.  LncRNA-p21 is one of the few that has been characterized and may be the “unexplained dark matter” of the Warburg effect in cancer and in aging.

Until recently, long noncoding RNA were not recognized as being major regulators of gene expression.  With the recent advent of next generation RNA sequencing (RNA-seq), almost 60,000 long noncoding “genes” have been found in the human genome. As of yet, only a handful of these have been characterized for their functional significance, such as HOTAIR, MALAT1, H19, KCNQOT1, ANRIL, etc.

One of these is “Long noncoding RNA at the p21 locus”, or lncRNA-p21, which is an independent gene downstream from the protein-coding gene, CDKN1A, or p21. When the CDKN1A gene is transcribed, the lncRNA-p21 is also transcribed.  Thus although this gene is a separate gene, it is co-expressed with its protein-coding gene.

The lncRNA-p21 does not appear to regulate the CDKN1a gene, however.  Instead, it appears to be a lncRNA with wide-ranging effects, regulating distant genes via “trans-regulatory” mechanisms.  LncRNA-p21 is a hypoxia-inducible gene.  More-over, HIF-1a is responsible for the hypoxia-inducible expression of lncRNA-p21.

In addition, lncRNA-p21 then stabilizes HIF-1a by disrupting the binding of HIF-1a to VHL.  The net effect is a “positive feedback cycle“, where HIF-1a induces lncRNA-p21, which then prevents the degradation of HIF-1a by VHL.  This “positive feedback cycle” may be the greatest factor that determines cancer formation.

Hypoxiapq1

Image source

References:

2014  Reciprocal Regulation of HIF-1α and LincRNA-p21 Modulates the Warburg Effect (agstract)

2013 Differential Expression of Long Noncoding RNAs in the Livers of Female B6C3F1 Mice Exposed to the Carcinogen Furan

3. CRNDE – this lncRNA name is an Acronym for “Colorectal neoplasia differentially expressed” lncRNA.  As expected, it was discovered in colon cancer cells, but like UCA1 and lncRNA p21, appears to be universally expressed in many cancers and noncancerous tissues.  There are many splice variants of the CRNDE lncRNA, but the key splice variants include a specific sequence in intron 4 called the “gVC-ln4” sequence. gVC-ln4 copies of CRNDE induce Warburg type metabolism. Interestingly, CRNDE is regulated by insulin signaling. Thus, CRNDE may be the molecular mediator of why the Insulin/IGF-1 pathway causes aging (as opposed to sugar being the cause of aging).

Reference: 2014 CRNDE, a long non-coding RNA responsive to insulin/IGF signaling, regulates genes involved in central metabolism

Conclusions: At least 3 lncRNAs have now been discovered that explain why both aging and Cancer exhibit Warburg-type metabolism. There may well be more such, given that the “Wild West” of long non-coding RNAs is only now being explored. The evolutionary selection pressure for the development of UCA1, lncRNA p21, and CRNDE all are probably for cellular survival or organismal survival. These lncRNAs are one of the farthest “upstream “causes” of aging and cancer that I have seen.

About HOTAIR

Can we identify interventions which can affect the expression of such IncRNAs?  I think the answer is Yes.  At least this is the case for one non-coding RNA for which expression can be altered by curcumin.  This lncRNA is called “HOTAIR and is a critical regulator of cancer metastasis and cancer survival. HOTAIR is over-expressed by 100-fold in breast cancer cells and the level of its over-expression correlates with both metastasis and survival in breast cancer.  Although work in this area has just begun, one report already shows that the metastasis-preventing effects of curcumin in renal cell carcinoma (RCC) may be mediated via HOTAIR.  No other lncRNA has been linked to polyphenol mechanism of action to date, however.  Because knowledge of what IncRNAs do is unfolding so rapidly now, we will not be surprised to hear soon of other examples.

Reference:  Influence of Curcumin on HOTAIR-Mediated Migration of Human Renal Cell Carcinoma Cells

III Metabolic reprogramming from the Warburg Effect

A.  Cells can be weaned off from Warburg metabolism back to mitochondrial respiration via DCA  and TMZ.  (and off Glutaminolysis with Arsenic Trioxide)

I am fascinated with the idea of using safe, nontoxic compounds for reprogramming our cells to be “weaned off” of aerobic glycolysis and to switch them back to glucose oxidation.  I am now convinced this can be done with plant polyphenols, assuming they are effectively delivered tp cells.  First, I focus focus on two drugs that are well known to do this,  DCA and TMZ.  Then I discuss the potential use of plant polyphenols in Subsection B to follow.

DCA and TMZ have been shown to have amazing effects on the heart, especially for heart failure with preserved ejection fraction (HFPEF).  However, in addition to treating HFPEF, these compounds appear to have great potential for treating or preventing cancer. Here is what I have learned so far about them.

1. Systemic Factors that “drive” the development of the heart’s dependence on free fatty acids (paradoxic effect….opposite of a ketogenic diet)

Cardiac-intrinsic causes of HFPEF

With diabetes, there is a 2-fold higher risk of heart failure in men and a 5 fold higher risk of heart failure in women.  This cannot be explained by advanced glycation end products, coronary artery disease (and MI), and hypertension alone.  There are also “cardiac intrinsic mechanisms” that are at work which are responsible for this dramatic increase in HF in diabetics.  In addition, non-diabetics develop a type of heart failure where there is no pre-existing heart attack and even in some cases, no hypertension.  This is called “normal ejection fraction heart failure”, or HFPEF.

An article on this is: 2003 Heart Failure – The frequent, forgotten, and often fatal complication of diabetes

Metabolic Reprogrammiong in the Heart – “mhs isoform switching”

With HFPEF in diabetes and with aging, there appears to be a metabolic energy substrate switching in the heart from the use of glucose as fuel to the use of free fatty acids as fuel.  This gene expression change is referred to as “mhc isoform switching”.  Here there is an increase in myosin V3 gene expression and the sarcoplasmic reticulum Ca++ pump ATPase genes.  This results in cardiac hypertrophy and metabolic reprogramming.  In addition the decreased glucose availability in the heart that occurs with insulin resistance and a high fat diet in diabetes drives this process.  As a result, gfat2 is expressed instead of gfat1.  Another systemic factor besides glucose and high free fatty acids is the stress hormones produced from the HPA axis and the sympathetic nervous system (SNS).  Specifically,  the epinephrine  (from the adrenal gland) and norepinephrine (from the sympathetic nervous system) that is produced when we are psychologically stressed, and with normal everyday waking hours (i.e. HPA axis hormones  and SNS stimulation).  In addition to triggering genes that switch fuel use (metabolic reprogramming), the SNS and HPA axis triggers gene expression of “fetal gene programs”.    Interestingly, dietary medium chain triglycerides (MCT oil) can prevent this occurrence.

Here are some articles on this:

2007 Proposed Regulation of Gene Expression by Glucose in Rodent Heart

1986 Diabetes mellitus and hypothyroidism induce changes in myosin isoenzyme distribution in the rat heart–do alterations in fuel flux mediate these changes?

1994 Modification of myosin isozymes and SR Ca(2+)-pump ATPase of the diabetic rat heart by lipid-lowering interventions

1995 Dietary medium-chain triglycerides can prevent changes in myosin and SR due to CPT-1 inhibition by etomoxir

1982 Cardiac alpha- and beta-adrenergic receptor alterations in diabetic cardiomyopathy

2.  Systemic Factors that “drive” cardiac fibrosis, cardiac remodeling, and age-related heart failure with preserved ejection fraction (HFPEF)

With diabetes and with aging, there is a second factor that is responsible for the cardiac remodeling that occurs with HFPEF.   This is the renin-angiotensin system (RAS), which triggers the expression of extracellular matrix genes (collagen genes and MMP genes), which cause the stiffness of the heart with this disease.  Fortunately, there is evidence that this cardiac remodeling is reversible.  Ironically, the extract from broccoli, brussel sprouts, cabbage, and cauliflower,  Indole-3-carbinol, can trigger this cardiac remodeling!   In the study below, withdrawal of I3C reversed the effects of the cardiac remodeling caused by the RAS system.  This is very puzzling.

Here are some articles on this:

htt2013 Cardiac remodeling during and after renin–angiotensin system stimulation in Cyp1a1-Ren2 transgenic rats

2007 The Renin-Angiotensin Aldosterone System: Pathophysiological Role and Pharmacologic Inhibition2012 Reversible cardiac remodeling after renin-angiotensin system stimulation in CYP1A1-Ren2 transgenic rats

3.  Once cardiac remodeling induces heart failure,   ACE inhibitors, beta-blockers, and Angiotensin II receptor blockers do not reverse the heart failure.

Here are some articles on this:

2003 Effects of candesartan in patients with chronic heart failure and preserved left-ventricular ejection fraction: the CHARM-Preserved Trial

2008 Advances in the treatment of heart failure with a preserved ejection fraction

2014 Association Between Use of β-Blockers and Outcomes in Patients With Heart Failure and Preserved Ejection Fraction

2014 Searching for Treatments of Heart Failure With Preserved Ejection Fraction – Matching the Data to the Question

2011 Treatment of Heart Failure With Preserved Ejection Fraction: Have We Been Pursuing the Wrong Paradigm?

4.  Even after heart failure has occurred with HFPEF, Cardiac Metabolic Reprogramming with DCA or TMZ can dramatically improve cardiac index and improve mechanical efficiency of the heart

Dichloroactetic acid, a simple, cheap compound that is well absorbed orally, induces a “metabolic switching” of cardiomyocytes from the utilization of fatty acids to the utilization of glucose.  This glucose utilization is NOT aerobic glycolysis (i.e. the Warburg effect), but instead is true “glucose oxidation” in the mitochondria. This increases the amount of ATP that can be generated and reduces the workload of the heart to generate ATP.   The dose is relatively high (50 mg/kg body weight in humans). This means for an average person, they would need to take 3-4 grams of DCA.

Here are some articles on sodium dichloroacetate. References:

1994 Improved hemodynamic function and mechanical efficiency in congestive heart failure with sodium dichloroacetate

2010 Sodium dichloroacetate selectively targets cells with defects in the mitochondrial ETC

There is a second compound that is a drug widely available in Europe (and Canada) but not in the US.  In fact, it is widely available in over 80 countries.  This drug is called “trimetazidine”, or TMZ, an is another “metabolic reprogrammer” for the heart.  It also makes the heart switch from utilizing free fatty acids to utilizing glucose.  DCA and TMZ do not carry out “metabolic reprograming” the same way, however.  TMZ does this by inhibiting long chain 3-ketoacyl coenzyme A thiolase, whereas DCA targets Pyruvate dehydrogenase kinase (PDK) by acting as a “pyruvate mimetic”.  PDK is an inhibitor of Pyruvate dehydrogenase (PDH). By inhibiting PDK, PDH is maintained in its active catalytic form and mitochondrial pyruvate consumption is increased.  Whereas DCA has to be taken in large doses,  TMZ is only a 20 mg three times per day dosage and has been used in Europe for over 40 years.  It does have one significant rare side effect of causing drug-induced Parkinsonism.

Reference: 2006 Clinical Trial of Trimetazidine , a Partial Free Fatty Acid Oxidation Inhibitor, in Patients With Heart Failure

5.  Metabolic reprogramming can also have wonderful anticancer effects by inhibitingWarburg metabolism, inducing differential apoptosis in cancer cells vs normal cells (preserved)

Warburg metabolism is seen in about 90% of cancers.  The remaining ones either utilize the glutaminlytic pathway to generate ATP, or still use free fatty acids or glucose oxidation.   However, it is thought that more than 99% of cancer cells either use Warburg type metabolism or Glutaminolytic metabolism.  There is strong evidence that DCA alone or DCA in combination with genotoxic drugs can make a huge difference in cancer  cells that are dependent on aerobic glycolysis.  Because cancer cells have been “hardwired” to undergo aerobic glycolysis (Warburg metabolism), it is very difficult to get them to  change their metabolism.  There are several reasons for this metabolic “hardwiring” in cancer cells.  The obvious one is that cancer  cells have lots of mitochondrial mutations so they cannot generate the components of electron transport/oxidative phosphorylation that are expressed only in mitochondrial encoded genes (mtDNA).  A second reason is that cancer cells have unregulated or stabilized HIF-1a, which encodes for all of the glycolytic enymes.  This up regulation of HIF-1a is due to the Insulin/IGF/PI3K/Akt/mTOR pathway being unregulated, the C-myc pathway being unregulated, and the ROS oncogenic pathway being unregulated.  The third reason is the loss of p53 gene expression, either due to mutations or due to epigenetic silencing in the p53 gene promoter by CpG methylation and histone-based silencing.

In summary, there are so many overlapping reasons why cancer cells display “Warburg Metabolism” that this form of metabolism appears to be “hardwired” and not changeable by dietary factors, oxygen, fasting, drugs, exercise, or stress reduction.  In other words, when it comes to cancer cell metabolism, if they have Warburg-like metabolism, they are stuck with it.  And the good news is that this is not necessarily so for normal cells.

DCA has been shown to act as a “pyruvate mimicker” and targets pyruvate dehydrogenase kinase (PDK).  PDK normally inhibits PDH.  When DCA inhibits PDK, PDH continues to consume mitochondrial pyruvate, which then metabolically reprograms” the mitochondria to utilize glucose for generating ATP, rather than utilizing free fatty acids for generating ATP (i.e. beta fatty acid oxidation).  This results in a reduction in serum lactate.  This is why DCA is used to treat congenital lactic acidosis.   Interestingly, DCA induces apoptosis in cancer cells but does not induce apoptosis in normal cells.

There are now numerous reports from cancer laboratories that show amazing benefits of DCA in experimental models.  There is also anecdotal evidence from patients who have self-medicated themselves with DCA and had long term survival associated with with daily use of DCA.  The main side effect of DCA is peripheral neuropathy.  This side effect can be prevented by keeping the dose below 10 mg/kg/day and by taking oral Thiamine, NAC, and CLA.

References:

2008 Dichloroacetate (DCA) as a potential metabolic-targeting therapy for cancer

Sodium dichloroacetate selectively targets cells with defects in the mitochondrial ETC

2012 Novel molecular mechanisms of antitumor action of dichloroacetate against T cell lymphoma: Implication of altered glucose metabolism, pH homeostasis and cell survival regulation

2011 Dichloroacetate induces apoptosis of epithelial ovarian cancer cells through a mechanism involving modulation of oxidative stress

DCA Site: Cancer papers by Archer and Michelakis

Dichloroacetate (DCA) Sensitizes BothWild-Type and Over Expressing Bcl-2Prostate Cancer Cells InVitroto Radiation

2008 Pyruvate kinase M2 is a phosphotyrosinebinding protein

2008 The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth

2011 In vitro effects of an in silico-modelled 17β-estradiol derivative in combination with dichloroacetic acid on MCF-7 and MCF-12A cells

2011 Targeting metabolism with arsenic trioxide and dichloroacetate in breast cancer cells

6.  Glutaminolysis – the “black sheep” metabolic pathway of cancer cells that is often forgotten.

Anther key metabolic dysfunction in cancer cells is the utilization of glutamate as an alternative energy source.  Although this does not occur in all cancer cells, it occurs in chemotherapy-resistant tumors.  For instance, promyeloid leukemia and certain subsets of pancreatic cancer utilize the glutaminolytic pathway to generate ATP.   There is a very old “metabolic inhibitor” of the cancer cell’s glutaminolytic pathway of generating ATP – Arsenic Trioxide (ATO).  ATO is very cheap but hard to get.  Alone, it has amazing but toxic effects on certain cancers.  When used in combination with DCA, it has a synergistic effect, inhibiting cancer even more than DCA or ATO can do alone.

References:

2011 Targeting metabolism with arsenic trioxide and dichloroacetate in breast cancer cells

2002 Molecular targets of arsenic trioxide in malignant cells

2002 Mechanisms of action of arsenic trioxide

2011 Combination of Poly I:C and arsenic trioxide triggers apoptosis synergistically via activation of TLR3 and mitochondrial pathways in hepatocellular carcinoma cells

2010 Genistein synergizes with arsenic trioxide to suppress human hepatocellular carcinoma

2009 A novel combination therapy with arsenic trioxide and parthenolide against pancreatic cancer cell

Conclusions:   Forced metabolic reprogramming may be a novel strategy for cancer cell apoptosis and for age-related heart failure with preserved ejection fraction. Although little has been done in this arena, I believe that this would be a huge opportunity to explore new ways of attacking aging phenotypes in many organs – heart, skeletal muscle, liver, etc.  Unfortunately, little has been done in this area in the anti-aging research field.

B  Countering the Warburg Effect with Phytosubstances

Here are some natural compounds I have found that may inhibit aerobic glycolysis:

  1. Green tea (EGCG) inhibits glycolysis – Inhibits the 6th step of glycolysis at the enzyme Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). EGCG from green tea undergoes “auto-oxidation”, forming “electrophilic quinones” inside cells.  This EGCG-quinone fits into the active enzyme pocket of the 6th enzyme in glycolysis, called Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).  In the enzyme pocket of GAPDH, the EGCG-quinone then forms a covalent bond with a cysteine thiol amino acid on GAPDH.  Mutatgenesis studies that substituted this cysteine with another amino acid eliminated the inhibitory effect of EGCG on GAPDH enzyme activity.

The inhibition of GAPDH by the EGCG-quinone is irreversible, which means that the cell has to synthesize more GAPDH to continue with aerobic glycolysis activity (i.e. Warburg-type metabolism).

Reference:  2008 Covalent modification of proteins by green tea polyphenol (-)-epigallocatechin-3-gallate through autoxidation

Conclusion:  Oxidized EGCG, referrred to as “EGCG-quinone”, forms a covalent bond with a cysteine amino acid side chain in the active enzyme pocket of GAPDH, an important enzyme in the glycolytic pathway.  Because this covalent bond formation results in irreversible inhibition of the GAPDH enzyme, this is one molecular mechanism by which green tea can reduce or reverse Warburg-type metabolsim.

  1. Green tea (EGCG) inhibits the conversion of pyruvate to lactate – the conversion of pyruvate to lactate by LDH and the export of lactate to the plasma for hepatic conversion to glucose are key metabolic alterations that allows cells to survive Warburg-type metabolism.  EGCG from green tea undergoes “auto-oxidation” and then inhibits another key enzyme in Aerobic glycolysis – LDHA. Lactate dehydrogenase (LDH) has two isoforms, LDHA and LDHB.   LDHA is not under the transcriptional control of HIF-1a.  LDHA can be inhibited by oxamate, a specific inhibitor of this isoenzyme.  As a result, less lactate is produced and the Warburg-type metabolism is disrupted in cancer cells.  As a consequence, pyruvate accumulates in the cancer cells and can induce cancer cell death or inhibit cancer cell growth. In the study quoted below, EGCG was found to have the same inhibitory effect on LDHA that oxamate produces.

Reference:  2014 Metabolic consequences of LDHA inhibition by epigallocatechin gallate and oxamate in MIA PaCa-2 pancreatic cancer cells

Conclusion:  The inhibition of LDHA is a second molecular mechanism by which oxidized EGCG can inhibit Warburg-type metabolism.

  1. Green tea (EGCG) inhibits HIF-1a, which would decrease Warburg-type metabolism– Although there are a few studies (two of them) which suggest that catechins from green tea increase HIF-1a stability, most studies have come to the opposite conclusions.  Below are some of them:
  • EGCG has been shown to inhibit hepatocellular carcinoma by inhibiting VEGF/VEGFR axis (see below).
  • EGCG has been shown to inhibit colorectal cancer by inhibiting the VEGF/VEGFR axis (see below)
  • ECGC has been shown to inhibit HIF-1a and VEGF in human cervical cancer cells and in human hepatoma cells.
  • EGCG from green tea was studied to see what effects the EGCG polyphenol would have on HPV-infected cells.  Cells infected with HPV produce oncoproteins E6 and E7, which induce Warburg-type metabolism and cancer formation.  This is one of the molecular mechanisms by which HPV infection can cause cancer – by activating Warburg-type metabolism and angiogenesis.
  • They found that EGCG inhibited angiogenesis in these HPV cells, both in vitro and in vivo, and also inhibited HIF-1a protein expression in HPV cells.
  • EGCG also reduced the secretion of VEGF and IL-8 from these HPV-infected cells.  In this same study, however, EGCG had no effect on inhibiting HIF-1a expression.

Several similar studies are listed below. References:

2006 Green tea extract and (-)-epigallocatechin-3-gallate inhibit hypoxia- and serum-induced HIF-1alpha protein accumulation and VEGF expression in human cervical carcinoma and hepatoma cells.

2013 (-)-Epigallocatechin-3-gallate inhibits human papillomavirus (HPV)-16 oncoprotein-induced angiogenesis in non-small cell lung cancer cells by targeting HIF-1α.

2009 (-)-Epigallocatechin gallat`e suppresses the growth of human hepatocellular carcinoma cells by inhibiting activation of the vascular endothelial growth factor-vascular endothelial growth factor receptor axis

2010 (-)-Epigallocatechin gallate inhibits growth and activation of the VEGF/VEGFR axis in human colorectal cancer cells

2009 (-)-Epigallocatechin gallate suppresses the growth of human hepatocellular carcinoma cells by inhibiting activation of the vascular endothelial growth factor-vascular endothelial growth factor receptor axis

Conclusions: EGCG inhibits HIF-1a, VEGF, and IL-8 production.  And oh yes, in addition to all of the above, lets not lose sight of the fact that EGCC promotes the expression of Nrf2 which is going to activate the NQ01 gene which will tend to normalize the NAD+/NADH ratio which, as pointed out earlier, will work against the Warburg effect.  .

  1. Resveratrol inhibits HIF-1a and VEGF expression-here is an example of another natural compound that inhibits Warburg-type metabolism via HIF-1a.  Resveratrol is a stilbene found in red wine, red grape skins, Itadori tea, and many other natural compounds.  Resveratrol was shown in this study to have major effects on both baseline HIF-1a protein accumulation as well as hypoxia-inducible HIF-1a protein expression.  Unlike some of the other compounds listed here, the molecular mechanism for resveratrol-induced decrease in HIF-1a protein appears to be due to an increase in the degradation rate of HIF-1a by the 26S proteasomal unit,  not a decrease in the gene  expression of HIF-1a.  Thus, there should be a synergistic effect of combining a compound like resveratrol with some of the other compounds that inhibit gene expression of HIF-1a

References:

2006 Resveratrol inhibits hypoxia-induced accumulation of hypoxia-inducible factor-1alpha and VEGF expression in human tongue squamous cell carcinoma and hepatoma cells

2004 trans-3,4,5′-Trihydroxystibene inhibits hypoxia-inducible factor 1alpha and vascular endothelial growth factor expression in human ovarian cancer cells

Conclusion:  Resveratrol reduces HIF-1a by increasing its degradation rate of HIF-1a by the ubiquitin proteasomal system (UPS), not by down regulating its gene expression.

  1. Curcumin directly inhibits HIF-1a and VEGF by down regulating their gene expression

There are clearly multiple mechanisms of action for natural products.  Curcumin, the active ingredient in turmeric, derived from the roots of the turmeric plant is the main active ingredient in curry spice.  Here is the first of two mechanisms that may have anti-Warburg metabolism effects in human cells:

Curcumin has been shown to have major effects on tumor suppression, but all of the mechanisms have not been fully elucidated.  In the study below, curcumin was shown to have a direct effect on down-regulating gene expression for HIF-1a.  Curcumin also down-regulated gene expression for Vascular endothelial growth factor, VEGF, the downstream target gene of HIF-1a.  This effect occurred both under hypoxic conditions (normal anaerobic glycolysis) and under normoxic conditions (Warburg metabolism).

Reference: 2oo6 Curcumin inhibits hypoxia-induced angiogenesis via down-regulation of HIF-1

Conclusions:  Curcumin shouild have major direct effects in inhibiting or reversing Warburg-type metabolism, both under hypoxic and normoxic conditions

  1. Curcumin indirectly inhibits HIF-1a by inhibiting STAT3– Although curcumin has major anti-inflammatory effects due to its ability to inhibit NF-kB signaling, not as much is known about its ability to inhibit Warburg-type metabolism.  However, it may do this via STAT3. Curcumin is an inhibitor of the pro-inflammatory transcription factor,Signal transducer and activator of transcription 3, aka STAT3/ STAT3 is a central regulatory of tumor metastasis, but plays an integral role in inducing Warburg metabolism due to its effects on activating HIF-1a.  Curcumin inhibits STAT3, which then inhibits HIF-1a, which is a down stream target gene of STAT3.  Thus, curcumin indirectly affects aerobic glycolysis or Warburg-type metabolism via STAT3 inhibition.

Reference:  2009 STAT3 as a Central Regulator of Tumor Metastases

Conclusions:  Curcumin should have major indirect effects on Warburg-type metabolism via STAT3 inhibition.

  1. Vitexin directly inhibits HIF-1a protein and also down regulates HIF-1a and VEGF gene expression– this interesting natural flavanoid has a dual mechanism of action on reducing HIF-1a activity.  Vitexin is a flavanoid found in many different plants, including Anthurium versicolor(Aquino et al., 2001), Ficaria verna Huds. (Ranuncu- laceae)       (Tomczyk et al., 2002), and Cucumis sativus L. (Cu- curbitaceae) (McNally et al., 2003).  It has also been shown to be present in Kombucha tea.  It was shown to be a potent inhibitor of HIF-1a protein activity in phenochromcytoma cells.  Others showed it to be a potent inhibitor of hepatocellular cancer cells via HIF-1a.

References:

2006 Vitexin, an HIF-1α Inhibitor, Has Anti-metastatic Potential in PC12 Cells (abstract)

2006 Vitexin, an HIF-1α Inhibitor, Has Anti-metastatic Potential in PC12 Cells (PDF full text)

2007 Hypoxia and hepatocellular carcinoma: The therapeutic target for hepatocellular carcinoma

2011 Effect of solvent fractions of kombucha tea on viability and invasiveness of cancer cells—Characterization of dimethyl 2-(2-hydroxy-2-methoxypropylidine) malonate and vitexin

 IV.  SIRT1 and inflammation

NAD+ and SIRT1 availaility play key roles in both acute and chronic inflammation.  The 2013 publication Deacetylation by SIRT1 Reprograms Inflammation and Cancer summarizes key issues in the SIRT1-inflammation story.  The abstract reports: “NAD+-dependent deacetylase SIRT1 is a master regulator of nucleosome positioning and chromatin structure, thereby reprogramming gene expression. In acute inflammation, chromatin departs from, and returns to, homeostasis in an orderly sequence. This sequence depends on shifts in NAD+ availability for SIRT1 activation and deacetylation of signaling proteins, which support orderly gene reprogramming during acute inflammation by switching between euchromatin and heterochromatin. In contrast, in chronic inflammation and cancer, limited availability of NAD+ and reduced expression of SIRT1 may sustain aberrant chromatin structure and functions. SIRT1 also influences inflammation and cancer by directly deacetylating targets like NFκB p65 and p53. Here, we review SIRT1 in the context of inflammation and cancer.”  Five diagrams in that publication lay out the story.

  1. There are Two Types of Inflammation: Acute and Chronic

According to the article, chronic infections contribute up to 20% to cancer, inflammatory diseases contribute up to 20% of cancer causation, and obesity contributes up to 20% of cancer causation.

  • Chronic infection, inflammatory disease, and obesity all produce extracellular signaling compounds that are Toll-like receptor activators, or cytokines, or ROS, or RNS.
  • Whereas infections and obesity have other signaling mechanisms, TLR and cytokine signaling are the principle mediators of inflammation and trigger inflammation via three pathways:
  1. STAT1– This pathway is activated by the IFN family of cytokines. IFN induces STAT1phosphorylation, which results in cytoplasmic-to-nuclear translocation of STAT1, where STAT1 binds to and turns on inflammatory genes.  Unlike acetylation of NF-kB,  the acetylation of STAT1 inactivates the transcription factor, whereas HDAC3s (not SIRT1) deacetylate STAT1 and thereby allows for phosphorylation and inflammatory gene activation.  This process has nothing to do with NAD or SIRT1

Reference:A phosphorylation-acetylation switch regulates STAT1 signaling

How to “fix” this problem:  take a polyphenol that is an HDAC inhibitor (Ex: EGCG), also take a STAT1 inhibitor,  and reduce IFN signaling

  1. STAT3– This pathway is activated by the IL-6 family of cytokines (IL-6, IL-10, etc.)   IL-6 induces the Janus kinase, called JAK in the cytoplasm, which then phosphorylates  STAT3.(This is why it is called the “JAK/STAT3 pathway”).  Phosphorylation of STAT3 by JAK results in cytoplasmic-to-nuclear translocation of STAT3, where STAT3 can bind to gene promoters to produce a transient activation of inflammatory genes.  IL-10 also activates STAT3, but paradoxically seems to have opposite effects.   IL-10 activation of STAT3 produces a sustained expression of genes that are mostly anti-inflammatory.  Thus STAT3 signaling can be pro or anti-inflammatory.

HDACs deacetylate STAT3 and disrupt the JAK/STAT3 signaling pathway.  These are not NAD-dependent deacetylases, however.

References:

STAT3 activation in response to IL-6 is prolonged by the binding of IL-6 receptor to EGF receptor

Activation of STAT3 by IL-6 and IL-10 in primary human macrophages is differentially modulated by suppressor of cytokine signaling 3

Cytokine response is determined by duration of receptor and STAT3 activation

Stat3/Socs3 Activation by IL-6 Transsignaling Promotes Progression of Pancreatic Intraepithelial Neoplasia and Development of Pancreatic Cancer

How to “fix” this problem:  take a polyphenol that is an HDAC inhibitor (Ex: EGCG), also take a STAT3 inhibitor, and reduce IL-6 signaling

  1. NF-kB– This pathway is activated by TLR signaling (double stranded RNA, single stranded RNA, bacterial flagellin, bacterial and viral CpG motifs, malarial pigment hemozoin, lipopeptides, lipoproteins, hyaluronan breakdown products such as short hyaluronic acid chains, endotoxin (LPS), amyloid-beta 42, viral proteins, and many other extracellular proteins).  TLR signaling phosphorylates the binding partner of NF-kB, called IKB-alpha.

The phosphorylation of IKBa results in the degradation of IKBa, which leaves NF-kB free to translocate into the cell nucleus.  NF-kb then binds to gene promoters of the inflammatory gene network.  Part of this includes the activation of the NAMPT gene, which increases NAD levels in the acute phase of inflammation.  The NAD stabilizes SIRT1a nd activates SIRT1.  Thus there is an increase in SIRT1 activity with acute inflammation.

Reference:  2010 TLR-signaling Networks: An Integration of Adaptor Molecules, Kinases, and Cross-talk

SIRT1 activity increases as acute inflammation evolves. The following diagram illustrates the cascade of events that occurs with TLR signaling:

SIRTINFLAMM1

   Reference for diagram:  Deacetylation by SIRT1 Reprograms Inflammation and Cancer

“Gain of SIRT1 functions during acute inflammation. TLR responses increase NAMPT-dependent NAD+ regeneration and activate SIRT1, which represses inflammation, glycolysis, and apoptosis and increases lipolysis, mitochondrial biogenesis, autophagy, and antioxidants. This sequential process restores homeostasis.”

Whereas bacterial infections, viral infections, surgery, accidents, and other triggers induce acute inflammation, chronic inflammation is typically triggered by disease and aging. For instance, obesity induces chronic inflammation which induces a number of inflammatory diseases, including HTN, type II diabetes, cancer, and atherosclerosis.

Chronic Inflammation produces a different form of gene expression than acute inflammation, however.  The differences in acute vs chronic inflammation includes different forms of chromatin (euchromatin vs heterochromatin).  The different “chromatin signatures” for each type of inflammation are shown below:

SIRTINFLAMM2

Illustration Reference:  Figure 2.  Deacetylation by SIRT1 Reprograms Inflammation and Cancer

“Inflammation phenotypes. Acute inflammation modifies the chromatin structure to switch from initiation to adaptation and resolution. Chronic inflammation sustains proinflammatory chromatin.”

With chronic inflammation, several things occur connected with SIRT1.  First of all, a high fat diet cleaves SIRT1 and low NAD levels reduce SIRT1 synthesis (see diagram below and article reference for diagram for more info on this).The net result of a high fat diet and lower NAD levels is a loss of SIRT1 function.  This results in hyperacetylation of the p65 subunit of NF-kB, which produces pro-inflammatory products.  These proinflammatory mediators have a “positive feedback” on the formation of obesity, diabetes, and aging.

The direct downstream effects of reduced SIRT1 include an increase in adiponectin, a decrease in PPARalpha, an increase in insulin and a lowering of UCP2, as well as an increase in telomerase expression, a decrease in UCP2 expression, and a decrease in FoxO1 and FoxO3 mediated pro-survival gene expression.

Reference for diagram below:  Deacetylation by SIRT1 Reprograms Inflammation and Cancer

SIRTINFLAMM3

Figure 4.

“Loss of SIRT1 functions during chronic inflammation. High fat diet reduces NAD+ availability and deactivates SIRT1, which promotes inflammation, lipogenesis, insulin resistance, and DNA damage. This unadaptive process prevents a return to homeostasis.”

When it comes to Cancer, SIRT1 has been a major puzzle.  Well the puzzle has now been solved – SIRT1 is both a tumor suppressor and a tumor activator.  Here is a diagram that illustrates that:

SIRTINFLAMM4

Diagram reference:  Figure 5.  Deacetylation by SIRT1 Reprograms Inflammation and Cancer  “Dual effects of SIRT1 on cancer: (A) inflammation and (B) modifying specific proteins.”

There is a diagram (below) that summarizes the factors that affect SIRT1 gene expression.

SIRTINFLAMM6

Diagram reference:  Deacetylation by SIRT1 Reprograms Inflammation and CancerFigure 1.

“SIRT1: (A) structure and (B) functions and regulation.”

I have been uncovering more and more paradoxical factors associated with NAMPT.   eNAMPTappears to be both a “bad thing” and a “good thing”. The difference may have to do with the dose (i.e. Biphasic dose-response curve) or it may have to do with the zip code (i.e. What cell or what tissue), or eNAMPT may have what is called “pleiotropic effects”. What is clear to me is this fundamental fact: eNAMPT has both a “cytokine effect” and an “enzyme effect”. The inhibitor FK866 and NMN inhibit the “enzyme effect” but do not inhibit the “cytokine effect”.

The “cytokine effect” is mostly pro-inflammatory, but also promotes survival of cells such as cancer cells and macrophages. The “enzymatic effect” may indirectly affect inflammation/immunity by providing NMN substrates for conversion to NR, and subsequent plasma membrane uptake by cells that are NAD deficient (NMN does not appear to be taken up directly, but is usually converted into NR which is absorbed).

What is upsetting the apple cart even more is the fact that much of the “dogma about biomarkers of aging and biomarkers of cellular senescence” may be wrong!  Specifically, the Japanese as well as others showed that IL-6, TNF-alpha, and CRP all predict all cause mortality and lifespan more accurately than age or gender.  However, I have found that all of these 3 “biomarkers of aging” are triggered by increased levels of eNAMPT (IL-6, TNF-a, and CRP). Likewise, the classic biomarkers of cellular senescence (I.e. The SASP components…IL-6, IL-8, MCP-1, etc.) are all expressed from NON-SENESCENT CELLS in response to eNAMPT signaling! This makes me question the use of these cytokines as biomarkers for aging and biomarkers for cellular senescence, since their levels are easily altered by eNAMPT expression! We know that eNAMPT expression is NOT the entire picture of aging (aging includes DNA damage/mutations, epigenetic dysregulation, cellular senescence, increased mTOR signaling, mitochondrial dysfunction, proteotoxicity, autophagy failure, etc).

This raises many questions about the validity of inflammatory cytokines as true biomarkers of aging, since eNAMPT activity/signaling can be inhibited by NMN and by FK866. It also calls into question the link between cellular senescence and these same cytokines (IL-6, IL-8, MCP-1). Here are some more facts about eNAMPT and a list of the proteins that are secreted by cells in response to eNAMPT signaling.

This quote from the 2010 publication Pre-B Cell Colony Enhancing Factor/NAMPT/Visfatin in Inflammation and Obesity- Related Disorders telegraphs some key points which will be covered here: “Whereas prototypic adipocytokines such as adiponectin or leptin are mainly derived from adipocytes, others such as pre-B cell colony enhancing factor (PBEF)/nicotinamide phosphoribosyl transferase (NAMPT)/visfatin or resistin are produced by various cell types throughout the body. Although first discovery of this molecule as PBEF suggested primarily a cytokine function, its rediscovery as the key enzyme in nicotinamide adenine dinucleotide (NAD) generation has considerably widened its biological perspective. Finally, the same molecule was introduced as visfatin claiming an insulin-mimetic effect which has been questioned. Both extracellular (cytokine-like) and intracellular (enzymatic) functions are responsible for its relevance in immune, metabolic and stress responses. Its cytokine functions are mainly pro-inflammatory as it induces potently various other pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) or interleukin-6 (IL-6). Its intracellular functions concentrate on the regulation of the activity of NAD-consuming enzymes such as various sirtuins thereby also affecting TNFα biosynthesis, cell life-span and longevity. Biochemical neutralization of PBEF/NAMPT/visfatin has been proven effective in various models of inflammation including sepsis/arthritis and in various models of cancer. Patients with non-alcoholic fatty liver disease (NAFLD) exhibit increased serum concentrations of PBEF/Nampt/visfatin and weight loss is associated both with a decrease in serum levels and reduced liver expression. Many of the biological functions of this “cytokine-enzyme” have been characterized in the last years, however, its definite role in various metabolic, inflammatory and malignant diseases has yet to be defined.”

  1. The pro-inflammatory effects of eNAMPT 

One of the most unusual molecules in nature is the secreted form of NAMPT, called “eNAMPT,”  eNAMPT is secreted in response to inflammation and also causes inflammation. Thus it is both a “cause” and an “effect” of inflammation. Many other names have been attached to eNAMPT before they were all found to be the same molecule. They include “Pre-B cell colony enhancing factor (PBEF) and Visfatin. (Here we shall just call it eNAMPT). eNAMPT increases circulating levels of pro-inflammatory cytokines, including IL-6, TNF-alpha, IL-1B, and TGF-B1. It also increases the chemokine receptor CCR3, VEGF, VEGFR, and MCP-1.  eNAMPT activates the Toll-like receptor 4 (TLR4) in a unique way, triggering NF-kB mediated gene expression in cells. In conclusion, eNAMPT may be the link between visceral fat and systemic inflammation. It also may be the major molecular mechanism behind “sterile inflammation” and “inflammaging”, which are ways of describing the phenomena seen with aging and age-related diseases.

References:

2013  Pre-B cell colony enhancing factor (PBEF), a cytokine with multiple physiological functions“Extracellular PBEF has been shown to increase inflammatory cytokines, such as TNF-α, IL-1β, IL-16, and TGF-β1, and the chemokine receptor CCR3. PBEF also increases the production of IL-6, TNF-α, and IL-1β in CD14+ monocyctes, macrophages, and dendritic cells, enhances the effectiveness of T cells, and is vital to the development of both B and T lymphocytes.”

eMAMPT0

Image source  Note that eNAMPT creates these terrible problems traveling here when using the alias PBEF

2009  Nicotinamide phosphoribosyltransferase (Nampt): A link between NAD biology, metabolism, and diseases  “New interest in NAD biology has recently been revived, and enzymes involved in NAD biosynthetic pathways have been identified and characterized in mammals. Among them, nicotinamide phosphoribosyltransferase (Nampt) has drawn much attention in several different fields, including NAD biology, metabolism, and immunomodulatory response. The research history of this protein is peculiar and controversial, and its physiological function has been a matter of debate. Nampt has both intra- and extracellular forms in mammals. Intracellular Nampt (iNampt) is an essential enzyme in the NAD biosynthetic pathway starting from nicotinamide. On the other hand, an extracellular form of this protein has been reported to act as a cytokine named PBEF, an insulin-mimetic hormone named visfatin, or an extracellular NAD biosynthetic enzyme named eNampt. This review article summarizes the research history and reported functions of this unique protein and discusses the pathophysiological significance of Nampt as an NAD biosynthetic enzyme vs. a potential inflammatory cytokine in diverse biological contexts.”

  1. eNAMPT is secreted by neutrophils, microglia, macrophages, and visceral fat cells when these cells are stimulated by LPS or pro-inflammatory cytokines.

Pro-inflammatory cytokines trigger eNAMPT and eNAMPT triggers the gene transcription of more pro-inflammatory cytokines from the neurotrophils, macrophages, microglia, and visceral fat. With LPS stimulation of neutrophils, eNAMPT expression occurs 5 hours later. This is what is classically called a “positive feedback loop” in molecular biology, or could be called a “self-perpetuating fate” in theology. Regardless of the description, eNAMPT becomes a mediator of ongoing, chronic inflammation, long after the original trigger of inflammation is gone. This has often been referred to as “sterile inflammation”.

References:

From the book 2016 The Stress Response of Critical Illness: Metabolic and Hormonal Aspects 

2004  Pre–B cell colony–enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis

  1. eNAMPT has a protein sequence that “mimics” bacterial lipopolysaccharide (LPS) which triggers Toll-like receptor 4 signaling. 

Lipopolysaccharide (LPS) is the molecular name for bacterial endotoxin. When lab experimenters want to create inflammation in a mouse or rat, they usually use  LPS to do that. LPS triggers inflammation by binding to cells via the mammalian cell Toll-like receptor 4 (TLR4). Although early reports suggested that TLR2 also mediated LPS toxicity, more up-to-date findings have confirmed that TLR4 is the sole molecular receptor for LPS. TLR4 receptor triggering leads to the secretion of many pro-inflammatory, pro-angiogenic, and anti-apoptotic compounds from neutrophils, activated lymphocytes, macrophages, and visceral fat cells.

References:

2004  Pre–B cell colony–enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis

2007 Pre-B-cell Colony-enhancing Factor (PBEF/Visfatin) Gene Expression is Modulated by NF-κB and AP-1 in Human Amniotic Epithelial Cells

2004  Tlr4: central component of the sole mammalian LPS sensor.

2012 Regulation of neutrophil function by NAMPT

  1. Inflammation up-regulates eNAMPT and eNAMPT up regulates inflammation. 

In experimental studies where cells are activated by LPS, many genes are upregulated via NF-kB. One of these is NAMPT, which is transcribed, translated, and secreted from the cell as eNAMPT. Thus eNAMPT can be considered both the “cause” and the “effect” of Inflammation. Since bacterial infections (LPS) are common triggers of inflammation, eNAMPT can be consIdered an “amplifier of infectious inflammation” or in the absence of LPS, eNAMPT can be considered a mediator of “sterile inflammation.”  Unfortunately, chronic inflammation acts as a “damper” on NAD production in the liver, in adipose tissue, in the pancreatic beta-islet cells, and in the brain. Thus the “true enemy” is inflammation, NOT eNAMPT! The diagram below from Imai’s recent 2013 paper illustrates this well.

References:

2000  Role of MD-2 in TLR2- and TLR4-mediated recognition of Gram-negative and Gram-positive bacteria and activation of chemokine genes

2007 Pre-B-cell Colony-enhancing Factor (PBEF/Visfatin) Gene Expression is Modulated by NF-κB and AP-1 in Human Amniotic Epithelial Cells

2012 Regulation of neutrophil function by NAMPT

2013  The importance of NAMPT/NAD/SIRT1 in the systemic regulation of metabolism and ageing

eMAMPT1

Image and legend source.  “The concept of the NAD World and the possible effect of chronic inflammation. Pancreatic β-cells and neurons (the brain) are two major frailty points in the NAD World because these two cell types have very low levels of intracellular nicotinamide phosphoribosyltranferase (iNAMPT). These particular cell types likely depend on extracellular nicotinamide mononucleotide (NMN), which is speculated to be synthesized by extracellular nicotinamide phosphoribosyltranferase (eNAMPT) secreted by adipose tissue, and maintain optimal nicotinamide adenine dinucleotide (NAD) levels for their functions. Chronic inflammation, which is caused by inflammatory cytokines and oxidative stress, decreases NAMPT and NAD levels in multiple tissues, contributing to the pathogenesis of age-associated metabolic complications, such as type 2 diabetes. It still remains unclear whether chronic inflammation in adipose tissue also decreases plasma eNAMPT levels and remotely affects the functions of ‘frailty’ cell types.”   So is eNAMPT good or bad?  Like many other key molecules we have studied, the answer appears to be “both.”

  1. TLR4 signaling by eNAMPT triggers both NF-kB and AP-1 mediated signaling.

Whereas NF-kB is the classic “master switch” for inflammatory genes, AP-1 is a lesser known transcription factor that also triggers inflammatory genes. Because of the dual transcription factor signaling pathways for eNAMPT, blocking NF-kB alone with molecules like steroids or certain phytosubstances will fail to fully block the pro-inflammatory and pro-angiogenic effects of eNAMPT signaling.  Of course, certain phytosubstances like curcumin do block both NF-kB and AP-1 inflammation(ref).

Reference:  2007 Pre-B-cell Colony-enhancing Factor (PBEF/Visfatin) Gene Expression is Modulated by NF-κB and AP-1 in Human Amniotic Epithelial Cells

  1. SIRT1 deacetylates iNAMPT, which enhances NAMPT activity and drives the secretion of eNAMPT from visceral fat cells. 

This is one of the “paradoxical aspects” of the eNAMPT story. SIRT1 is typically thought of as a CR pathway enzyme that has anti-inflammatory effects.  But here SIRT1 is playing a positive role in the secretion of eNAMPT from fat cells by deacetylation of lysine 53 (K53) on the NAMPT protein. This is the “secrete me” signal that triggers the export of the deacetylated eNAMPT  out of the cell.  In the plasma, fat cell-derived eNAMPT secretion circulates systemically and affects the hypothalmus. In the hypothalmus, the circulating eNAMPT increases NAD+ in the hypothalmus. Decreased eNAMPT decreases NAD+ in the hypothalmus, whereas increased eNAMPT in the plasma increases NAD in the hypothalmus.  In NAMPT knock-out mice, NMN “rescues” the defect and the mice become more physically active. This is very puzzling until you put it in the context of fasting (CR) vs times of food abundance. Specifically, iNAMPT is normally acetylated at lysine 53 in both white fat and brown fat.  However with fasting or starvation, SIRT1 deacetylates iNAMPT at lysine 53 (K53) which promotes its export. Thus fasting is an iNAMPT export signal leading to eNAMPT and its possible sequela as outlined here.  Another example of the multiple good-bad roles played by eNAMPT.

References: 

2015 SIRT1-Mediated eNAMPT Secretion from Adipose Tissue Regulates Hypothalamic NAD+and Function in Mice

2012 The Pathophysiological Importance of Nicotinamide Phosphoribosyltransferase as a Key NAD Biosynthesis Enzyme in Metabolic Homeostasis

eNAMPT2

Image and legend source.  “Nicotinamide phosphoribosyltransferase (NAMPT), the key NAD+ biosynthetic enzyme, has two different forms, intra- and extracellular (iNAMPT and eNAMPT), in mammals. However, the significance of eNAMPT secretion remains unclear. Here we demonstrate that deacetylation of iNAMPT by the mammalian NAD+-dependent deacetylase SIRT1 predisposes the protein to secretion in adipocytes. NAMPT mutants reveal that SIRT1 deacetylates lysine 53 (K53) and enhances eNAMPT activity and secretion. —“

  1. eNAMPT triggers systemic Insulin resistance and IGF-1 receptormediated PGE2-induced arthritis 

The link between insulin resistance and eNAMPT is among the most fascinating things I have learned recently. This finally explains the links between fat (eNAMPT source), the regulation of the Insulin/IGF-1 pathway, and NMN. For quite some time, eNAMPT (also called PBEF or Visfatin) has been linked to inflammation, but exactly how eNAMPT was linked was unclear. This has largely been cleared up in the last 3-4 years.

  1. eNAMPT, IGF-1 signaling, and inflammation

In 2012, Jacques and colleagues from Marie Curie University in Paris showed that in joints, eNAMPT triggers inflammation by inducing PGE2 synthesis by chondrocytes. In cells with no IGF-1 receptors, eNAMPT triggered PGE2 biosynthesis. In cells with two functional copies of the IGF-1R, eNAMPT did not trigger nearly as much PGE2 release. However, Jacques and colleagues were not able to show if eNAMPT regulated the insulin receptor. They did show that inhibiting eNAMPT with FK866 gradually decreased PGE2 release and administering exogenous Nicotinamide increased PGE2 release.

Reference:  2012 Proinflammatory actions of visfatin/nicotinamide phosphoribosyltransferase (Nampt) involve regulation of insulin signaling pathway and Nampt enzymatic activity  “We conclude that the proinflammatory actions of visfatin in chondrocytes involve regulation of IR signaling pathways, possibly through the control of Nampt enzymatic activity.”

  1. eNAMPT is the molecular cause of Insulin Resistance

The exact mechanism of how fat-induced inflammation causes insulin resistance has long remained a mystery. The mystery may have been solved in 2015 when a large, multi-center research group from Canada and China showed that eNAMPT induces the translocation if the insulin receptor out of lipid micro domains (lipid rafts) into non-lipid raft regions of the cell membrane. This had the net effect of making the insulin receptor resistant to insulin signaling (I.e. It did not trigger Akt phosphorylation). This effectively reduced insulin signaling by moving the IR to the non-raft region, which caused cells to become insulin resistant.  This has explained the mystery of why insulin receptor density in cells does not decrease with fat-Induced inflammation, but that insulin resistance still occurs with fat-induced inflammation.

References: 

2015 Pre-B cell colony enhancing factor induces Nampt-dependent translocation of the insulin receptor out of lipid microdomains in A549 lung epithelial cells   “We conclude that PBEF can inhibit insulin signaling through the IR by Nampt-dependent promotion of IR translocation into the nonraft domains of A549 epithelial cells. PBEF-induced alterations in the spatial geometry of the IR provide a mechanistic explanation for insulin resistance in inflammatory states associated with upregulation of PBEF.”

2013 Pre-B cell colony enhancing factor (PBEF), a cytokine with multiple physiological functions

Conclusion re, eNAMPT and insulin resistance

The circulating molecule by which obesity (mainly visceral fat) can induce insulin resistance in distant organs (muscle) has now been identified – it is eNAMPT.  eNAMPT is secreted mainly by visceral fat and inflammatory cells and circulates in the bloodstream as an enzyme and a pro-inflammatory, pro-angiogenic cytokine. In distant cells (such as muscle), it triggers insulin receptor movement from lipid rafts to non-lipid raft regions, effectively inactivating the intracellular signaling cascade that centers around Akt phosphorylation. Thus eNAMPT is the molecular “cause” of insulin resistance. eNAMPT also plays a role in triggering bioactive lipid-mediated inflammation (I.e. PGE2) in inflammatory diseases such as arthritis. For eNAMPT to trigger PGE2 release, there must be IGF-1 receptors (IGF1R) present. IGF1Rs are required for full eNAMPT-mediated PGE2 release. This IGF-1 and IGF1R play vital roles in the mechanism by which eNAMPT triggers PGE2 release in arthritis. This effect can be blocked with FK866, an eNAMPT inhibitor, or NMN, which inhibits eNAMPT by “feedback inhibition”.

  1.  IL-8

IL-8 is secreted by the eNAMPT target cell by eNAMPT binding to the TLR4 receptor on the target cell and triggering IL-8 gene expression. 

IL-8 is a classic biomarker of cellular senescence, proposed by Mayo Clinic and Buck Institute researchers. However, mRNA for IL-8 is increased 2-8 fold with eNAMPT over-expression in cells that are NOT senescent. The cell surface receptor activated by eNAMPT on the target cell is the Toll-like receptor 4 (TLR4). The two transcription factors that mediate this eNAMPT effect in the target cell are NF-kB and AP-1, classical villains of unwanted inflammation. These are not the transcription factors that trigger IL-8 secretion in senescent cells. (There it is JAK/STAT signaling pathway). Thus IL-8 may NOT always be due to senescent cell secretion….it may just be due to eNAMPT binding to a non-senescent cell and triggering AP-1 mediated IL-1B secretion, which then triggers IL-8.

References: 

2008 A critical role of PBEF expression in pulmonary cell inflammation and permeability

2009 Regulation of Inflammatory Cytokine Expression in Pulmonary Epithelial Cells by Pre-B-cell Colony-enhancing Factor via a Nonenzymatic and AP-1-dependent Mechanism

2004  Pre–B cell colony–enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis

2002 Pre-B-cell colony-enhancing factor, a novel cytokine of human fetal membranes

  1. IL-6

eNAMPT triggers the production of IL-6 in target cells in humans. 

References:

2002 Pre-B-cell colony-enhancing factor, a novel cytokine of human fetal membranes

2010 Pre-B Cell Colony Enhancing Factor/NAMPT/Visfatin in Inflammation and Obesity- Related Disorders

  1. IL-1Beta

IL-Beta expression is triggered by eNAMPT

eNAMPT also triggers IL-1Beta secretion by cells. (IL-1B actually then triggers IL-8 secretion). While IL-1B is not a classic biomarker for cellular senescence, it is probably the #1 cytokine associated with osteoarthritis (OA), rheumatoid arthritis (RA), and many other chronic inflammatory diseases. Is OA and RA inflammation due to eNAMPT?   Probably not!  However I have read some articles that suggest that eNAMPT does play a role in arthritis. Here ls one:

Reference:  2004  Pre–B cell colony–enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis

  1. IL-16

IL-16 expression triggered by eNAMPT

eNAMPT signaling also stimulates IL-16 gene expression and production of the IL-16 cytokine. Here there appears to be a clear difference from cellular senescence. With cellular senescence, there is an increase in IL-6 and other cytokines, but the literature related to senescence-stimulated cytokines does not appear to mention IL-16 to our knowledge.

References:

2008 A critical role of PBEF expression in pulmonary cell inflammation and permeability

2009 Regulation of Inflammatory Cytokine Expression in Pulmonary Epithelial Cells by Pre-B-cell Colony-enhancing Factor via a Nonenzymatic and AP-1-dependent Mechanism

  1. CCR3

CCR3 expression triggered by eNAMPT

CCR3 is a chemokine

References:

2008 A critical role of PBEF expression in pulmonary cell inflammation and permeability  “PBEF expression also affected the expression of two other inflammatory cytokines (IL-16 and CCR3 genes). These results suggest that PBEF is critically involved in pulmonary vascular and epithelial inflammation and permeability, which are hallmark features in the pathogenesis of acute lung injury. This study lend further support that PBEF is a potential new target in acute lung injury.”

2009 Regulation of Inflammatory Cytokine Expression in Pulmonary Epithelial Cells by Pre-B-cell Colony-enhancing Factor via a Nonenzymatic and AP-1-dependent Mechanism*

  1. eNAMPT may be the major molecular cause of normal, spontaneous labor and infection-induced pre-term labor. This was a surprising finding reported in 2002.

Reference:  2002 Pre-B-cell colony-enhancing factor, a novel cytokine of human fetal membranes

  1. Cancer cells secrete eNAMPT

Strong expression of eNAMPT has been seen in breast, colorectal, brain, stomach, thyroid, endometrial, ovarian, multiple myeloma, astrocytoma, and prostate cancer. This is not surprising, since cancer cells seem to up-regulate inflammatory pathways independently and autonomously from their environment.  Since there is evidence that both iNAMPT and eNAMPT are pro-survival factors, there may be more than one reason why cancer cells up regulate the NAMPT gene expression.  The transcription factor that upregulates eNAMPT in these cells is cMYC, an oncogene transcription factor critical in embryogenesis.

References:

Nicotinamide Phosphoribosyltransferase Promotes Epithelial-to-Mesenchymal Transition as a Soluble Factor Independent of Its Enzymatic Activity*

2011 The Role of Visfatin in Prostate Cancer

Nicotinamide Phosphoribosyl Transferase (NAMPT) Inhibitors: Novel Modulators of Cancer-Related Inflammation

  1. eNAMPT triggers one of the major steps in carcinogenesis – the epithelial-to-mesenchymal transition. Cancer cells must go through a critical transformation where they go from epithelial cells to mesenchymal cells that resemble mesenchymal stem cells. This step is most often triggered by chronic inflammation and may be one reason why aspirin inhibits cancer formation. eNAMPT has been shown in vitro, to promote the epithelial-to-mesenchymal transition in breast cancer. It did this via the TGG-Beta1 signaling pathway.  Thus eNAMPT plays a major role in cancer formation.

References:

Nicotinamide Phosphoribosyltransferase Promotes Epithelial-to-Mesenchymal Transition as a Soluble Factor Independent of Its Enzymatic Activity*

Nicotinamide Phosphoribosyl Transferase (NAMPT) Inhibitors: Novel Modulators of Cancer-Related Inflammation

  1. eNAMPT is secreted from cardiomyocytes and causes cardiac hypertrophy and ventricular remodeling.

This is a surprising finding. NAMPT transgenic mice developed cardiac hypertrophy at 6 months of age. Cultured cardiomyocytes secreted eNAMPT in response to H2O2.  The downstream signaling pathways in the cardiomyocytes that were exposed to eNAMPT were the transcription factors, JNK, p38, and ERK. There was also increased calcineurin and NFAT translocation into the nucleus in response to eNAMPT.

References:

2013 Nampt secreted from cardiomyocytes promotes development of cardiac hypertrophy and adverse ventricular remodeling

2013 Resistin and Visfatin Expression in HCT-116 Colorectal Cancer Cell Line

  1. eNAMPT is a major molecular mediator of cartilage destruction in RA and OA.

This was not known until 2013. eNAMPT levels are very high in both plasma and in the synovial fluid of patients with RA. Adding eNAMPT to fibroblast cultures triggers the synthesis and secretion of cartilage breakdown enzymes like MMP-3. This effect could be reversed with the eNAMPT inhibitor, FK866.

Reference:  2013 Investigating the role of Nicotinamide phosphoribosyltransferase (NAMPT) in cartilage catabolism

  1. eNAMPT inhibition reduces ROS secretion by neutrophils but did not inhibit their ability to kill bacteria

Many researchers were predicting that eNAMPT would be an essential mediator of innate immunity due to its ability to activate neurotrophils.   For this reason, it was somewhat surprising to find out that the inhibition of eNAMPT with FK866 decreased ROS production by neutrophils but did not reduce their ability to destroy bacteria. eNAMPT inhibition did decrease TNF-alpha mediated gene expression, however.

Reference:  2012 Regulation of neutrophil function by NAMPT

  1. eNAMPT inhibition by NMN prevents pro-inflammatory destruction of pancreatic islet cells.This is very good news for Type II Diabetics.

Reference:  2011 Nicotinamide mononucleotide protects against pro-inflammatory cytokine-mediated impairment of mouse islet function.  This article describes a situation where administration of exogenous NMN comes to the rescue.  “— We hypothesised that altered NAMPT activity might contribute to the suppression of islet function associated with inflammation, and aimed to determine whether NMN could improve cytokine-mediated islet dysfunction. — Acute effects of NMN on cytokine-mediated islet dysfunction were examined in islets incubated with TNFα and IL1β, and in mice fed a fructose-rich diet (FRD) for 16 weeks. Changes in iNAMPT, eNAMPT and inflammation levels were determined in FRD-fed mice.  Results:  FRD-fed mice displayed markedly lower levels of circulating eNAMPT, with impaired insulin secretion and raised islet expression of Il1b. NMN administration lowered Il1b expression and restored suppressed insulin secretion in FRD-fed mice.  NMN also restored insulin secretion in islets cultured with pro-inflammatory cytokines. The changes in islet function corresponded with changes in key markers of islet function and differentiation. The anti-inflammatory effects of NMN were partially blocked by inhibition of sirtuin 1.”

  1. eNAMPT promotes macrophage survival via an IL-6/STAT3 survival mechanism and is differentially expressed in M1 vs M2 macrophages. 

An interesting finding is that eNAMPT helps keep macrophages alive. This pro-survival effect is not due to the enzymatic activity of eNAMPT, but instead is due to the cytokine activity of eNAMPT. This pro-survival effect of eNAMPT could not be mimicked by adding NMN. It did not require the presence of Nicotinamide and it was not blocked by FK866. Thus it is clear that eNAMPT promotes survival as a cytokine in macrophages, not as an enzyme.  Given the long list of “bad” things we have had to say here about eNAMPT, it is nice to identify this potentially “good” thing.

Another interesting finding is that the NAMPT gene is markedly upregulated in M1 macrophages but is down regulated in M2 macrophages. M1 macrophages are the phenotype associated with chronic inflammation whereas M2 phenotype macrophages inhibit inflammation. Exercise can phenotypically “switch” M1 macrophages to M2 macrophages, even in obese mouse models of inflammation. (See ref below). PPARgamma activation is at least one of the exercise-induced molecular mechanisms responsible for this phenotypic switch. PPARgamma is also a suppressor of inflammation and has been associated with down-regulation of eNAMPT expression.

References: 

2008 Extracellular Nampt Promotes Macrophage Survival via a Nonenzymatic Interleukin-6/STAT3 Signaling Mechanism

Nicotinamide Phosphoribosyl Transferase (NAMPT) Inhibitors: Novel Modulators of Cancer-Related Inflammation

Exercise training inhibits inflammation in adipose tissue via both suppression of macrophage infiltration and acceleration of phenotypic switching from M1 to M2 macrophages in high-fat-dietinduced obese mice

2007 PPARγ Activation Primes Human Monocytes into Alternative M2 Macrophages with Anti-inflammatory Properties

  1.  eNAMPT makes cerebral ischemia worse. In a recent mouse model of cerebral ischemia, the non-enzymatic effect (cytokine effect) of eNAMPT was shown to exacerbate the oxygen/glucose deprivation injury. Exogenously administered eNAMPT triggered TNF-alpha release from glial cells and it appeared that this was the mechanism of injury. No enzymatic activity of eNAMPT was necessary for this effect. This eNAMPT is a “cytokine” and not an enzyme when it comes to ischemia-reperfusion injury.

References:

2013 Cerebral Ischemia Is Exacerbated by Extracellular Nicotinamide Phosphoribosyltransferase via a Non-Enzymatic Mechanism

2008 Extracellular Nampt Promotes Macrophage Survival via a Nonenzymatic Interleukin-6/STAT3 Signaling Mechanism

RECAP

In recap, here are some key facts I have covered:

  1. eNAMPT has a protein moiety on the protein that “mimics” endotoxin and activates the TLR4 receptor, essentially creating a “sterile inflammation.”
  2. eNAMPT is secreted by “angry microglial cells”, angry macrophages, and visceral fat.
  3. eNAMPT signaling via TLR4 triggers two pro-inflammatory intracellular cascades/transcription factors, NF-kB and AP-1.  Simply inhibiting only one of these pathways like NF-kB does NOT work!
  4. eNAMPT expression triggers the expression and secretion of practically every SASP biomarker/cytokine, such as IL-8, IL-6, MCP-1, IL-16, TNF-a, IL-1B, CCR3, etc.  Thus eNAMPT triggered secretions “mimic” cellular senescence!
  5. eNAMPT can be inhibited by NMN.
  6. eNAMPT can be inhibited by FK866 and MSO, two small molecule inhibitors of the enzyme.
  7. eNAMPT induces “insulin resistance” by making the IR translocated out of the lipid raft (i.e. it “pushes IR off the raft.”)
  8. eNAMPT induces “IGF-1 resistance” in joints by making the IGF-1R unable to trigger cell growth and cartilage ECM generation.
  9. eNAMPT is the “ISIS of Molecular Biology”