Cancer a Mitochondrial Disease – Keto diet and NAD+ therapy

This theory of cancer is more than 100 years old, but it didn’t become the dominant view until the 1950s, when, after Watson and Crick, genes assumed an exalted position in the study of biology.  The “somatic mutation theory” continues to dictate the course of cancer research and treatment today.

It is uncontested that cancer cells have abnormal chromosomes.  Dozens of different mutations have been found in malignant cells.  They have been catalogued as different oncogenes, and because they are so different in their functions, cancer has been re-conceived from a single disease to a category containing many different diseases with similar symptoms.

Are mutated genes the root cause of cancer?  Toxins that commonly break DNA (teratogens) are also found to cause cancer (carcinogens).  Radiation, ditto.  “Ionizing” radiation packs enough wallop in each photon to break a chemical bond, and is associated with cancer, while non-ionizing radiation (visible, infrared, and radio waves) is not mutagenic and generally not carcinogenic*.  This has been taken as powerful circumstantial evidence for the prevailing theory.

A direct answer to the question of whether cancer originates in the nuclear DNA is available from an experiment that is simple in principle: Swap nuclei between two cells, one normal and one malignant.  Take the mutated DNA out of a cancer cell and put it in a normal cell, to see if it becomes malignant.  Take the un-mutated DNA out of a normal cell and put it in a cancer cell to see if the cell is rescued and restored to health.

This experiment has been technically feasible for more than 30 years, and indeed Barbara Israel and Warren Schaeffer actually performed both experiments at UVM and wrote them up in 1987 [ref, ref].  The results were exactly the opposite of what was expected: The cell with normal cytoplasm and cancerous nucleus was normal; the cell with normal nucleus and cancerous cytoplasm was cancerous.

This result has been confirmed in other labs[reviewed by Seyfried, 2015].  Still, the genetic paradigm has a stubborn grip on cancer research and treatment to this day.

An alternative theory of cancer as a metabolic disease was put forth by the Nobel polymath Otto Warburg in the 1930s.  The principal proponent of this theory today is Thomas Seyfried of Boston College.  Seyfried cites evidence that damage to the nuclear DNA, conventionally thought to be a root cause of cancer, is actually an effect of the damaged mitochondria and irregular metabolism.  “The metabolic waste products of fermentation can destabilize the morphogenetic field of the tumor microenvironment thus contributing to inflammation, angiogenesis and progression.”


Respiration and Fermentation

Every cell in our bodies (and almost every cell in all eukaryotes everywhere) makes uses of energy in the form of ATP, adenosine triphosphate.  ATP is manufactured in the mitochondria, usually by a controlled burning of sugar to form CO2 and H2O.

Highly energy-intensive cells such as muscles and nerves have thousands of mitochondria in each cell.  The word “respiration” in this context is used to mean burning sugar in an efficient energy conversion process, yielding 38 ATPs for every sugar molecule.

But when oxygen is scarce, perhaps because you’re breathing as fast as you can or sprinting in deep anaerobic mode, another process can be used to rapidly convert available sugar stock to lactic acid, requiring no oxygen at all, but yielding only 2 ATPs per sugar molecule.  The latter process is called “fermentation”.  (This observation explains the extraordinary effectiveness of interval training (sprints) for weight loss.)

Warburg was among the first to notice [1931] that most cancer cells use fermentation rather than respiration as an energy source.  Metabolic studies pointed to damaged mitochondria in tumor cells that had become inefficient in producing sufficient energy through respiration.

He theorized that impaired mitochondrial function is the root cause of cancer.  In fact, Warburg did some of the early work establishing the role of mitochondria as cellular energy factories.

So most cancer cells are sugar addicts.  They consume enormous amounts of sugar, both because they are actively growing and dividing, and also because they use sugar so much less efficiently than normal cells.  A PET scan can be used to visualize concentrations of sugar in the body, and PET technology is often used to locate tumors.

Sugar is easily made from carbohydrate foods, and when you eat a diet containing carbs, sugar is the fuel of choice.  Ketones are an alternative fuel used by the body when burning fat, either stored fat or ingested animal fat or vegetable oils.  (Medium chain saturated fatty acids like coconut oil seem to be most effective in inducing metabolic ketosis.)  Unlike sugar, ketone bodies cannot be fermented.  They generate ATP energy only through oxidative respiration in the mitochondria.

The logical question:

Are zero-carb diets an effective treatment for cancer?

Some well-known cancer drugs (Gleevec, Herceptin) already target the fermentation metabolism.  Acarbose has been proposed but not yet tried.  But might it be safer and more effective to starve cancer cells by cutting carbohydrates in the diet to zero?  There is a robust literature suggesting, “yes” [e.g., ref, ref, ref, ref, ref, ref, ref] but so far the results have been less than earth-shaking.

A search of yields 25 trials of ketogenic diet variants for cancer treatment.

Most are in early stages, 5 have been completed, 2 have results.  In this study, the ketogenic diet, with or without chemotherapy, did not cure glioma.  This small study found modest benefits in a variety of advanced cancers.

These results are consistent with many mouse studies, in which some benefit was recorded from the ketogenic diet, but not a dramatic difference.  The most encouraging results I have found was a study in which 9 of 11 mice treated with a combination of radiation and a ketogenic diet were cured of brain cancer.

Clearly, this is no miracle cure, but it’s too early to give up–we’re just figuring out how to make the diet work, and it has not yet been tried except at late stages, after all else has failed.

Fasting shows more promise than ketogenic diets.  (Perhaps fasting lowers blood sugar even more than ketogenic diets.)  A series of studies by Valter Longo make the case that fasting simultaneously sensitizes cancer cells to chemo or radiation and de-sensitizes normal cells.

Seyfried has proposed a “press-pulse” system based on this vulnerability, targeting the glucose metabolism and the glutamine metabolism with hyperbaric oxygen.

Besides glucose, glutamine is also a major fuel for tumor cells.  Drugs will be required to target glutamine, as glutamine is the most abundant amino acid in the body and can be easily synthesized from glutamate.  Hyperbaric oxygen requires a patient to be enclosed in a pressurized oxygen chamber or room filled with pure oxygen at 2.5 x atmospheric pressure.

There is one highly encouraging case report for the success of this triple combination—hyperbaric oxygen, glucose inhibitors, and low-dose chemo—in which a late-stage, resistant breast cancer is driven to total remission.

Last week, a research paper from Duke U suggested a target for attacking the fermentation metabolism of cancer cells, and a marker for identifying which cancers are likely to be sensitive to it.

The research group of Jason Locasale found a protein called GAPDH which switches to the fermentation metabolism, and a compounded called koninjic acid, extracted from fungi, that inhibits GAPDH.  They have tested koninjic acid extensively in cell lines, and have begun testing in live mice.  Whether such drugs are more effective than simply restricting glucose is a topic for investigation.

Explanatory diagram from the Duke study of GAPDH


Mito-targeted Cancer Prevention

 Supplements that promote mitochondrial health include NMN, NR, CoQ10, PQQ, mitoQ/SkQ, alpha lipoic acid (ALA), carnitine, and melatonin.  Can they lower risk of cancer?  So far, we have just a few hints; this is a promising area for research.

CoQ10 was studied in the 1990s as a cancer treatment, with some encouraging results [ref].  PQQ has been shown to kill cancer in vitro [ref].

One mouse experiment looked at ALA as part of a cancer treatment [ref].  Use of carnitine remains theoretical [ref].  Most has been written about melatonin [ref, ref, ref], but even here, there is no epidemiological evidence.


The Bottom Line

All the evidence for radiation and other mutagens causing cancer might be re-interpreted in terms of mutations to mitochondrial DNA.  (Mitochondria live in the cytoplasm, outside the cell nucleus, but they have a bit of their own DNA and ribosomes for transcribing it.)

Damaged mitochondria can also cause cancer even when their DNA is intact, and Seyfried (after Warburg) makes a strong case that mitochondrial damage is the root cause of cancer.  Inflammation is probably the single worst source of mitochondrial damage. Do we need one more reason to minimize inflammation?

Viruses often target mitochondria for their own ends, and this may explain cases in which viral infections are associated with etiology of cancer.

The insight that mitochondrial damage is the root cause of cancer (preceding nuclear mutations) also has broad implications for cancer prevention.

As for treatment, there have been a few disappointments and also some promising pilot studies, especially in combining glucose deprivation with radiation or chemo to finish the job (“press-pulse”).  This is a research field that deserves much more attention.

Ben Greenfield on NAD+

This is the transcript from Ben’s podcast you can listen to here:

Ben: Hey folks, it’s Ben Greenfield, and I wanna tell you about an article I recently read in Scientific American.  This article was called “Beyond Resveratrol: The Anti-Aging Nad Fad”, and NAD referring to NAD.  And what this article includes, among many other things, is, for example, quote, “Recent research suggests it may be possible to reverse mitochondrial decay with dietary supplements that increase cellular levels of a molecule called NAD.”  And there’s another part of the article that says, quote, “The mitochondria in muscles of elderly mice were restored to a youthful state after just a week of injections with NMN, a molecule that naturally occurs in cells and boosts levels of NAD.”  Well, since that article was published a few months ago, I have received an onslaught of questions about this mysterious molecule called NAD.

And it just so happens that a friend of mine, named Thomas Ingoglia, he knows one of the best NAD scientists on the face of the planet.  He’s in contact with one of the best NAD clinicians on the planet, both with decades of experience, second to none when it comes to NAD, and I actually consider Thomas himself to be one of the most knowledgeable and frequent users of NAD who I’ve ever met.  He’s one of the few guys that’s been playing around with it in combination with things like cryotherapy, and blueberry extract, and hyperbaric oxygen, and all these other biohacks that have allowed him to turn completely around from being bedridden sick and losing half his family in a car crash, to being in the best health of his life, including crushing his first Spartan race with me last year, prior to which he actually took high doses of NAD.

And the problem is a lot of NAD clinical researchers seem to mostly be underground at the moment.  The FDA doesn’t look kindly at NAD supplement companies and integrative doctors who use NAD.  They’re skeptical of naturopathy, and the first impulse is to turn these type of compounds into patentable drugs because that’s a language that the FDA speaks, and NAD can be dangerous.  I’ve spoken with Thomas and he knows a guy personally who has poisoned himself while using NAD incorrectly and hospitalized himself with the same substances we’re gonna be talking about in this podcast episode.

So, yeah.  You need to proceed with caution and with the type of formal clinical information that Thomas has opened my eyes to, and Thomas is actually on the call with me today, but I don’t just have Thomas here with me today.  Along with Thomas, first of all, we have Dr. Ross Grant, PhD., and Dr. Grant is one of the most prolific authors in the field of NAD, and he specializes in the effect of NAD on the brain.  He’s been researching it since 1994, back when nobody was doing NAD research.  He’s the clinical associate professor at the University of Sydney Medical School, and the CEO of the Australasian Research Institute, and a biochemical pharmacologist himself.  So he’s a smart cookie, and he specifically researches NAD and its role in oxidative stress, and the human cellular response to oxidative stress and how NAD affects that.  And in addition to Dr. Grant, we’re also, as if that weren’t enough, joined by Dr. Philip Milgram, who is an M.D., and Philip Milgram is based out of the NAD Treatment Center in San Diego, California, and he specifically helps people in recovery from addiction using NAD protocols.

So, between Dr. Grant, Dr. Milgram, and Thomas, we have quite a few folks who specialize in NAD.  So if you’re curious about this stuff, you are in the right place.  Now before I jump in, and I’m gonna jump in starting, with Thomas, and Thomas telling us his story and how he first kinda discovered NAD, I wanna tell you that all the show notes for everything that we’re gonna talk about, including a link to that article that first kinda sparked my own interest in this, you can find at  That’s  So, with that being said, let’s just go through here real quick so everybody can know everybody else’s voices.

Dr. Grant, welcome to the show and thank you for coming on at 4 A.M., Australian time.


Ben:  Alright.  Well, fantastic.  Well, Thomas, like I mentioned, you’re the guy who kinda first became my outlet into the wide world of NAD.  So tell me a little bit about how you first discovered NAD, and how you got to the point where you were kind of dug deep into a health hole, so to speak when it came to needing some serious healing.

Thomas:  Well, I’d like to start by saying that this all really began when I lost half my family in a car crash a few weeks after I lost them, old friends to drugs and alcohol, I had a friend who knocked on my door the day that he killed himself, and he had abused drugs, and he had severe psychological problems because of it.  But I had lost my dad, and my brother, and my nephew instantly in a head-on collision, and it was very hard for me because I was chronically ill for about seven years.  I was suffering from what doctors called Fibromyalgia and Chronic Fatigue Syndrome, and I took opiate drugs at that time.

My illness was brutal.  I spent a lot of time in bed.  I had trouble standing for periods of, let’s say, over 45 minutes.  I was very sick.  I would get headaches, there were so many different symptoms that I had, and I was very fatigued all the time.  And going to the funerals, because we had a funeral in Costa Rica, and I had to go see my mom in Hawaii where the accident took place, the car crash, it was just, it was very hard on me, and I was very angry.  And I made a point that at the funeral, to myself, that I would do anything and everything to cure myself.

Ben:  So, do you know what exactly it was that was causing you to be in a position where like you couldn’t stand up for 45 minutes, or for more than 45 minutes?  Or what exactly the infection was?

Thomas:  Well, it’s crazy because when people ask me, I’m forced to tell them that, you know, I don’t know.  I mean, the Mayo Clinic couldn’t figure out what was going on.  I do know that there were some tests for Lyme that showed positive results.  I do believe I had a lot of the symptoms for antibiotic adverse reaction, and I put some of the claim on that.  And then also, there’s a concept called opiate-induced hyperalgesia which is this idea that longer term use of opiates will cause pain throughout your body.  So, you know, I think it was probably sort of a mixture of those things, and we can go down a rabbit hole on this great deal and I’d rather, maybe save that for another time, but needless to say that it was a nightmare, and I was looking for something that would be beneficial for me, no matter what I had.

Ben:  So you were more or less addicted to opiates and extremely sick?

Thomas:  Yeah, and tired.

Ben:  Okay, and so what did you do from there?

Thomas:  Well, I was up late at night in one of my support groups, and someone would mention NAD, and I was like, “Okay.  I’ll give it a shot.”  And I called Springfield Wellness Clinic, which was the longest running NAD clinic in the country for 15 years.  They may have treated over a thousand patients, and they told me, “You know what?  We have a place in San Diego where it’s being done.”  And so, I was like, “Great!  I live in San Diego.”  So, I started the treatment and it’s intravenous NAD+, and that’s very important that you get the right molecule.  It’s not NADH, which is completely different.

Ben:  Okay.  So, when you say intravenous, you’re actually getting this stuff injected?

Thomas:  Yeah, it’s into your vein.  It’s a lengthy treatment.  I ended up going for 12 days.  It might be like seven hours a day, and then I was a noisy patient, I was not easy to be around.  They asked me to give up opiates and I was scared and, you know, just kind of grumpy.  But after that, about the day seven, the day nine, things began to change.  I remember Anne Rogers saying, “Your eyes are shining.  You’re changing.  I can see it,” and I was a little bit surprised that she could see that I was changing physically, and that happens to quite a lot of chronically ill patients.  Their face and cheeks just start to get color, it doesn’t happen with everyone, and then a day later, they get more color and gloss in their eyes.  Their eyes no longer look, like dull.  So…

Ben:  I mean, I’ve done IV injections before, like Myer’s cocktails where you get high dose glutathione, and vitamin C, and a host of different vitamins, but with this NAD, is it just NAD that you’re injecting when you do something like this intravenously?  Or are you including other things like vitamin complex, or things along those lines?

Thomas:  You know, the NAD doctors that are involved with the Mestayer model, where Mestayer is the doctor in Springfield.  They worked to customize the protocol, her patient, and so, other things that you might see in Myer’s cocktails might be added at some point, just so you know that the supplements aren’t mixed in the same bag.

Ben:  Okay.  Gotcha.

Philip:  Let me jump in real quick.  There are definite protocols that Dr. Mestayer from Louisiana has worked on, and we’re also working on on improving and yes, it is similar to getting Myer’s cocktail.  It’s an intravenous slow drip at the rate that the person can tolerate it, and then we add other minerals, and we individualize the treatment according to testing and what the patient’s personal needs are to enhance the NAD experience.  The NAD is the most active ingredient but we use these other compounds, because there’s an art to this, it’s not just starting an IV and running NAD in.

Ben:  Okay.  Gotcha.  Now, I do wanna definitely ask you about some of the protocols that you do there Dr. Milgram, but back to you Thomas, in terms of starting into this, you’re literally saying within days of beginning NAD injections that your health completely turned around?

Thomas:  Yeah, I mean, I remember being in the ocean, swimming in the ocean about a week afterwards, and I just remember looking around and everything I sensed was just richer and full of more color, and my pain went down by 50% in the first 10 days of treatment, and then I started to go down further over the next six months and a year, and it said it might be beneficial to keep coming in every so often to get a booster, and I was diligent in doing that.  And I felt healthier, and less pain, and more energy, you know, every time I came in.  So…

Ben:  Interesting.

Thomas:  At that point, I went back to old doctors and just sort of talk to them, and some of these doctors were like, they said, you have no idea what you just stumbled upon.  We just came to a conference, we were just at a conference, I was just talking about this, and you should pursue this and then they gave the whole “you-should-go-to-medical-school” thing, but it was really motivational, and they encouraged me go to medical conferences which I did, and I got the same responses that these integrative medical conferences that I went.  And I also remember meeting with a scientist who was working in the NAD field for coffee, and he asked me about my story.  I remember telling him, I was like, “You know there’s one thing that I gotta tell you about, and that’s the music sounds beautiful.  Like, I can really have an emotional response to music.  I can really hear all the nuances in it.”  And he responded, “Well, it’s because your nerves are revived.”

Ben:  Wow.

Thomas:  I dissected mice on NAD and we noticed a difference in their nerve connections to their ears, and I don’t know, I was sort of, at that point, that was incredible.  But it was six months after I did the NAD that I saw the article by David Sinclair.  Well, I saw it in Time magazine, the journal article he did and so, at that point I was like, “What did I stumble upon?”  They’re using a precursor, and we’re using, you know…

Philip:  The actual NAD.

Thomas:  The actual NAD.  Yeah.

Ben:  Okay.  Gotcha.  So there is a difference, I know, and I wanna delve into that here in a moment, but Dr. Milgram, in terms of you and your story, are you the person who Thomas actually hooked up with, in terms of his first experience with NAD?

Philip:  Yes.  He hooked up with this lady that I worked with together with Dr. Mestayer in Louisiana, and her name was Anne Rogers, she was looking to bring it to San Diego, and she found me as an addiction medicine doctor, and also somebody interested integrative medicine and nutrition, and individualized medical care with metabolic nutrition, and just serendipity, she found me.  I’ve been doing addiction medicine for 25 years.  I’ve been in treatment myself for opiates, in recovery since March 23rd, 1988.  Then I studied intervention with Vernon Johnson, the guy that invented intervention back in 1991, and I’ve dedicated my life to this because this really changes people’s lives.  I’ve been involved in Tom’s therapy, I was trained by Dr. Mestayer in Louisiana, and who’s been doing it since 2001, and then I’ve been involved with administering NAD to Tom, yes.

Ben:  Okay.  Gotcha.  And you yourself, if I’m not mistaken, didn’t you used to have some kind of like addiction issues that you overcame with NAD as well?

Philip:  I didn’t overcome them with NAD.  I’ve been medical director of several detox places but originally, I was just exposed to the old, white knuckle type of detox where they prescribe high levels of drugs to counteract withdrawal symptoms like valium, benzodiazepines, and other heavy duty drugs, and I’ve been doing that for 25 years.  When we came to NAD, it’s a game changer.  There’s virtually no withdrawal symptoms, the hyperalgesia is decreased, and…

Ben:  What’s hyperalgesia?

Philip:  What Tom referred to that opiates, in a way makes it a cycle that makes you use more opiates.  It actually increases your sensation of pain, then you require more opiates.

Ben:  Okay.  Gotcha.

Philip:  It’s a vicious cycle.

Ben:  Gotcha.  How is NAD actually breaking addiction?  Like how does this work?

Philip:  Well, I can’t give you the answer in 25 words or less, but it actually works at the epigenetic level to decrease withdrawal symptoms.  Okay?  It also works at the mu-opioid receptor level to decrease this exaggerated response of pain through the sodium and calcium-gated channels.  And it also decreases anxiety by having an effect on the bundled up chromatin that comes from lack of NAD.

Ben:  Okay.  So someone who’s like anxious, who’s having withdrawal symptoms, and who has like a chemical addiction to opiates.  It’s working on all three of those different platforms to decrease the addiction to opiates, and let me know if I’m correct about that, but also decrease addiction to other substances as well?

Philip:  Yes.  And in fact, it has the greatest effect on alcohol addiction.  It’s amazing.  It actually acts at the metabolism and genetic expression of alcohol predisposition.  Again, I can go as deep into this as you’d like.

Ben:  Well, what I’m curious, I guess the first thing and I’m sure a lot of our listeners are wondering this too, and perhaps this is a question for you, Dr. Milgram, or for Dr. Grant, but what exactly is NAD?  Like, what is it and what does it do?

Philip:  Well, it’s like we’re on a phone call talking about relativity, and we have Einstein on the phone with us.  Why don’t we have Dr. Grant, who’s one of the world’s experts on this, how he answers this question.

Ross:  Yeah.  Look, I’m happy to make a few comments and I’ll try and make them fairly brief, but NAD probably, is what could be considered one of the master regulators of cell metabolism and often when people talk about different molecules having this amazing impact, they’re often sort of talking at a level which is on molecules functioning at a fairly high level in a cell.  But here, we actually do have a molecule which is fundamental to the way the cell functions, and its levels as it goes up and down has an influence on multiple different areas.  So NAD itself, I mean apart from the clinical benefit as Dr. Milgram has talked about, and obviously Tom has experience, but at its fundamental level it’s actually involved in a number of different things.

It’s a co-factor, which we’ve known for many decades now.  So, you know, alcohol dehydrogenase as you need NAD, in fact it’s the NAD that runs out when people are trying to metabolize alcohol, and so they’re ending up with high levels of alcohol as a result, but it’s needed as a co-factor.  Another one is to lactate dehydrogenase, et cetera, which is also involved with that buildup of lactate, when you’re using your muscles.  It’s an electron transporter.  Now what that means is that you need to, when you’re turning the food you eat into the energy that the body needs to the muscles, et cetera, you need to be able to transfer the electrons from that food that you took down, what we call a respiratory chain in the mitochondrion, and that’s how we generate the energy, ATP.

Ben:  Okay.

Ross:  NAD is critical for that.  You know, it’s also needed for DNA repair, so, our DNA is getting damaged all the time, and I guess we’ll talk about aging a little bit later on, but it’s a really important molecule.  Well for now call a substrate, so it’s actually used up by the enzymes, or one of the key group of enzymes, there are a number of them, but basically it repairs particularly, and it’s actually used by those enzymes in order to help prepare the DNA.

Ben:  So, your body makes NAD on its own?

Ross:  It makes NAD on its own, and it makes it from a few different precursors.  So I know that was mentioned before, about nicotinamide mononucleotide or NMN, but there’s a number of precursors that it can actually make it from.  It can make it actually, originally also, from the molecule tryptophan, which is an amino acid people would have linked up in their minds to things like serotonin, which is one of these neurotransmitters, but happy to get into that a little bit later on, the epigenetic signaling its involved with as we know, the switching on and switching off genes by changing acetylation patterns, and these are sorta linked to people might have heard in a sort of aging medicine, or aging sort of like chemistry with the sirtuins, and NAD is a precursor to that, so sirtuins might be important.

And I guess a lot of the enthusiasm about resveratrol is the fact that it was theoretically driving sirtuin activity.  What’s interesting is that sirtuins are like a factory, like any enzyme, they won’t function unless they have, if you like, the raw material for that factory to work on, and NAD is that raw material.

Ben:  Okay.  Gotcha.  Now, I took physiology in college, like physiology and biochemistry, and what we were basically told was that NAD, or this nicotinamide adenine dinucleotide was basically, all it does is it carries electrons, right.  Like it’s involved in redox reactions in the cell, and you basically have it, this NAD+ gets an electron from other molecules, it gets reduced, and that makes this stuff called NADH, which then can donate electrons, and it’s just essentially one of the ways that the human body kinda keeps working as a giant battery.  We never learned about any of this stuff regarding like anti-aging, or addiction, or it acting on these sirtuin pathways you’re talking about, and so, I guess that’s my first question, especially after hearing you and reading this Scientific American article about how this could be the new anti-aging drug of the future.  How exactly is it working when it comes to NAD?  I know that you just mentioned this sirtuin pathway, and I know that’s intimately involved with aging, so can you go into, not only what this sirtuin pathway is, but what’s the proposed mechanism between, or as to how NAD would actually help someone when it comes to anti-aging?

Ross:  Yeah.  I mean, it’s a very good question and all of the answers aren’t known in here.  What we do know is that when it comes to aging, so if you think of what’s actually happening when somebody is aging, they’re actually getting an accumulation of damage within the cell, and that accumulation of damage particularly around the DNA, is affecting the way those cells function and therefore the way the organ functions.  And a couple of the key things that characterize aging is that part from the cumulative damage is also this decrease in energy, and just a decrease in what we might call the viability of the cell.

So very simply, where does NAD set in?  NAD, we know, can improve energy efficiency because one of the key things that’s needed in order for the mitochondrion to work well, in order for the energy to be produced, is that we need to have an efficient supply of these NAD molecules, as you said, to be able to transfer those electrons so that we end up having a mitochondrion being able to convert literally the energy we take in, mix it with the oxygen that we also breathe, and finally produce ATP at the end.  If you don’t have NAD, the mitochondrion doesn’t work.  Therefore, you don’t generate the energy.

Now, at the epigenetic level, then we have again NAD as a master regulator at that point.  If NAD levels drop, then the epigenetic switches, and people would be aware that now we know we have methylation patterns, and acetylation patterns, and what these are is just molecules that set on and off, both the DNA as well as the chromatin that sits around with the proteins, to allow the genes to either be switched on or switched off.  If you’ve got lots of NAD around, it is able to switch on, so sirtuins act, they deacetylate things, and they’re able to switch on pathways that are linked to improving cell viability, and in other words improving the health of the cell.  Now, remember this is at a global level, so it’s not just happening in one organ, it’s actually happening across the body, including the brain, as well as the muscles, as well as other tissue.  And so this increases things like a radio oxidant production, and basically keep cells working in what we would consider to be a younger state of metabolism.

Ben:  Okay.  Gotcha.  So essentially all we’re doing is we’re allowing our mitochondria to function more efficiently when we have adequate NAD, and we’re also fighting a lot of the oxidation that could cause aging, but by giving our body extra NAD via something like the intravenous injections that Thomas and Dr. Milgram were talking about, we’re not just able to, for example, fight off some of the bad things that can happen when we’re addicted to a substance, but we can literally activate these anti-aging pathways.

Ross:  Correct, and I guess the other important thing is that it looks like we were able to increase the repair of DNA that might get damaged in the course of normal metabolism.

Philip:  Ben, can I chime in here?

Ben:  Yeah.  For sure.

Philip:  I’ve got a list, you know, I want you to get out of thinking that it’s just NAD to NAD+ in the Krebs cycle. That’s what we learned, making ATP.  That’s what we learned back in Biology 101, but this is much greater than this.  It’s an epigenetic co-factor in blocking over-reactive genes that are not being expressed properly.

Ben:  So what do you mean by that?

Philip:  In histone acetylation, methylation, phosphorylation, and DNA methylation, and micro RNA, it has an effect to allow the histones to get tightened up in the chromatin, and of course, very greatly simplifying this, and the NAD actually helps unbundle this.  Though it actually works to remodel your chromatin by its effect on these sirts and the PARPS, it actually affects DNA repair.  It affects the CD38 gene for increasing immune function.  It causes the mitochondrial biogenesis.  It increases the production, and effectiveness of each individual mitochondria.  It actually offers a neuroprotective device qualities protecting the nerves from demyelination and other things.  It being explored at Harvard for use in ALS, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease.  It also again, it’s very important that the sensation of pain by the gated calcium and sodium channels, the voltage-gated chain, the NAD helps that function properly so you don’t have this hyperalgesia.  It may act itself as a neurotransmitter, and it has an effect on autophagy.

Ben:  Really?  So it can also act as an actual neurotransmitter?

Philip:  That’s still being studied.  That’s sort of nebulous, probably beyond the scope of this discussion.

Ben:  Okay, but basically it could potentially be involved in like cell-to-cell communication?

Philip:  Yes.

Ben:  Okay.  Interesting.

Ross:  Yeah.  And I can jump in there and yeah, it certainly support that, it looks like NAD probably does serve as a neurotransmitter by systemically as well as in the central nervous system.

Ben:  Interesting.  Okay.  So, this Scientific American article it, obviously, based on some of the cool things about NAD that you guys have just talked about, it goes into how there’s a lot of different NAD sources being created right now.  Like there’s this stuff called Niagen.  There’s something called Basis by I think a pharmaceutical company Elysium.  They talked in this article about things that can assist with your body’s own production of NAD, like resveratrol, that we’d find in red wine or in supplement form, or something called pterostilbene.  Can you help us cut through all this clutter, Dr. Grant, as far as what the best way is to actually get NAD into our bodies, and what exactly the status is as far as the development of pharmaceuticals or the development of supplements?

Ross:  Yeah, sure.  Look, to get it into the body, and this is one of the reasons why clinics like Dr. Milgram’s and Dr. Mestayer will use IV NAD, if you want to get NAD into the body efficiently, what we call a 100% bioavailability, you know, when it’s all getting in, then the best way is to take an IV.  The unfortunate thing is, and even though there’s a lot of work that needs to be done still in this area, it looks like if you take NAD orally, and I’ve seen supplemental tablets for NAD out there, but taking it orally, unfortunately, you’re pretty much not going to absorb it across the gastrointestinal tracks.  So from the stomach and the gut, you’re not gonna absorb NAD very efficiently.  So, to what to get NAD up efficiently, it’s intravenous.

Now, there are a few other ways in which the body can actually make NAD, so that what we call precursors, and you mentioned one of them, niagen.  Niagen’s a fairly new one, and that was identified actually originally from milk in around about 2007, but that is called nicotinamide riboside.  And nicotinamide, yeah, nicotinamide riboside actually gets converted into NMN, which is one of the other precursors that are also available, the one you talked about in one of the other article.  So you can get those two, and those two will be absorbed across the gap.  So you can get effectively NAD increased from taking those orally.

The other two ways of getting them is by the classic vitamin B3’s that you’ll get from over-the-counter at the drugstore, and that would be nicotinic acid which is the acid form of vitamin B3, and this is the one that the…

Ben:  You said B like boy?

Ross:  Yeah, B like boy.

Ben:  Okay.

Ross:  So, vitamin B3.  So the nicotinic acid form is the one that’s been used for actually quite a few decades now to reduce cholesterol.  It’s the one unfortunately that has a side effect that gives you flushing.  We can talk about why that happens.  But then the other molecule that you’ll also get, again across the counter and in many supplements, is the amide form called nicotinamide.  Now, you’ll notice that NAD is nicotinamide adenine dinucleotide.  So that is the nicotinamide version.  Now nicotinamide can also be, what we call, recycled.  It’s in what we call the salvage pathway.  It can be recycled through to NAD.  The negative thing with having too much nicotinamide, as opposed to the other types of NAD precursor that I’ve mentioned, is that nicotinamide is a byproduct.  Now, you’ve heard we’ve talked about CD38, we’ve talked about sirtuins, and talked about the PARPS.  So CD38, the immune modulators, the Sirts, the epigenetic modulators, and PARPS, the DNA repair enzymes.  Now all of those three, when they use NAD, they will actually generate nicotinamide as a byproduct.

Ben:  Okay.

Ross:  And the important thing about that is that that nicotinamide, as the levels start to go up in the cell, the nicotinamide starts to inhibit those enzymes, the CD38s, the sirtuins, the PARPs.  And so too much nicotinamide, unfortunately, can stop the very reactions that you want to have acting.

Ben:  Interesting.  Okay.

Ross:  So, we would probably suggest that getting it from some of the other sources, the nicotinamide riboside, NMN, or nicotinic acid would be better.

Ben:  Okay.  And the NMN, is that something that one can, for example, purchase in like a supplement form, or this nicotinamide riboside?

Ross:  Both NMN and nicotinamide riboside can be purchased.

Ben:  Okay.  And the absorption of those in the gut is sufficient to actually increase NAD levels inside the human body?

Ross:  Yes, they do.  And there has been some studies in animals particularly, in fact we’re in the middle of preparing to conduct what we call a head-to-head on these so that we can see which one is actually the better one.  And most of them there is evidence that both of them will significantly increase NAD.  We’re probably favoring the riboside at the moment as opposed to the NMN, but both of them seem to be able to do it.

Ben:  Okay.  So…

Philip:  And again, Ben, it’s dose related.  You know, think of the breakdown of nicotinamide adenine dinucleotide into nicotinamide and adenine, okay?  That reaction of breaking the NAD down to nicotinamide is where the Sirt and the CD38, and the PARPs are generated and activated.  So if you have too much of the nicotinamide, it can actually force that to not happen.  So I would say like people that are taking too much nicotinamide is probably not a good idea.

Ben:  And how much would be too much? ‘Cause if you look on like Amazon, right, like if you go to Amazon, you do a search for like nicotinamide supplements, you’ll find like NOW Foods, and Life Extension, and all these other companies selling like 500mg capsules of nicotinamide, like that’s about the dose you’ll see in a lot of these.  When you say, “If you take too much, you’re gonna shut down those positive pathways that you’re trying to activate,” how much would be too much if someone were just gonna like try to get a nicotinamide supplement from one of these companies?

Philip:  Well, usually they come as 250 or 300mg, and they suggest two a day, and I think that’s the proper dose.  But people will think, “Oh, wow.  If a little bit is good, a lot is better,” and they’re taking more than that, I think they may actually be doing themselves disfavor.

Ben:  Okay.  So once you’re exceeding, like close to about 600mg, you’re saying that that could be Bad News Bears?

Philip:  Correct.  Just like too much vitamin D.

Ben:  Okay.  So, nicotinamide, we can buy nicotinamide, also known as NMN supplements on a website, like Amazon for example, from a good company, and we could take anywhere from 300 to 600mg of that to activate some of these anti-aging, or antioxidant pathways, or new mitochondrial-building pathways.  Now this nicotinamide riboside, which is not the NMN form, is that also something that you can find in supplemental form?

Ross:  Yes, you can find nicotinamide riboside as a supplement, and nicotinamide riboside, like NMN, will be converted through to NAD.  In fact, nicotinamide riboside is converted first to NMN, and then through to NAD, and for reasons that we still don’t understand, it seems to be [0:41:35] ______ way of getting there, and you can take, unlike the nicotinamide, which is actually having to be recycled through the pathway, and it recycles through NMN as well, but nicotinamide riboside, you probably can take up to, I would think, easily 500 to even possibly 1,000mg a day without too much concern ’cause you’re not gonna be generating nicotinamide directly.  If you’re supplementing with nicotinamide on its own, so that’s the vitamin B3 that’s often out there, if you supplement that on its own, you’re already increasing the body’s nicotinamide, and that’s doing the inhibition.  But taking the other two, the NR, the nicotinamide riboside, or the NMN, neither of those will produce nicotinamide until it goes through NAD.

Ben:  Okay.  Gotcha.  Now this article for example, the one in Scientific American talked a lot about resveratrol, which you find in wine, and the fact that resveratrol can kinda like rev up the sirtuin pathways.  And when we rev up the sirtuin pathways, we can do things like form new mitochondria, or keep mitochondria running smoothly.  Now, when it comes to resveratrol, is that something that you would take in conjunction with something like NAD injections, or one of these nicotinamide supplements, or is that something that one would use on its own?  What are your thoughts on the use of something like a resveratrol supplement?

Ross:  Yeah, I’m happy to make a comment.  I think that there is some positives with certainly the antioxidant effects that come from the stilbenes, which resveratrol is one, and…

Ben:  What’d you call, the stil, the pterostilbene?

Ross:  Yeah.  The pterostilbenes.  They’re a class of molecules that are what we call in the phytonutrient class.  So, there’s mention of them coming from things like the skin of red grapes, and from blueberries, and in fact, you even get them from things like peanuts, et cetera.

Ben:  Is that why Thomas that you told me that you were using like a blueberry extract in conjunction with your NAD injections?

Thomas:  That was just something that I had originally gotten from a brain conference that I went to, but as soon as I found that out, yeah, I’m very pretty religious on taking my blueberry extract.

Ben:  Okay.  And that’s because this pterostilbene that Dr. Grant was talking about, you’re gonna find that in addition to resveratrol in things like blueberries, and grapes, and dark purple, and blue type of fruits, or berries?

Ross:  Mhmm.

Thomas:  Yeah.

Ben:  Okay.  Gotcha.

Philip:  And we’ve found that NAD really is the most important thing to increase your Sirt 1 through 7 activity, but resveratrol specifically, you need that for Sirt 2.

Ben:  Okay.  Gotcha.  So there’s different sirtuin pathways that are activated by different forms of supplements and you would, for example, take resveratrol in conjunction with NAD to get the best of both worlds.

Philip:  Correct.

Ross:  Yeah.  Though theoretically, there seems to be a fairly good benefit.  I mean all the sirtuins need NAD, some of them get switched on a little more efficiently than others in different parts of the cell.

Ben:  Now, in terms of the best delivery mechanism, it sounds like intravenously that you can really get very, very high levels of NAD compared to some of these supplements that we’re talking about, but how much of a difference is it?  I mean, are we talking about like a night and day difference between intravenous injections versus someone using a supplement, like a resveratrol mixed with an NAD supplement?  Are we talking about a slight difference?  I mean, in terms of comparison between intravenous delivery versus oral delivery, how do these different delivery mechanisms vary?

Thomas:  In my personal experience, I just think that intravenous NAD is, you know, I haven’t seen anything that’s like it in comparison.

Philip:  Let me respond on a clinical level.  You know, we suffer from lack of NAD because we’re using it up and not replenished in proper levels, and it’s the cause of underlying aging, and other disease processes.  So I think that you have to sort of flood the body with NAD to start off with, but then you can supplement, and that’s why we use intravenous.  Then you can supplement NAD with a pure source of NAD that we give intranasally that I think is a very good method of continuing to substitute to boost your NAD levels.

But, you know, the NAD business has been around actually for many, many years.  But because of human greed, it’s sort of been kept underground.  People wanted to make money on it, so they called it different things.  They called it Coenzyme 1, they called it amino acid therapy when Dr. Hitt was doing it down in Mexico.  But then it was analyzed by Dr. Mestayer and his friends in Louisiana, and found that the active ingredient was actually pure NAD.  And they now have the pure source of, you wanted NAD that doesn’t have a contamination in it, you want it so that it has a high level, it’s not very stable at room temperature for a prolonged period of time, so it has to be gotten fresh, and be made up fresh and used quickly, and that’s why we have these protocols, and then we also use it with other nutrients to individualize therapy for people’s individual needs.

Ben:  Okay.  Gotcha.  So, in terms of other things, are you talking about things aside from like blueberry extract and resveratrol that play well with something like NAD when it comes to the anti-aging or the addiction mitigating effect?

Philip:  Correct.  Not only that we need to add things to enhance the NAD experience, and we know what those are, certain minerals and, but also, it seems like a lot of the things that we’ve been doing for detox for years actually go and work against these…

Ben:  Like what?

Philip:  Like benzodiazepines.  We do not give benzodiazepines when we do NAD therapy.

Ben:  You mean like valium, and stuff like that?

Philip:  It’s like anti-NAD.

Ben:  Would that be, for example, like a valium?

Philip:  Valium, Xanax.

Ben:  Okay.  So basically, a lot of these will actually have an effect that inhibits mitochondrial function?

Philip:  Correct.

Ben:  Okay.  Gotcha.  And what are some of the   things  that  combine  well  with  NAD?    You mentioned minerals as one.

Philip:  Well, magnesium, calcium, potassium, but we also handling the side effects, there’s drugs that we use and drugs that we don’t use that we’ve found to not inhibit NAD, but decrease some of the withdrawal symptoms, and enhance the NAD thing. I mean, we’ve developed this over the years.  Dr. Mestayer has been working on it, and we’ve develop these and increased these even more.

Ben:  It’s really interesting because I did a podcast with a guy named Dr. David Minkoff a few weeks ago where we talked about cancer and mitochondria, and anti-aging, and it seems that a lot of this kinda overlaps in terms of, you know, we spoke of hyperbaric oxygen therapy to enhance oxygen availability for enhancing mitochondrial function.  I mean, I know that’s something that Thomas, you told me that you had experimented a little bit with.  I’ve talked about cold therapy, and cold thermogenesis for enhancing mitochondrial function and the amount of nitric oxide that you have in your body.

You know, you guys are now bringing up other ways to enhance mitochondrial function such as mixing NAD with resveratrol, with minerals.  It seems to me that when we’re talking about like anti-aging, that there are all these different things that seem to kind of be going after the same effect, which is, more or less, improving mitochondrial health, while also improving the ability of DNA to repair, in the case of NAD.  But I know that, and I have a question for Dr. Grant actually about this, I know that the effect of this stuff can go beyond anti-aging and, for example, could kinda reach into the realm of fitness, which I think a lot of our listeners might be interested in.  As far as the research on NAD and anything such as VO2 Max, or lactate tolerance, or time to exhaustion, or anything like that, Dr. Grant, have there been studies on NAD, and how it affects the actual fitness or exercise performance?

Ross:  There’s been very few and I suspect that that’s going to rapidly change.  We’re involved in a study a little while ago now where we were looking at the influence of a particular supplement that was being provided to athletes, and seeing what the effect was, that will be getting a 15% improvement in what they called ‘Time to Fatigue’, as you’ve mentioned, so they could exercise about 15% minutes longer, and it was the exercise physiologists who brought the problem to our lab, and asked us to have a look, and see what we thought could be the issue.

So we checked around various pathways and it looked like NAD levels were actually increased with this supplement.  Now we thought that that was probably functioning as a preservation of NAD.  So it makes sense because of NAD’s role in being able to efficiently or make the mitochondrion where you’re producing the energy, where you’re making that happen efficiently.  And, as I mentioned, with lactate dehydrogenase being able to convert that more efficiently through the pyruvate.  So increasing NAD, it makes sense that you would have the potential for increase in fitness.

Ben:  Okay.  Gotcha.  So, what they’re saying is it may improve time to exhaustion by somehow working on the ability to turn over lactic acid more quickly, but it’s not actually something that’s been fleshed out in actual human research?

Ross:  In great detail.

Ben:  Okay.  Gotcha.

Thomas:  But then also, by acting through specifically the Sirt1, we found some of the things that you and your experience have shown to increase, improve the human condition.  Things like fasting, mechanically stretching muscles, exercise, low glucose diets, proper circadian rhythm sleep cycles, actually act to let the Sirt1 and the NAD work properly.  And that’s why they work, it works through the NAD and Sirt pathway for these things that you can do to be a healthy person, that’s why they work.  A lot of the better diets, the ketogenic diet, Atkin’s diet, Paleo diets, that’s how they work, they also work to increase Sirt1.

Ben:  Right.

Ross:  And that’s true, and the good thing with an activation of things like Sirt1 is it looks like we’ll end up getting an increase generation of mitochondrions.  You actually increase the number of mitochondrion as well as the efficiency of energy productions.  So, it [0:53:01] ______.

Philip:  And especially in the brain.

Ross:  Mhmm.  In the brain and in the muscles as well.  But, yes, certainly both.

Ben:  We’re talking now about, like, you know, some different lifestyle strategies that could, for example, enhance these Sirt1 pathways, you know, in a similar way as NAD, such as some of the things you were talking about, like ketosis, we mentioned calorie restriction, we mentioned cryotherapy, hyperbaric oxygen chambers.  What are some of the best ways to actually raise NAD levels naturally with other food strategies, or other lifestyle strategies, or other biohacks, for example?

Ross:  Yeah.  Look, I’m happy to jump in there with some of the lifestyle elements, which I think are particularly important.  If you want to turn over your NAD fast, in other words, if you want to drop your NAD, which is not what we’re gonna do and I’m just using that, then you’re gonna have any condition which has an increase in inflammation and what we call oxidative stress, or free radical damage.  Both of those, not only does it signal through to increase the CD38 activity, but it also increases PARP activity, which is the DNA repair.  So inflammation, free radical damage, both of those are gonna decrease your NAD, which is what you don’t want.

So, very simply, any of the lifestyle behaviors that are going to improve that is going to increase your NAD naturally.  And this includes taking in less calories, we know that using whole foods and some of the work in the brain that we’ve done which is interesting, showing that the caroteniods particularly, so these are the molecules that are coming from the red, yellow, and green leafy sort of veggies, these are particularly high in caroteniods and these can actually help preserve, particularly in the brain, NAD levels.  Exercising, eating whole grains, I mean these are gonna be able to provide things like your nicotinic acid and vitamin B3, et cetera, on their own.  But all of these, essentially doing those things that you’d be [0:54:52] ______ , most people would recognize were healthy for them is going to help to increase their NAD and maintain high NAD levels, and therefore high sirtuin activity, et cetera.

Ben:  Okay.  Gotcha.  What about light therapy?  You hear a lot about like infrared light and your infrared intranasal light, things along those lines, how about light?  Does it play a role here?

Ross:  Look, I don’t know, nobody’s done any work on that directly, as far as I’m aware, not specifically with NAD, but there is work that’s been done on light therapy of different wavelengths, even through the blue wavelengths decreasing inflammation within certain parts of the body, particularly in people with pain.  So, where there’s benefit that’s there, and I guess there’s still a lot of work still going on there, that photomodulation, that if it’s going to decrease pain, which is associated with cytokine signaling and increase in inflammatory cytokines drive or increase your pain, then there is going to be benefits with NAD.  And that probably works both ways, so the higher NAD is able to shut down some of those pathways.

Ben:  Okay.  Gotcha.  What about you, Thomas?  I know you’ve experimented with some things when it comes to enhancing your response to NAD therapy and supplementation.  What are some ways that you’re using food or lifestyle strategies to increase your own levels of NAD?

Thomas:  Personally, I had a very substantial decrease in pain and a very strong uptake in energy in that 12 days I initially did NAD therapy, but I wasn’t a 100% back to recovery and so, I was still desperately trying to make some changes and I think, sort of accidentally, I was really cutting down on my caloric intake, I was eating more vegetables, and my carbohydrate intake had come down, and I think that that may have contributed to sort of the positive impact that sort of accentuated the high levels of NAD that I had.  And when that happened to me in over the course of, let’s say, a year, I had lost about 40 pounds in fat ’cause I did a body comp.  And then it was a lot, I think it was actually a year and a half, and then at that point, I met up with you at the Spartan race in Las Vegas and it was my first Spartan, and I ended up placing at the top one and a half percent.

Ben:  Prior to that race, you actually took some NAD.  I remember we were in the condo, and you were taking something, but you didn’t do an IV, right?  You just basically were using like one of these nicotinamide type of supplements we were talking about?

Thomas:  No, actually I did IV NAD a few days before I arrived, and I wanted to see what kind of impact that had.  I don’t, I’m not sure, I mean this is just anecdotal, I definitely, I mean it’s obvious, I already said that, yeah, I mean, I had no energy before, and now I’m doing all these things.  But as far as me personally, having this immediate effect, I don’t know where but that’s something that needs to be looked at a little bit more.

Ben:  Okay.  Gotcha.  Now, I wanna get into anti-aging here real quick.  Dr. Grant, I know that you mentioned that NAD levels will drop when we age, which is perhaps why NAD is being proposed as an anti-aging supplement, but how much?  Like is this a significant drop?  Has this been studied in terms of the actual decrease?

Ross:  Yeah, absolutely.  I mean, we did some early studies back in 2011, we were just looking at animals, and across the age range you can see, while you know, accumulation of damage within the body, and things like PARPs go up the enzyme that’s actually trying to repair the DNA go up, NAD drops.  And then we said, “Alright, what we saw in animals, we thought we’d better have look at that in humans”.  So we looked at it in, with the pelvic, non-sun exposed skin, all the eight way from, sort of eight day old circumcisions up to 79 year old hip replacement, and you could see again the damage sort of accumulating with time, and particularly after the age of 60.  But then you could see the NAD drop fairly consistently with inflammation and oxidative damage, and in 2014 we published some work showing the same sort of decline within the brain, and this can be anywhere up to 40%.  So there is a significant drop that can happen…

Ben:  Forty percent is how much it could decrease as you age.

Ross:  Yeah.

Ben:  Is that just due to everything from oxidation to DNA damage, to living in an industrialized area where you’ve got lots of inflammation, free radicals, and things like that?

Ross:  Yeah, it’s predominantly what causes that inflammation and oxidative damage.  So as were saying lock-in formation of cell will drive oxidative damage inflammation which then damage the DNA, so you’ve got an increase in [0:59:51] ______ that drive the CD38.  Now, CD38 is definitely involved in the immune function as Dr. Milgram is saying earlier, but CD38 probably has some other function.  We have some other what we called NAD like a hydrolases that function in ways that were not really quite sure in a cell but these seem to go up to certain CD38 when we have inflammation going on and that seems to be also a primary driver of decreasing NAD as we get older.

So, there is a significant drop in NAD and it depends where you’ll look for I mean, whether or not it’s enough in the plasma or whether it’s in the tissue itself.  So it can vary depending on the different tissues, but certainly in the plasma they can give quite some significant shift.  And also in the fluid that drains the brain called the cerebral spinal fluid which is a good indicator of what actually happening clinically within the tissues.

Ben:  Okay.

Philip:  I remember reading some studies that they tested people under 45 and over 45 for their NAD levels and they found that 300% increase in the average between the pre- 45 and post-45.  Also, another study shows that it seems to exponentially increase after age 60 the loss of NAD.

Ross:  The loss of NAD that’s certainly what we found after the age of 60.  So, there seems to be an acceleration of damage and therefore an acceleration of the rapid loss of NAD as a result.

Ben:  Can you actually test your own NAD levels, like is there a blood test for that?

Ross:  Yeah look, we have actually offered that to one or two of our clinics here in Australia or any because we know that they can actually get sample to us in a particular way which preserve NAD.  The trouble with doing it and sending it through a normal pathology lab is a) they won’t do the test, and b) the samples itself once it collected needs to be preserved extremely quickly otherwise NAD will change very rapidly, and so you’ll get abnormal results.  So it is possible to do it but it’s a bit tricky to do it sort of in a regular basis in a normal lab at this stage.

Philip:  We are gonna be dealing clinical trials in our facility and in conjunction with Dr. Mathai in Louisiana to do this the right way, and actually test people with the special testing that Dr. Grant is talking about.  We had to purchase this special centrifuge and we have to handle the samples in a certain way, and we are gonna be deforming clinical studies on this.

Ben:  Well, vitamin B3 is also known as nicotinic acid or niacin or nicotinamide, couldn’t you just measure your vitamin B3 levels?

Ross:  Vitamin B3 is an important precursor to NAD and as we mentioned nicotinamide, if you take nicotinic acid, yes you’ll be able to generate NAD through that pathway.  If you take nicotinamide, nicotinamide is that sort of complicated situation where it’s both can be made back into NAD but it’s actually a byproduct of NAD.  So nicotinamide is going up doesn’t necessarily mean that NAD is going up.  Nicotinamide going up could actually mean that you’re turning over NAD quite a lot.  So, difficulty there.

Ben:  Okay, gotcha.  And I’ve got a few other questions: the first is I know that a lot of people listening in and Thomas kinda briefly mentioned that he thought that some of his symptoms may have been indicative of something like Lyme disease, and I’ve heard a few snippets here and they’re about the use of NAD for the use of something like Lyme which I know is a frustrating issue for a lot of people who have chronic fatigue is this Lyme infection.  What’s the link between NAD and the treatment of Lyme disease?

Ross:  Well, I’m not sure if Dr. Milgram wants to make comment there.  I can sort of begin that one off.  Lyme disease is originally as a Borrelia infection which is a spirochetal bacteria.  And people think to end up with chronic fatigue, reduced energy, and there are some central nervous system dysfunction that can happen with people at a chronic stage, and one of the things that NAD will do as we’ve talked about in essentially from the beginning of our discussion is that it has a significant improvement in being able to increase the efficiency of energy production.  So it’s probably working on at that level, I don’t know of anybody who’s done any specific studies to look at mechanism and I guess that’s where we probably still lacking a bit of information but improving energy production, improving the way the immune system probably functions which is the primary driver often behind and this is sub-clinical so it’s hard to test a little bit.  It seems almost certain that you’ve got disregulated immune function.  Again, maybe through CD38, some of these [1:04:50] ______ maybe out of half ways but we probably able to modulate more efficiently the immune function as well as increasing mitochondrial function.

Ben:  Okay, got you.

Philip:  It’s a controversial subject, Ben, about treating Lyme disease, and I know doctors, well intentioned, wonderful doctors that have lost their medical license by treating Lyme disease.  But I can say that for sure the NAD in many cases can help decrease some of the symptoms of Lyme disease.  We’re not treating the cause of Lyme disease which is antibiotics treating a bacteria but some of the symptoms can be lessened with NAD.

Ben:  Now Dr. Milgram and Dr. Grant, as we’re coming up towards the end here, is there anything else that you want to throw in as far as things that you find exciting in the realm of NAD research or the use of NAD as an anti-aging drug or for the use in other conditions?

Philip:  Well, even the MTHFR gene has improved with NAD.  The…

Ben:  When you say the MTHFR gene has improved with NAD, what do you mean?

Philip:  Well, again, this unspooling of the DNA, it may make the expression of the MTHFR gene change through a snip called C677T.

Ben:  And for people who are listening, can you explain this MTHFR gene?

Philip:  I’d rather like to talk about, more about some other things that I’m really excited about NAD.

Ben:  Okay.

Philip:  I have been doing addiction treatment for 25 years, and I’ve treated hundreds of patients, the good old-fashioned way of white knuckling it, and seeing them go through terrible withdrawal symptoms, through cravings, and the NAD virtually stops that.  It’s just absolutely amazing.  They have like a brain, people say they feel like they’ve never used before, or drank.  Their cravings are decreased.  It’s just amazing, and I’m really excited about, not only the anti-aging things that they’ve talked about, and there’s people like Dr. Watson, and if you have a chance to hear Dr. Watson talk from UCLA, he is extremely excited about the effects of NAD for an anti-aging.  And also, these different disease processes that we’ve before had no way to treat them.  Things like fibromyalgia, or chronic fatigue syndrome, Alzheimer’s disease, Parkinson’s disease, ALS.  They’re finding that NAD can make a difference in the therapy of these patients.  I think, in my opinion, and this is just an opinion, NAD will turn out to be one of the greatest advances in medical science since Fleming invented penicillin.

Ben:  Wow.  That’s quite the claim.  And in terms of people using NAD, you’re thinking that it’s gonna be just like these type of injections that you’re doing, that are gonna get the most efficacy?  Or are you just talking about NAD in general use as a supplement?

Philip:  I think you need to flood the body with NAD to start off with, and then we’re looking that this place that found out where, what is actually was in Dr. Hitt’s amino acid therapy from Mexico, it was pure NAD.  They are manufacturing a pure source of NAD that we are now using as supplementation.  And then you can also use the nicotinamide riboside, and other dietary things to enhance this.  And also it’s important that you do all of the things that, you’re expert in Ben, with the fasting, the mechanical stretch, the exercise, the low glucose, proper circadian rhythms, and sleep, all these things will enhance the effectiveness of your own NAD to work.

Ben:  Yeah, and I think that’s the key here whenever we talk about like a new anti-aging drug, or we talk about like this Scientific American article is they’ll talk about the use of things like resveratrol, or NAD, or nicotinamide, or many of these other compounds, and they’ll say that in terms of like mice and mitochondria, that the effects of NAD simulate the effect of calorie restriction, for example.  And the way I like to think about things is that you first do the lifestyle strategies, right.  Like consume more dark berries, blueberries, or grapes, or resveratrol containing compounds, engage in calorie restriction, expose your body to good amounts of oxygen, pay attention to things like air, and light, and water, and electricity, and all those other things that can affect mitochondrial variables.

I’m not saying you cover up a bad lifestyle with something like NAD injections, or the use of some of these NAD supplements that we’ve talked about, but it sounds like, especially as you age, and especially based off of what Dr. Grant said as you get past 60, if you want to engage in some type of anti-aging protocol, it sounds like this might be a smart one to throw into the mix based on actual research that’s been done on what this can do to everything from DNA to cellular damage.

Thomas:  Correct.

Ross:  Yeah.  Absolutely.

Philip:  And also, when Dr. Grant is the world’s expert on oxidative stress and NAD, and he brought up a point as we were talking about preparing for this conference.  He talked about, you know, if you have a lot of rust around, you have a lot of oxidation going around, the NAD’s not gonna be as effective.  So you have to have a healthy lifestyle which will enhance your NAD expression.

Ben:  Yeah.  Yeah.  Exactly.  As with a lot of these things we talked about here, you can’t cover up a bad lifestyle with a pill.  Well guys, first of all, Dr. Grant, I wanna thank you so much for coming on the show, as I know it’s extremely early over in Australia.

Ross:  You’re most welcome.

Ben:  And Dr. Milgram also.  Thank you for coming on and devoting your time.  And Thomas, thank you for your introduction to Dr. Milgram and Dr. Grant, and for kinda opening my eyes to NAD.

I have a couple more quick questions.  As far as any further resources Thomas, for folks, you had mentioned to me a book written by a guy named Dr. Nady Braidy, called “NAD+ Metabolism In Neurodegeneration and Ageing,” which I know is available on Amazon.  Is that what you would consider to be the best resource for people to delve more into NAD, or are there other places you would point people to?

Thomas:  I think that’s a great resource.  It’s heavy reading.  That was written by Dr. Grant’s student, and now colleague of Dr. Grant, and I think that would that’s a great place to start if you wanna go heavy into the science.  Dr. Milgram also has a blog at his website,, and the blog gets updated with new science as it unfolds.

Ben:  Okay.

Thomas:  So that’s

Ben:  Cool.  I’ll link to that, and I’ll also link to everything else that we talked about including this Scientific American article, some of the resveratrol supplements that we discussed, the book by Nady Braidy, et cetera if you just go to  And if you go to, that is also where you can leave any questions, any comments, any feedback that you have about today’s episode, or anything else that you wanna pipe in on in terms of your own personal experience with NAD, or other anti-aging protocols that we discussed in today’s show.  So gentlemen, thank you all for joining on the show today.

Philip:  Thank you.

Ross:  You’re welcome.

Thomas:  My privilege.

Ben:  Alright, folks.  So, this Ben Greenfield, along with Dr. Grant, Dr. Milgram, and Thomas Ingoglia signing out from  Once again, you could check out the show notes at  Thanks for listening and have a healthy week.

The Ketone body β-Hydroxybutyrate a key signaling metabolite in disease and aging

However, in its oxidized form (NAD+), NAD is also a cofactor for sirtuin enzymes and poly-ADP-ribose polymerase (PARP), both of which consume NAD in the course of removing acyl groups from and adding poly-ADP to proteins, respectively. Sirtuins and PARP thereby regulate cellular functions ranging from gene expression and DNA damage repair to fatty acid metabolism (133). During times of fasting, or relative scarcity of cellular energy, more NAD is in the oxidized state, and sirtuins and PARP can be more active.

NAD+, a simple energy carrier, thereby acts as a fulcrum around which many cellular processes can be regulated in response to changes in the external environment. Such signaling metabolites include acetyl-CoA (coenzyme A), another carrier of high-energy bonds that is also substrate for a widely prevalent protein posttranslational modification (lysine acetylation), and S-adenosylmethionine, which similarly is substrate for a common posttranslational modification of histones and other proteins (methylation) (43).

Independent manipulation of these signaling molecules can recapitulate, or abrogate, some of the broader biological effects of environmental changes such as fasting or dietary restriction. For example, long-term dietary restriction can prevent the onset of common age-related hearing loss in C57BL/6 mice. However, dietary restriction in mice that carry a genetic knockout of the NAD-dependent sirtuin gene SIRT3 has no such beneficial effect (121).

Inhibition of the TOR (target of rapamycin) signaling complex by rapamycin (46), activation of AMPK by metformin (81), or provision of NAD+ precursors (159) recapitulates some of the beneficial effects of dietary restriction on diseases of aging and longevity. Understanding the specific molecular actions of these signaling pathways and signaling metabolites that link changes in the environment to broad regulation of cellular functions will permit researchers to more precisely capture the therapeutic potential of metabolic or dietary changes to treat disease. It might also help explain the heterogeneous responses of individuals to such environmental changes, depending on their genetic or epigenetic capacity to generate and respond to these signaling metabolites.

Here, we review the signaling activities of the endogenous metabolite β-hydroxybutyrate (BHB). BHB is the most abundant ketone body in mammals. Ketone bodies are small molecules synthesized primarily in the liver from fats that circulate through the bloodstream during fasting, prolonged exercise, and when carbohydrates are restricted.

They are taken up by tissues in need of energy, converted first to acetyl-CoA and then to ATP. Emerging evidence, however, shows that BHB not only is a passive carrier of energy but also has a variety of signaling functions both at the cell surface and intracellularly that can affect, for example, gene expression, lipid metabolism, neuronal function, and metabolic rate. Some of these effects are direct actions of BHB itself. Some are indirect effects governed by downstream metabolites into which BHB is converted, such as acetyl-CoA. We focus this review on BHB itself, referring to ketogenic diets only when necessary for translational context.

Although ketogenic diets have been widely used both for research into the effects of ketone bodies and as therapeutics for conditions ranging from epilepsy to obesity, a ketogenic diet is a complex physiological state with many possible active components of which BHB is only one. Still, the signaling effects of BHB we summarize are likely relevant to the molecular mechanisms of interventions such as fasting, dietary restriction, and ketogenic diets. Altogether, these observations present a picture of a powerful molecule that offers both opportunities and cautions in its therapeutic application to common human diseases.


Ketone bodies are small, lipid-derived molecules that provide energy to tissues when glucose is scarce, such as during fasting or prolonged exercise. Over 80% of the human body’s stored energy resides in the fatty acids contained in adipose tissue (7). During fasting, after muscle and liver stores of glycogen are depleted, fatty acids are mobilized from adipocytes and transported to the liver for conversion to ketone bodies. Ketone bodies are then distributed via blood circulation to metabolically active tissues, such as muscle or brain, where they are metabolized into acetyl-CoA and eventually ATP (7).

In humans, serum levels of BHB are usually in the low micromolar range but begin to rise to a few hundred micromolar after 12–16 h of fasting, reaching 1–2 mM after 2 days of fasting (13, 108) and 6–8 mM with prolonged starvation (12). Similarly, serum levels of BHB can reach 1–2 mM after 90 min of intense exercise (64). Consistent levels above 2 mM are also reached with a ketogenic diet that is almost devoid of carbohydrates (60). The term ketone bodies usually includes three molecules that are generated during ketogenesis: BHB, acetoacetate, and acetone. Most of the dynamic range in ketone body levels is in the form of BHB.

When ketogenesis is activated, such as during fasting, blood levels of BHB rise much faster than either acetoacetate or acetone (74).

Regulation of Ketone Body Metabolism 

The biochemistry of ketone body production and utilization is well understood and has been recently summarized both in the literature and in textbooks (e.g., 7, 66, 92) (Figure 1). Two points are particularly relevant to understanding the signaling activities of BHB. First, the same enzyme, β-hydroxybutyrate dehydrogenase (BDH1; EC, interconverts BHB and acetoacetate in both the final step of ketogenesis and the first step of BHB utilization.

BDH1 imparts chirality to BHB, as described below. Second, regulation of BHB synthesis is controlled via two principal mechanisms: substrate availability in the form of fatty acids and expression and activity of the enzyme HMG-CoA synthase (HMGCS2; EC Ketogenesis occurs mostly in the liver (7), although expression of HMGCS2 may be sufficient to produce ketogenesis in other tissues (127, 158). Insulin and glucagon regulate ketogenesis primarily by modulating the availability of fatty acid substrates at the levels of mobilization from adipose tissue and importation into hepatic mitochondria (71).

HMGCS2 gene expression is regulated by insulin/glucagon via acetylation and deacetylation of the FOXA2 transcription factor (143, 144, 135). FOXA2 deacetylation is controlled in part by the NAD-responsive enzyme SIRT1 (135). HMGCS2 gene expression is also regulated indirectly by the target of rapamycin complex mTORC1; mTORC1 inhibition is required for the activation of peroxisome proliferator-activated receptor alpha (PPARα) and fibroblast growth factor 21 (FGF21), both of which are required to induce ketogenesis (3, 4, 49, 116).

The activity of HMGCS2 is regulated posttranslationally by succinylation and acetylation, regulated by the mitochondrial desuccinylase SIRT5 (104) and deacetylase SIRT3 (117), respectively. Altogether, this network of regulation centered on HMGCS2, involving substrate availability, transcriptional control, and posttranslational modification, lends tight temporal and spatial precision to BHB synthesis.

BHB Transport 

BHB transport is relatively less well understood than its synthesis and utilization. As a small, polar molecule, BHB is readily soluble in water and blood (7). Several monocarboxylic acid transporters, including MCT1 and MCT2, carry BHB across the blood-brain barrier (99), and their expression can regulate brain BHB uptake (5). The monocarboxylate transporter SLC16A6 may be the key transporter for exporting BHB from the liver (52), but the putative transporters that facilitate the uptake of BHB into target tissues or its intracellular movement remain to be identified.

BHB Chirality 

BHB is a chiral molecule at the 3′ hydroxyl group, an important feature in consideration of its signaling activities and possible therapeutic applications. There are two enantiomers, R/D and S/L. R-BHB is the normal product of human and mouse metabolism. This chiral specificity is introduced by BDH1, which catalyzes the final step in BHB synthesis by reducing the nonchiral 3′ carbonyl group of acetoacetate to the chiral 3′ hydroxyl group of BHB.

BDH1 is also required for the utilization of BHB, by catalyzing the same reaction in reverse. As a result of the chiral specificity of BDH, only R-BHB is produced by normal metabolism and only R-BHB can be readily catabolized into acetyl-CoA and ATP. Fasting, exercise, caloric restriction, ketogenic diet, and any other state that results in endogenous production of BHB will produce only R-BHB.

S-BHB itself is not a normal product of human metabolism. However, S-BHB-CoA is a transient intermediate in the final round of β-oxidation of fatty acids. Under normal circumstances, it should not persist long enough to leave the mitochondrion or circulate in the blood. Experiments involving infusions of labeled R-BHB, S-BHB, or mixtures thereof into rats or pigs found that S-BHB is converted mostly to R-BHB (74); the molecular pathway for this is not known, but it may occur through conversion of S-BHB to acetyl-CoA and then production of R-BHB from acetyl-CoA.

At least some of the S-BHB is eventually converted to CO2, presumably also after being metabolized to acetyl-CoA. S-BHB is metabolized much more slowly than R-BHB is (138), so that infusion of the same amount of S-BHB may result in higher and more sustained blood levels of S-BHB compared with a similar infusion of R-BHB (19). We discuss the chiral specificity of BHB signaling activities below.


Several direct signaling actions of BHB have been described, including binding to cell-surface receptors, competitive inhibition of enzymes, as a substrate for protein posttranslational modification, and modulation of ion channel activity (Figure 2).

Schematic of direct and indirect signaling functions of the ketone body BHB. Indirect signaling functions require catabolism to other molecules, whereas direct signaling functions are actions of BHB itself. The major downstream effects of signaling functions are noted. Abbreviations: BHB, β-hydroxybutyrate; CoA, coenzyme A; FFAR3, free fatty acid receptor 3; GABA, γ-amino-butyric acid; HDAC, histone deacetylase; HCAR2, hydroxycarboxylic acid receptor 2; NAD, nicotinamide adenine dinucleotide; VGLUT, vesicular glutamate transporter.

BHB Inhibits Class I Histone Deacetylases 

BHB inhibits class I histone deacetylases (HDACs) (41), a family of proteins that have important roles in regulating gene expression by deacetylating lysine residues on histone and nonhistone proteins (reviewed in References 85, 90, and 150). Class I HDACs (e.g., HDAC1, HDAC2, HDAC3, and HDAC8) are small, mostly nuclear proteins that consist primarily of a deacetylase domain, and are usually found in large regulatory multiprotein complexes. Histone hyperacetylation is generally associated with activation of gene expression; so as a broad generality, class I HDAC activity suppresses gene expression. Many nonhistone proteins, including NF-κB, TP53, MYC, and MYOD1, among others (34), are also subject to HDAC-mediated deacetylation and regulation.

BHB inhibits HDAC1, HDAC3, and HDAC4 (classes I and IIa) in vitro with an IC50 of 2– 5 mM. BHB treatment of certain cultured cells induces dose-dependent histone hyperacetylation, particularly at histone H3 lysines 9 and 14 (118). Fasting, which increases plasma BHB levels, is associated with increases in histone acetylation in a number of mouse tissues by Western blot analysis (118), as well as in the liver by quantitative mass spectrometry (147). BHB can also increase histone H3 acetylation in vitro in macrophages (153) and neurons (120).

Treating mice with BHB via an osmotic pump increases histone hyperacetylation, particularly in the kidney, and causes specific changes in gene expression, including induction of forkhead box O3 (Foxo3a), the mammalian ortholog of the stress-responsive transcriptional factor DAF16 that regulates life span in C. elegans (59). Induction of Foxo3a appears to be a direct effect of HDAC inhibition, as HDAC1 and HDAC2 are found on its promoter, knockdown of both relieves HDAC-mediated Foxo3a repression, and BHB causes hyperacetylation of histones at the Foxo3a promoter that results in increased Foxo3a expression (118).

BHB regulates Bdnf (brain derived neurotrophic factor) expression in the mouse brain by a similar mechanism after exercise. Exercise increases BHB levels and Bdnf expression in the hippocampus, and Bdnf expression is increased by treating hippocampal slices with BHB or by infusing BHB intraventricularly. Treating primary neurons with BHB increases histone H3 acetylation, reduces Bdnf promoter occupancy by HDAC2 and HDAC3, and increases Bdnf expression (120).

Inhibition of class I HDACs appears to be a direct effect of BHB. Enzymatic inhibition in vitro is observed using immunopurified FLAG-tagged HDAC proteins with a synthetic peptide substrate, which should provide no opportunity for BHB metabolism or secondary effects (118). Competitive inhibition of the catalytic site is the likely mechanism.

Crystal structures of the human class I HDAC8 bound to several hydroxamic acid inhibitors (122, 130), as well as modeling of other inhibitors, show that a carbonic or hydroxamic acid group of an inhibitor is commonly bound to the catalytic zinc at the bottom of the hydrophobic active site channel of the HDAC (137). BHB is structurally similar to the canonical HDAC inhibitor butyrate.

Butyrate demonstrates the kinetics of a competitive inhibitor (115), suggesting that its carboxylic acid group might chelate the catalytic zinc in a manner similar to that of other acidic moieties on HDAC inhibitors. The structures of butyrate, BHB, and acetoacetate differ only in the oxidation state of the 3′ carbon. Increasing oxidation may be a barrier to binding within the hydrophobic channel of the HDAC active site, and as expected, the IC50 of the three compounds for HDAC1 increases with the oxidation state (115, 118).

β-Hydroxybutyrylation of Proteins 

In addition to inhibiting enzymes involved in the regulation of protein posttranslational modifications such as HDACs, BHB can itself modify proteins at the posttranslational level. Lysine β-hydroxybutyrylation [K(BHB)] was detected via mass spectrometry as a histone modification in yeast, fly, mouse, and human cells (147). Western blot analysis detected that histone K(BHB) increases in human cells in proportion to treatment with exogenous BHB, and increases in mouse liver in proportion to increases in plasma BHB levels with either fasting of normal mice or induction of diabetic ketoacidosis with streptozotocin. At least 40 BHB-modified histone lysine sites were detected in human cells or mouse liver by mass spectrometry, including sites critical for transcriptional regulation such as H3K9. Fasting increased the relative abundance of K(BHB) at these sites by up to 40-fold (147).

Chromatin immunoprecipitation (ChIP) with specific antibodies also revealed that K(BHB) of H3K9 is preferentially associated with the promoters of actively transcribed genes (147). A comparison of ChIP data and RNAseq expression data from fasted mouse liver found a strong correlation between genes with the greatest increase in expression and genes with the greatest increase in K(BHB) at the promoter.

H3K9 K(BHB) is correlated with other activation markers, such as acetylated H3K9 and trimethylated H3K4, but has an association with gene activation independent of these other two markers, suggesting an independent function (147). What that function is remains to be determined. The scope of β-hydroxybutyrylation as a posttranslational modification is not clear: It is not known whether nonhistone proteins are modified, or whether K(BHB) exists in organs other than the liver.

It is also not known whether any enzymes catalyze the addition or removal of K(BHB), providing opportunities for specific targeting and regulation. The wide diversity of lysine acylations removed by the various mammalian sirtuins (1) hints that hydroxybutyrylation might be removed by a specific sirtuin. In all, histone K(BHB) may be the nexus of an important new network of gene regulatory activity associated with fasting and other conditions linked to increase BHB production.

Cell Surface Receptors: HCAR2 and FFAR3 

BHB binds to at least two cell surface G-protein-coupled receptors (GPCRs), HCAR2 (hydroxycarboxylic acid receptor 2) and FFAR3 (free fatty acid receptor 3). These are among several GPCRs with fatty acid ligands that have important roles in metabolism and metabolic disease (9, 70). HCAR2 (also known as HCA2, PUMA-G, and Gpr109) is a Gi/o-coupled GPCR that was first identified as a nicotinic acid receptor (129). It was later shown to bind and be activated by BHB, with an EC50 of 0.7 mM (124).

HCAR2 activation reduces lipolysis in adipocytes (95, 124), which might perhaps represent a feedback mechanism to regulate the availability of the fatty acid precursors of ketone body metabolism. However, elevated levels of free fatty acids in plasma from dysregulated adipocytes are also thought to contribute to insulin resistance through a variety of mechanisms, including proinflammatory cytokines, oxidative stress, and endoplasmic reticulum stress (10).

Pharmacological agonists of HCAR2 reduce both plasma free fatty acids and plasma glucose levels in humans with type 2 diabetes (21). Infusion of BHB has long been observed to reduce the concentrations of nonesterified fatty acids in plasma, even when insulin levels (which also regulate fatty acid release from adipocytes) are held constant (108). In retrospect, this reduction of free fatty acids in plasma may be due to HCAR2 activation.

HCAR2 is also expressed in a variety of other cell types, including immune cells, microglia, and colonic epithelial cells, in which its activation induces anti-inflammatory effects (38). In the central nervous system, this is associated with neuroprotection mediated in part by the activation of IP3dependent intracellular calcium release, alterations in prostaglandin production, and downstream inhibition of NF-κB activation (reviewed in Reference 96).

More specifically, HCAR activates a neuroprotective subset of macrophages via the production of PGD2 by COX1 (103). The neuroprotective effect of ketogenic diet in a mouse stroke model is abrogated in Hcar2 knockout mice, and it appears that activation of HCAR2 on brain-infiltrating macrophages/monocytes is crucial to this neuroprotection (103). Finally, HCAR2 activation in neurons can potentiate glutaminergic signaling, which in one particular region of the brain helps regulate blood pressure and heart rate (106).

Activation of HCAR2 in the gut epithelium by short-chain fatty acids produced by bacterial fermentation of dietary fiber activates the NLRP3 inflammasome and maintains gut membrane integrity (79). The mechanism for inflammasome activation appears to be potassium efflux stimulated by intracellular calcium release. Inflammasome activation is beneficial in mouse models of colitis, and appears to explain the benefit of a high-fiber diet in these conditions, but stands in interesting contrast to the role of BHB in suppressing NLRP3 inflammasome activation described below.

BHB is also a ligand for FFAR3 (also known as GPR41). FFAR3 is another Gi/o-proteincoupled receptor that is highly expressed in sympathetic ganglions (62) throughout the body of mice (94). Ffar3 knockout mice have reduced basal oxygen consumption and body temperature but are then insensitive to the usual further sympathetic depression seen during prolonged fasting (62).

Antagonism of FFAR3 by BHB suppresses sympathetic tone and heart rate and may be responsible for the sympathetic depression commonly seen during fasting (62). However, an electrophysiological study in rats later reported that BHB in fact acts as an agonist of FFAR3 (145), which regulates voltage-dependent calcium channels. In fact, other endogenous short-chain fatty acids, including acetate, butyrate, and propionate, have been reported as agonists of FFAR3 (11). Further work is needed to confirm the activity of BHB on FFAR3, but a substantial literature is emerging on other biological functions of FFAR3 relevant to human health.

One such function is glucose homeostasis. Deletion of Ffar3 (together with Ffar2) in mouse pancreatic β cells improves insulin secretion and glycemic control on a high-fat diet (125). Genetic gainand loss-of-function neurotransmitter models of Ffar3 alone similarly show that lower FFAR3 signaling increases insulin secretion from
pancreatic β islet cells (132). An alternative route by which FFAR3 affects glucose metabolism
is via activation by gut-derived propionate in the periportal afferent neural system. This feeds

into a brain-gut signaling circuit that induces intestinal gluconeogenesis, with beneficial effects on glucose and energy homeostasis (17).

FFAR3 also regulates inflammation. Activation of FFAR3 by gut-derived propionate helps rescue allergic airway inflammation in mice by reducing the capacity of lung-resident dendritic cells to promote TH2 -mediated inflammation (128). Similarly, activation of FFAR2 and FFAR3 by acetate suppresses the expression of inflammatory cytokines in human monocytes, which suggests FFAR2/3 agonists may help ameliorate inflammatory bowel diseases (2). As these examples illustrate, whether activation or inhibition of FFAR3 is beneficial to health may be highly dependent on the specific tissue and disease contexts.

Membrane Channel and Transporter Regulation 

Two threads of indirect evidence imply a role for BHB in the modulation of potassium flux across the plasma membrane. Inhibition of potassium efflux is the suggested mechanism by which BHB inhibits activation of the NLRP3 inflammasome (153). An extensive series of careful in vitro experiments excluded other known direct and indirect signaling functions of BHB, including HDACs, HCAR2, and metabolism to acetyl-CoA. The event most upstream of inflammasome activation that is affected by BHB treatment is prevention of the decline in intracellular potassium in response to inflammasome-activating signals (153). This effect is the reverse of HCAR2 activation stimulating potassium efflux.

Application of acetoacetate or R-BHB to brain slices reduces the rate of firing from γ-aminobutyric acid (GABA)ergic neurons, and pharmacological inhibition of ATP-sensitive K (KATP) channels or knockout of the Kir6.2 subunit abrogates the effect (78). Although this might suggest that BHB could increase K channel opening, several lines of evidence indicate that the change in KATP activity is more likely an indirect effect of the absence of glucose on local intracellular ATP concentrations.

The nonmetabolized enantiomer S-BHB did not have a similar effect on neuron firing (78); a later study showed that the change in KATP channel opening is due to reduced glycolysis in the presence of BHB (76) and that providing sufficient BHB and oxygen to maintain normal cellular ATP production can prevent the change in KATP activity (77).

BHB appears to have a direct regulatory effect on the neuronal vesicular glutamate transporter VGLUT2 (56). Cl− is an allosteric activator of glutamate uptake in an in vitro system consisting of proteoliposomes containing purified VGLUT2. Acetoacetate, BHB, and pyruvate inhibit this Cl−-dependent glutamate uptake, with kinetics indicating competition for the Cl− binding site. All these effects require the anions to be exposed to the extravesicular side of the membrane transporter.

Acetoacetate is >10-fold more potent than BHB at inhibiting VGLUT2, but the IC50 of 3.75 mM for BHB still suggests that biologically relevant effects on glutamate uptake might occur at physiological low-millimolar BHB concentrations. Cl−-dependent activation of other VGLUT transporters were similarly inhibited by acetoacetate, but the vesicular GABA transporter VGAT was not.

Consistent with these biochemical findings, acetoacetate inhibits glutamate release from neurons in a manner suggesting a reduced vesicular quantity of glutamate (56). In other contexts, however, BHB enhances glutaminergic transmission by increasing neurotransmitter release (120). Nevertheless, VGLUT inhibition may be a mechanism by which BHB can reduce excitatory glutamate neurotransmission without affecting inhibitory GABA neurotransmission.

Enantiomeric Specificity of Direct Signaling Activities 

As noted above, BHB is a chiral molecule, and R-BHB is the enantiomer generated and readily consumed in normal mammalian metabolism. Signaling functions or other effects that depend on the rapid catabolism of BHB therefore are relevant only to the R-enantiomer. Signaling functions that are direct actions of BHB, however, might be recapitulated in part or in full by S-BHB depending on the stereoselectivity of the proteins involved. Indeed, several of the direct signaling functions described here have been reported to be nonstereoselective in the literature.

S-BHB can bind the GPCR HCAR2, albeit with somewhat lower affinity than R-BHB. An in vitro assay with modified Chinese hamster ovary cells expressing human HCAR2, using calcium flux as the readout, found that both R-BHB and S-BHB showed robust, similar receptor activation at concentrations of 15 mM. A quantitative binding assay using radiolabeled GTP to measure nucleotide exchange found that the EC50 was 0.7 mM for R-BHB and 1.6 mM for S-BHB with human HCAR2, and 0.3 mM and 0.7 mM, respectively, with mouse HCAR2. Acetoacetate was inactive (124). The chirality of BHB used to test activation of FFAR was not reported (62).

S-BHB can also block inflammasome activation (153). The use of caspase-1 activation in lipopolysaccharide-treated bone marrow–derived macrophage cells as an in vitro assay of NLRP3 inflammasome activation showed that S-BHB effectively blocks inflammasome activation, albeit perhaps with lower efficacy than R-BHB.

The stereoselectivity of HDAC inhibition by BHB remains to be explored, but if, as described above, the relevant biochemical action is chelation of a catalytic zinc at the base of a hydrophobic channel, then the chirality of the trailing 3′ hydroxyl group may not be a critical determinant of activity. There is at least one suggestion that both R-BHB and S-BHB can increase histone acetylation in vitro (154).

The chirality of histone K(BHB) may be important to its biological function. The method used to detect K(BHB) could not distinguish modification with R-BHB from modification with S-BHB (147), but it is likely that both K(S-BHB) and K(R-BHB) are present. First, K(BHB) was detected in yeast and flies, and although generation of S-BHB-CoA as an intermediate of lipid β-oxidation is a conserved pathway in these metazoans, mitochondrial ketogenesis of R-BHB is not. Nor is Saccharomyces cerevisiae known to use polymers of R-BHB as an energy store, as is common in prokaryotes (18).

Second, although fasting increases synthesis of R-BHB in mouse liver, it also increases flux of S-BHB-CoA as an intermediary of fatty acid oxidation. A number of factors probably influence whether K(R-BHB) or K(S-BHB) predominates. By analogy with other protein acylations, K(BHB) is likely formed from CoA-activated BHB. β-Oxidation produces SBHB-CoA from an acyl-CoA precursor.

Free R-BHB (as well as free S-BHB) must undergo enzymatic activation by a CoA synthase, which has long been observed to occur (108), although the precise CoA synthase involved and cellular compartment in which the activation occurs are unknown. The balance of K(R-BHB) versus K(S-BHB) in the liver might therefore depend on the rate and site of activation of R-BHB and on the efficiency of extramitochondrial transport of both activated forms.

The balance in extrahepatic tissue might also depend on the relative utilization of fatty acids versus BHB for energy. Neurons, for example, which do not utilize fatty acids (7), would likely favor K(R-BHB) under ketogenic conditions. Whether the chirality of K(BHB) matters depends on its biological function, which is not yet understood. If the function is simply to occupy the lysine site and prevent alternative modifications, the chirality may be irrelevant. If the function is to serve as a binding site for other proteins, the 3′ hydroxyl group of BHB should remain both chiral and accessible to influence the binding of any putative K(BHB) recognition proteins.


In addition to its direct signaling activities, BHB might exert additional signaling effects in the course of its catabolism to acetyl-CoA and ATP. These reflect the signaling activities of other intermediate metabolites, including acetyl-CoA, succinyl-CoA, and NAD. Finally, catabolism of BHB can alter the steady state of downstream metabolic pathways, such as those that regulate neurotransmitter synthesis. When considering the possible medical applications of BHB precursors, note that R-BHB should be much more potent at eliciting these indirect signaling actions than S-BHB is, as S-BHB is catabolized much more slowly and through a different route.

Production of Acetyl-CoA, Substrate for Protein Acetylation 

Catabolism of BHB into acetyl-CoA should raise intracellular acetyl-CoA levels, favoring both enzymatic and nonenzymatic protein acetylation. This effect complements HDAC inhibition by BHB but may have broader effects in multiple cellular compartments. Protein acetylation in mitochondria appears to be particularly sensitive to acetyl-CoA flux, as a variety of states associated with increased lipid utilization—including dietary restriction, fasting, and high-fat diets—increase mitochondrial protein acetylation. This effect on mitochondrial acetylation occurs despite the fact that neither acetyltransferases nor the HDACs that are inhibited by BHB enter mitochondria (48). Acetylation—and deacetylation by SIRT3—is widespread in mitochondria (105) and regulates the function of several mitochondrial enzymes, including HMGCS2 (117) and the long-chain acylCoA dehydrogenase (51).

Increased acetyl-CoA pools also affect nuclear protein acetylation. Mitochondrial acetylcarnitine is a source of acetyl-CoA for histone acetylation (80). Export of acetyl-CoA from the mitochondria is accomplished via a citrate shuttle mediated by citrate synthase inside mitochondria and ATP citrate lyase inside the cytoplasm (140).

ATP citrate lyase is a key enzyme in fatty acid biosynthesis, but its role in facilitating acetyl-CoA export from mitochondria is also required for the increase in histone acetylation that occurs with growth factor stimulation (140). An alternative pathway for acetyl-CoA export from mitochondria is via the enzymes carnitine acetyltransferase and carnitine/acylcarnitine translocase (89). Indeed, a muscle-specific knockout of carnitine acetyltransferase in mice compromises glucose tolerance and decreases metabolic flexibility (89), demonstrating the importance of intracellular acetyl-CoA transport to overall metabolic health.

Consumption of Succinyl-CoA, Substrate for Protein Succinylation 

Utilization of BHB in peripheral tissues uses succinyl-CoA to donate CoA to acetoacetate. This consumption of succinyl CoA may affect the balance of lysine succinylation, which, like acetylation, is widespread in mitochondria (104) and present across diverse organisms (139). A substantial fraction of these succinylation sites are regulated by the mitochondrial desuccinylase, the sirtuin SIRT5 (104). HMGCS2 has long been known to be succinylated, a modification that reduces its activity (102); HMGCS2 enzymatic activity in the liver is suppressed by succinylation and restored by desuccinylation mediated by SIRT5 (104). The mechanism of lysine succinylation is not clearly understood; succinyltransferase is not known to exist in mammalian cells, and because both liver succinyl-CoA abundance and succinylation of HMGCS2 are reduced in rats after treatment with glucagon (101, 102), it is possible that succinylation is primarily a nonenzymatic process dependent on local concentrations of succinyl-CoA. Although HMGCS2 itself is not expressed in the peripheral tissues that utilize BHB, enzymes in many other key mitochondrial pathways, including fatty acid oxidation, branched-chain amino acid catabolism, and the tricarboxylic acid (TCA) cycle, are heavily succinylated and regulated by SIRT5 (104). By analogy with acetylation (and the effect of succinylation on HMGCS2), these pathways may be activated by a reduction in succinylation. Consumption of succinyl-CoA during BHB utilization and consequent reduction in mitochondrial protein succinylation may therefore regulate many of these crucial mitochondrial pathways in peripheral tissues, perhaps favoring the switch to lipid-dependent energy usage.

Cytoplasmic and Mitochondrial NAD:NADH Equilibrium 

Cellular NAD balance is emerging as a crucial factor in metabolic disease and aging (123). NAD utilization during BHB metabolism differs from that during glucose metabolism in two important respects. Fewer NAD+ molecules are consumed per acetyl-CoA produced when BHB is used than when glucose is used, and the cellular compartment in which NAD+ is consumed is different. Metabolism of one molecule of glucose to two molecules of acetyl-CoA involves conversion of four molecules of NAD+ into NADH. Two of these molecules are converted in the cytosol during glycolysis; the other two are converted in the mitochondrion by pyruvate decarboxylase. The cytosolic NADH are shuttled into mitochondria, potentially depleting the cytoplasmic NAD+ pool with high glucose utilization.

By contrast, metabolism of one BHB molecule to the same two molecules of acetyl-CoA involves conversion of only two molecules of NAD+ into NADH, both in the mitochondrion by BDH1, thereby preserving the cytoplasmic NAD+ pool (7). The cytoplasmic and mitochondrial NAD pools are relatively distinct, so the preservation of cytoplasmic NAD+ by BHB may have important cellular effects. NAD+ is a cofactor for sirtuin deacylases (such as nuclear/cytoplasmic SIRT1) as well as poly-ADP-ribose polymerase (PARP) (133).

Consumption of NAD+ by PARP or overproduction of NADH may promote age-related diseases by decreasing the activity of sirtuins (53). Conversely, repletion of NAD+ by exogenous feeding with nicotinamide mononucleotide improves glucose tolerance in both high-fat-diet-fed and aged mice (152). The relative sparing of NAD+ by utilizing BHB vis a` vis glucose may therefore have important consequences for metabolic diseases and diabetes.

In the mitochondrion, BHB utilization is both determined by and changes the NAD:NADH equilibrium, with implications for signaling. The mitochondrial BHB:AcAc equilibrium is so closely linked to the NAD:NADH equilibrium that early metabolic studies of the liver used the former as a proxy for the latter (65). This tight relationship suggests that any influence that pushes the mitochondrial NAD:NADH equilibrium toward NADH (such as consumption of NAD by sirtuins or reduced activity of NADH dehydrogenase/complex I) will reduce BHB consumption. This reduction could increase the local concentration of BHB available for direct signaling functions within and around the mitochondrion, but also potentially reduce the flux of BHB transiting the cytoplasm and nucleus toward mitochondria.

However, BHB utilization also affects the mitochondrial redox state. Increased utilization of BHB is associated with pushing the NAD:NADH equilibrium toward NADH, as well as increasing the oxidation state of coenzyme Q (114). Owing to the increased heat of combustion of BHB compared with that of pyruvate, BHB also increases the efficiency of ATP production from the mitochondrial proton gradient and reduces the production of free radicals (131). Changes in free radical production might alter the activity of signaling networks that sense and respond to mitochondrial free radicals, such as p66Shc (33).

Neurotransmitter Synthesis 

The potential mechanisms of action of ketogenic diets in treating epilepsy remain complex and controversial and have been the subject of several thorough reviews (47, 83, 154). One mechanism consistently proposed, however, involves how the downstream effects of BHB catabolism on the abundance or flux of other intermediate metabolites might alter the biosynthesis of the inhibitory neurotransmitter GABA.

The biosynthesis of GABA in inhibitory GABAergic neurons begins with the synthesis of glutamine in astrocytes. Glutamine is exported from astrocytes to neurons, where it undergoes conversion to glutamate and then decarboxylation to GABA. An alternative fate for glutamate in neurons is donation of its amino moiety to oxaloacetate, producing aspartate and α-ketoglutarate. Studies of isotopically labeled BHB show that it is used as a substrate for the synthesis of glutamine and other amino acids (156). Data from clinical studies of children on ketogenic diet for epilepsy show that cerebrospinal fluid GABA levels are higher on ketogenic diet, and the highest levels correlate with best seizure control (16).

BHB may affect GABA production via increased synthesis and/or pushing the fate of glutamate toward GABA and away from aspartate. Studies in synaptosomes show that BHB increases the content of glutamate and decreases that of aspartate (25). Studies of ketogenic diet in rodents similarly show that less glutamate is converted to aspartate (156). In cultured astrocytes, the presence of acetoacetate reduces the conversion of labeled glutamate to aspartate (155). Infusion of labeled BHB into rats fed a normal diet rapidly increases the levels of all components of this pathway (glutamine, glutamate, GABA, and aspartate) (112).

Even when ketotic states are not associated with an increase in overall GABA levels, the proportion of glutamate shunted to GABA production is increased (84). The reason for this shunt may be the effect of BHB catabolism on TCA cycle intermediates. The relatively greater efficiency of BHB at generating acetyl-CoA compared with that of glucose, described above, increases the flux of acetyl-CoA through the TCA cycle. This increases the proportion of oxaloacetate required to condense with acetyl-CoA to permit its entry into the TCA cycle, reducing the availability of free oxaloacetate to participate in glutamate deamination to aspartate. By indirectly tying up oxaloacetate, BHB pushes the fate of glutamate toward GABA (154). Altogether, one effect of BHB catabolism, alone or as part of a ketogenic diet, appears to increase the capacity of GABAergic neurons to rapidly generate GABA from glutamate (154).

Although BHB is structurally reminiscent of GABA itself, evidence of a direct effect of BHB on activating GABA receptors is lacking. Neither BHB nor acetoacetate alters GABA currents in cultured rodent cortical neurons (22) or in rat hippocampal neurons (126). BHB did enhance the function of GABAA receptors expressed in Xenopus oocytes, but only modestly and at concentrations of 10 mM or higher (149), leaving the physiological significance of this effect unclear.

However, GABOB (γ-amino-β-hydroxybutyric acid) is an endogenous agonist of GABA receptors that differs structurally from BHB only in the presence of the γ-amino moiety. It is biochemically plausible that BHB might be a direct substrate for GABOB synthesis, but no such aminotransferase is known to exist. Nor has any pathway for conversion of GABA to GABOB yet been identified in mammals. Of interest in consideration of BHB precursors as therapeutics, the enantiomer of GABOB that would be derived from S-BHB has the more potent antiepileptic affect and is a stronger GABAB receptor agonist (148).


The integration of the various direct and indirect signaling functions of BHB appears to broadly help the organism adapt to a fasting state. The transition to a fasting state is already under way when ketogenesis is activated in the liver. BHB production might further promote that transition in extrahepatic tissues while also fine-tuning the control of lipid and glucose metabolism.

The combinatorial effects of BHB on gene expression, described below, might be expected to generally facilitate the activation of new transcriptional programs. The enrichment of histone K(BHB) at genes activated by fasting suggests that this might be particularly important for activating fasting-related gene networks, although this could also reflect a nonspecific association with activated transcription.

Inhibition of class I HDACs by BHB could play a major role in metabolic reprogramming, according to studies of HDAC knockout mice and of HDAC inhibitors. HDAC3 regulates expression of gluconeogenic genes (86), and HDAC3 knockout mice have reduced fasting glucose and insulin levels (8, 26, 63). In fact, chronic treatment with the HDAC inhibitor butyrate essentially keeps mice metabolically normal on a high-fat diet, with lower glucose and insulin levels, better glucose tolerance, reduced weight gain, and improved respiratory efficiency (32). Butyrate also provides some of these benefits even to mice already obese from being fed a high-fat diet (32).

Similarly, inhibition of class I HDACs, but not class II HDACs, increases mitochondrial biogenesis, improves insulin sensitivity, and increases metabolic rate and oxidative metabolism in a mouse diabetes model (31). The mechanism for these metabolic benefits of class I HDAC inhibition may be upregulation of PGC1α (Ppargc1a) in in a variety of tissues by relief of HDAC3-mediated transcriptional repression (31, 32). Transcription of Fgf21 is similarly upregulated via inhibition of HDAC3 by butyrate, activating ketogenesis in obese mice (72). Several single nucleotide polymorphisms in HDAC3 have been associated with an elevated risk of type 2 diabetes in a Chinese population (157).

Activation of HCAR2 reduces lipolysis in adipocytes; because the availability of fatty acids in the liver is a critical determinant of ketogenesis (and is strongly regulated by insulin), this may provide a self-feedback mechanism to limit the production of BHB. Why this mechanism is insufficient to prevent dysregulated BHB production in insulin-deficient states such as diabetic ketoacidosis, and whether it could be potentiated to treat such states, such as through HCAR2 agonists, is unclear.

HCAR2 was originally identified as a niacin receptor, spurring efforts to develop more specific agonists to capture the therapeutic benefits of niacin on cardiovascular risk or glycemic control that were thought to be due to HCAR2’s effects on the levels of free fatty acid in blood. An HCAR2 agonist, GSK256073, transiently reduces levels of free fatty acids in blood but has only a modest effect on glycemic control in type 2 diabetes mellitus (20). Two other HCAR2 agonists similarly lowered free fatty acids but without otherwise altering the lipid profile in humans, while niacin was found to produce beneficial changes to the lipid profile in Hcar2 knockout mice (69).

Thus, the model that niacin (and by extension BHB) exerts beneficial effects on cardiovascular risk through activation of HCAR2 in adipose tissue is probably too simplistic. It may even be the case that other cell types such as macrophages might mediate the therapeutic effects of HCAR2 agonists (30).

Interpreting the metabolic effects of BHB mediated by FFAR3 depends on whether BHB acts as an agonist or antagonist, and in which contexts. Strong evidence suggests that BHB antagonizes FFAR3 to reduce sympathetic activity, resulting in reduced heart rate, body temperature, and metabolic rate. If BHB also antagonizes FFAR3 in other contexts, it could improve insulin secretion from pancreatic β islet cells and impair intestinal gluconeogenesis. Altogether, these findings suggest that BHB would improve glycemic control, though a decrease in metabolic rate could be obesogenic.

The important role of the NLRP3 inflammasome in regulating obesity-associated inflammation and metabolic dysfunction has been extensively reviewed (15, 45). Briefly, the NLRP3 inflammasome appears to mediate an inflammatory response to nutrient excess and mitochondrial dysfunction. Mice deficient in NLRP3 are grossly normal when fed chow but are protected from obesity and insulin resistance when fed a high-fat diet. One proposed mechanism is through a reduction in inflammasome-induced IL-1β, which otherwise inhibits insulin signaling in adipocytes and hepatocytes while inducing pancreatic β-cell dysfunction.

While NLRP3 may have an important homeostatic role in the response to day-to-day nutrient fluctuations, its chronic activation by nutrient excess may contribute to the development of metabolic disease—and inhibition of the NLRP3 inflammasome by BHB might ameliorate these maladaptations.


Regulation of gene expression is the most common theme that emerges from the direct and indirect signaling functions of BHB. Histones are at the nexus of this theme, with their posttranslational modifications, including acetylation, succinylation, and β-hydroxybutyrylation. Histone acetylation is a well-understood mechanism for both broad and specific regulation of gene expression, and BHB can alter histone acetylation by directly inhibiting HDACs and by indirectly promoting acetyltransferase activity via acetyl-CoA flux.

The biology of histone β-hydroxybutyrylation largely remains to be elucidated but is an area that should receive strong interest for its potential relevance to gene expression reprogramming in response to metabolic stimuli. Lysine succinylation is another histone posttranslational modification (146) and is regulated by the sirtuin SIRT7 in the context of the DNA damage response (73). Finally, alterations in cytoplasmic and nuclear NAD+ levels can affect the activity of sirtuins, which deacylate a number of histone tail residues (54).

The net integration of all these effects might be expected to facilitate gene transactivation, particularly of quiescent genes that are activated in response to stimuli. Two well-characterized examples of how HDAC inhibition (and acetyltransferase activation) can activate gene expression include reactivation of HIV latency and activation of lineage-specific genes during muscle differentiation. The HIV provirus sits in a transcriptionally inactive state in resting T cells, providing a reservoir of potential virus production that is impossible for current antiretroviral therapy to eradicate (44).

The class I HDACs HDAC1, HDAC2, and HDAC3 help maintain this transcriptionally silenced state, as components of several transcriptional repressor complexes, by deacetylating histone tails. Ordinarily, a key step in the reactivation of HIV transcription is the recruitment of complexes containing the acetyltransferase p300 to acetylate histone tails. Inhibition of HDACs with vorinostat or panobinostat also promotes histone acetylation, resulting in the loss of latency and reactivation of HIV transcription (44). In vivo, such HDAC inhibitors broadly reactivate transcription from a diverse pool of latent proviruses (6).

The differentiation of a satellite cell (i.e., muscle stem cell) into the muscle lineage involves the transcriptional activation of a sequence of differentiation-promoting transcription factors (see Reference 119 for a review). One of these factors, MYOD1, further activates a wide range of muscle-specific genes. HDACs, including HDAC1 and HDAC2, reside at the promoters of many of these MYOD1 targets, maintaining histones in a deacetylated state.

Activation of the genes requires dissociation of the HDAC from the promoter, and often recruitment of a p300 complex to promote histone acetylation. HDAC inhibitors potentiate this process, much as in HIV latency, and both increase acetylation at MYOD1 target promoters and increase myogenic differentiation. One contrast with HIV latency is that many gene promoters in satellites cells are maintained in a poised or bivalent state, possessing both activating and repressing histone modifications (119).

Thus, whereas HIV latency is an example of HDAC inhibition waking a gene from deep silencing, muscle differentiation is an example of potentiating or lowering the threshold of activation for a gene already poised to do so. A similar example of activating poised genes may be relevant to cognition and dementia (see below).

How histone K(BHB) participates in epigenetic regulation largely remains to be determined. K(BHB) was particularly enriched after fasting at genes that were upregulated by fasting (147). Other stimuli or contexts expected to change gene expression were not examined, nor were cells or animals treated with BHB in the context of such stimuli.

Thus, we do not yet know whether K(BHB) is specifically associated with a gene network that is responsive to fasting, or whether it is generally associated with newly transactivated promoters. We also have yet to understand the biological meaning of K(BHB)—whether it is an active signal that stimulates transcription through regulatory or binding interactions with the protein machinery of chromatin remodeling and transactivation, or whether it is a bystander modification emplaced at fasting-activated promoters owing to the confluence of increased BHB-CoA levels and newly accessible histone tails.

An additional layer of regulation can occur on nonhistone proteins involved in gene expression, as these posttranslational modifications are not restricted to histones. Acetylation and succinylation occur throughout the proteome and can affect protein function. It is possible that β-hydroxybutyrylation will be found to as well. Acetylation of nonhistone proteins is a critical step in both HIV latency and muscle differentiation, where acetylation activates the transcription factors Tat (44) and MYOD1 (119), respectively. The multilayered effects of BHB on gene expression can be illustrated with HMGCS2 itself, the rate-limiting enzyme in BHB synthesis. As described above, the transcription factor FOXA2 helps control Hmgcs2 transcription.

FOXA2 is itself acetylated by EP300 in a reaction using acetyl-CoA as the acetyl group donor (increased by BHB). FOXA2 is deacetylated by a class I HDAC (inhibited by BHB) and/or by the sirtuin SIRT1, which requires NAD+ as a cofactor (increased by BHB). Initiation of Hmgcs2 transcription by activated FOXA2 might involve displacing or deactivating HDACs (inhibited by BHB) to permit emplacement of activating histone marks such as H3K9 acetylation by acetyltransferases (via acetyl-CoA increased by BHB). As this example illustrates, the overall impact of BHB might generally favor initiation or upregulation of transcription, but the effects on any specific loci could vary depending on the posttranslational regulation of the proteins involved at that site.


The ketogenic diet has been clinically used for decades to treat epilepsy and currently has a wide range of therapeutic applications, mostly in childhood epilepsies (142). Despite this extensive clinical history, the mechanism of action of the ketogenic diet remains controversial (107). In fact, whether BHB itself is necessary or even active in the therapeutic effect of ketogenic diets is controversial, and the evidence varies between animal models (107). The various possible mechanisms of the antiepileptic effect of ketogenic diets have been reviewed (83, 107).

Several of the signaling activities described above may be relevant, particularly modulation of potassium channels, FFAR3 activation, and promotion of GABA synthesis. Epigenetic modifications may also contribute to neuronal hyperexcitability and the long-term effects of epilepsy on the brain through persistent changes in gene expression (111). REST (RE1-silencing transcription factor) is a transcriptional repressor that recruits HDACs, among other chromatin-modifying enzymes, to help silence target genes.

REST expression is increased in neurons after seizures and promotes aberrant neurogenesis. However, whether REST activity is helpful or harmful for seizure control differs in different seizure models (111). The contribution of epigenetic modifiers, including those that are modulated by BHB, to epilepsies and their long-term effects requires much further study.

Ironically, given the decades of study on the role of ketogenic diets in epilepsy, more molecular detail is known about the potential mechanisms of BHB in ameliorating dementia. Two major threads link BHB signaling with dementia: epigenetic modifications and neuronal hyperexcitability. There is a growing literature on the importance of epigenetic regulation in learning and memory, specifically in mouse models of dementia. Age-related impairments in learning and memory in wild-type mice are associated with alterations in histone acetylation (98), and treatment with HDAC inhibitors improves memory performance in both young and aged mice (42, 98).

HDAC inhibitors also improve cognition in the CK-p25 dementia mouse model (28). HDAC2 appears to be the crucial mediator of these effects, as overexpression of HDAC2, but not HDAC1, impairs learning and memory in wild-type mice (42). Conversely, Hdac2 knockout mice show improved memory formation, which is not further improved by HDAC inhibitors (42). HDAC2 expression is increased in the brains of two mouse dementia models as well as the brains of humans with Alzheimer’s disease (39). One model of how HDAC inhibitors regulate cognition is via epigenetic priming, reminiscent of the poised transcriptional state of genes involved in muscle differentiation (40). The broader role of epigenetics in cognition and neurodegenerative disease has been reviewed (67, 68, 100).

The worlds of epilepsy and dementia have been linked through the finding that mouse models of Alzheimer’s disease show neuronal hyperexcitability and epileptiform spikes from dysfunctional inhibitory interneurons (97, 134). Epilepsy, an extreme manifestation of this hyperexcitability, is associated with more rapid cognitive decline in patients with Alzheimer’s disease (136). Promising treatments that reduce epileptiform spikes, including at least one commonly used antiepileptic drug, improve cognition in these models (113, 134).

The various signaling activities by which BHB acts in epilepsy may thus be relevant to ameliorating cognitive decline in Alzheimer’s disease. In small studies, provision of BHB precursor molecules improves cognition in an Alzheimer’s mouse model (58) and in a patient with Alzheimer’s disease (93). Further exploration of the links between BHB signaling, epilepsy, and dementia may prove fruitful in generating new translational therapies.

Inhibition of the NLRP3 inflammasome could also prevent cognitive decline and dementia. β-amyloid protein, which aggregates into the amyloid plaques characteristic of Alzheimer’s disease, activates the NLRP3 inflammasome in microglia, the resident macrophage population in the brain, releasing inflammatory cytokines including IL-1β (reviewed in References 29 and 35).

This activation is evident in the brain of humans with both mild cognitive impairment and Alzheimer’s disease, and Alzheimer’s mouse models that carry deficiencies in NLRP3 inflammasome components are protected from β-amyloid deposition and cognitive decline (50). Microglia, as critical mediators of brain inflammation, may be the site of integration of various BHB-related signals, including HCAR2 activation.


The hypothesis that BHB may play a broad role in regulating longevity and the effects of aging comes in part from the observation that many of the interventions that most consistently extend longevity across a wide range of organisms, such as dietary restriction and fasting, intrinsically involve ketogenesis and the production of BHB in mammals (92).

The effects of such regimens on invertebrate, rodent, and human health have been reviewed and can include extended longevity, cognitive protections, reductions in cancer, and immune rejuvenation (75, 82). More specific interventions that promote ketogenesis, such as transgenic overexpression of FGF21, also extend life span in rodents (161). BHB itself extends longevity in C. elegans (24), and whether it would do so in rodents remains to be investigated.

Several of the signaling functions of BHB described above broadly regulate longevity and diseases of aging pathways, most prominently HDAC inhibition and inflammasome inhibition. The data from invertebrate organisms showing that reduction in class I HDAC activity extends life span, and generally acts through similar pathways as dietary restriction, have been reviewed (92). Briefly, deletion of Rpd3, the yeast and fly homolog of mammalian class I HDACs, extends replicative life span by 40–50% in S. cerevisiae (61).

Rpd3 deletion enhances ribosomal DNA silencing (61), the same mechanism by which overexpression of the sirtuin Sir2 enhances replicative longevity in S. cerevisiae (57). Drosophilids heterozygous for a null or hypomorphic Rpd3 allele show a 30–40% extension of life span, with no further increase with caloric restriction (110). Both caloric restriction and reduced Rpd3 activity increase expression of Sir2 (110). Conversely, mutations in Sir2 block life span extension by either caloric restriction or Rpd3 mutations (109). In both organisms, then, modest reductions in HDAC activity (stronger reductions are lethal) extend life span via the same mechanisms as in dietary restriction and Sir2 expression.

Other possible longevity mechanisms downstream of Rpd3 in invertebrates include autophagy, which is regulated by histone acetylation of specific genes (151), and enhanced proteostasis through increased chaperone expression (162).

No life span data yet exist for reduced HDAC function in rodents. However, Hdac2 knockout mice display impaired IGF-1 signaling and are 25% smaller than normal (163), a potential longevity phenotype (87). Hdac2 knockout is also protective in models of tumorigenesis (163). Conditional knockouts in mouse embryonic fibroblasts and embryonic stem cells demonstrated roles for HDAC1 and HDAC2 in hematopoiesis (141) and stem cell differentiation (23). By analogy to the modest reductions in class I HDACs that enhance longevity in invertebrates, it may be of interest to determine whether heterozygous HDAC1/2 knockout mice, or mice treated with low-dose pharmacological HDAC inhibitors, have enhanced longevity.

An inducible compound heterozygote knockout of HDAC1 and HDAC2 does suppress one translatable age-related phenotype, cardiac hypertrophy, as do HDAC inhibitors (88). HDAC inhibitors ameliorate cardiac dysfunction in mouse diabetes models (14) and prevent maladaptive cardiac remodeling (160). The mechanism for the effect on cardiac hypertrophy appears to be inhibition of HDACs that suppress the activity of a mechanistic mTOR complex (88). This is one of several examples of intersections between BHB, its signaling effects, and mTOR/rapamycin, a canonical longevity-regulating pathway (55). As described above, mTOR is also a checkpoint in the activation of ketogenesis; inhibition of mTORC1 is required to activate the transcription factors and hormones that control ketogenesis (4, 116).

Inhibition of NLRP3 inflammasome activation might also have broad effects on aging and longevity, as reviewed in References 27 and 37. The NLRP3 inflammasome in particular has a wide range of activating stimuli, many of which accumulate with age such as urate, amyloid, cholesterol crystals, and excess glucose. The age-related phenotypes that may be ameliorated by its inhibition are similarly diverse: insulin resistance, bone loss, cognitive decline, and frailty.

In two such examples, BHB inhibited NLRP3 inflammasome activation in urate crystal–activated macrophages, and ketogenic diet ameliorated flares of gout arthritis in rats (36). Whether genetic or pharmacological inhibition of the NLRP3 inflammasome would extend mammalian life span remains unknown, but the potential certainly exists for translational application to human diseases of aging.


The ketone body BHB expresses a variety of molecular signaling functions, in addition to its role as a glucose-sparing energy carrier, that may influence a broad range of human diseases. There is sufficient evidence for several significant human diseases, including type 2 diabetes mellitus and Alzheimer’s dementia, in model organisms to justify human studies of BHB or a BHB-mimetic intervention. The diversity of age-associated diseases and pathways affected by BHB signaling suggests that therapies derived from BHB may hold promise for broadly enhancing health span and resilience in humans (91).

The translation of these effects into therapies that improve human health span requires the pursuit of two converging strategies: deeper mechanistic understanding of the downstream effects of BHB signals and improved systems for the targeted delivery of BHB for both experimental and therapeutic goals. Deeper mechanistic understanding would solidify some of the transitive connections described above. For example, BHB inhibits HDACs, and HDAC inhibition protects against cognitive decline in rodents; but does BHB protect against cognitive decline? Via HDAC inhibition? Which gene promoters are targeted? Establishing such links would permit rational design of human studies to test specific effects of BHB, with plausible biomarkers and intermediate outcomes. Improved delivery systems would facilitate both animal and human studies.

BHB-mimetic drugs, or ketomimetics, would recapitulate the desired activity of BHB. The key obstacles to exogenous delivery of BHB are its nature as an organic acid and the rapid catabolism of R-BHB. The quantity of exogenous BHB required to sustain blood levels over a long period would likely be harmful because of either excessive salt load or acidosis. Alternatively, approaches to ketomimetics include (a) the use of agents that activate endogenous ketogenesis in an otherwise normal dietary context, (b) the delivery of BHB prodrugs or precursors that avoid the acid/salt problem, and (c) the use of agents that phenocopy specific downstream signaling events.

The last approach, such as using HCAR2 agonists or HDAC inhibitors, is tempting, but a perhaps crucial advantage of adapting BHB itself is utilizing the existing endogenous transporters and metabolite gradients to bring BHB to its sites of action. Esters of BHB are a promising approach to delivering BHB as a prodrug, but the expense of synthesis is challenging. Confirming whether such synthetic compounds need be enantiomerically pure, or indeed whether S-BHB has better pharmacokinetics for the desired signaling function, might help reduce cost.

The ketone body BHB, a fasting fuel and fasting signal, is emerging as a poster child of the endogenous metabolite that transmits signals from the environment to affect cellular function and human health. Researchers have made important strides in understanding the signaling functions of BHB, many of which have crucial implications for the management of human diseases. A deeper knowledge of the endogenous actions of BHB, and improved tools for delivering BHB or replicating its effects, offers promise for the improvement of human health span and longevity.

PCSK9 protein a major driver of inflammation in cardiovascular disease

This is the fourth in what we expect will be seven or more blog posts concerned with chronic inflammation.  It relates to a recently discovered protein PCSK9 and tells a story of how age-related loss of LDL receptors leads to increase in LDL, associated uncontrollable cardiovascular inflammation, and atherosclerotic cardiovascular disease – the main killer of older people.  It lays out why the old standard of care for atherosclerotic cardiovascular disease – the taking of statins – is slowly in the process of being augmented and replaced with a new standard of care – inhibiting PCSK9.

Part 1 of the Inflammation series is the same as Part 5 of the NAD worldThat blog entry is concerned with The pro-inflammatory effects of eNAMPT(extracellular NAMPT, nicotinamide phosphoribosyltransferase).  Part 2 of the Inflammation series relates a) the “master” pathway network of inflammation (NF-kB) to two other pathway networks clearly implicated in aging and disease processes, b) Genomic Instability (p53), and c) Oxidative stress (Nrf2)Part 3 of the Inflammation series of blog entries is concerned with the all-important resolution phase of inflammation, how acute inflammation goes away under ideal conditions instead of hunkering down to lingering and dangerous chronic inflammation.  It is concerned with recently identified substances found in fish and flaxseed oils that play important roles in resolving certain kinds of inflammation – what they can do and how they work.

Atherosclerotic cardiovascular disease is the leading cause of morbidity and mortality worldwide (Gupta, Expert Review, Dove Press).  Wannah die later rather than sooner?   Then pay attention to this.

Image source

Many National and International though leaders concerned with the disease have refocused their efforts from lowering cholesterol to lowering LDL levels (Grundy,, 3rdReport of NECP). The reason for this is due in part to the discovery of PCSK9 protein, which is a circulating protein in blood that triggers the degradation of the LDL-receptor.

First, a little history about the discovery of the PCSK9 gene and why all the excitememnt about it.  PCSK9 is an abbreviation for Preprotein Convertase Subtilisin/kexin type 9, an extracellular protein that triggers the degradation of the LDL receptor, the LRP-1 receptor, and other receptors found on the surface of many cells (especially the liver).  In 2003, French researchers reported two mutations in the PCSK9 gene that caused familial autosomal dominant hypercholesterolemia (Abifadel,, Nature Genetics).  In 2008, Spanish researchers found a new mutation in the promoter of the PCSK9 gene that increased gene expression of PCSK9 mRNA and plasma levels of PCSK9 (Blesa, J Clin Endocrin Met). About the same time, researchers at UT Southwestern in Dallas reported a series of PCSK9 gene nonsense mutations found in 2.6% of a large group of African Americans that reduced LDL plasma levels by 15% and reduced coronary artery disease risk by 47% (Cohen,, NEJM).  In the ensuing 10 years, monoclonal antibodies that target the circulating PCSK9 protein have been developed, tested in over 25 R clinicaltrials, and FDA-approved.

What these clinical trials have shown in their “size effect” of reducing all-cause mortality (ACM) is nothing short of astounding – much more than statins ever did.  All cause mortality is reduced 55% in patients with hypercholesterolaemia.  So, we go on now to discussing how this all works.


It has long been known that there is a positive correlation between circulating LDL, atherosclerosis and heart disease.  Why is this?  The most harmful aspect of high LDL levels is most likely due to the fact that LDL is oxidized to “oxidized-LDL” (oxLDL) by free radicals (ROS) which triggers a pro-inflammatory receptor found on vascular endothelial cells called the “Lectin-like oxidized low density lipoprotein receptor 1” (LOX-1).  Here is a diagram of how this works:

Image source and reference: Role of Oxidized LDL in Atherosclerosis

Oxidized LDL lipoproteins bind to the LOX-1 receptor and are then internalized into endothelial cells, vascular smooth muscle cells, as well as monocyte/macrophages.  Inside the cell, this process triggers free radical production (ROS) and activation of NF-kB (the master switch for inflammation). NF-kB then turns on the transcription of hundreds of inflammatory genes, including cytokines (IL-8) and chemokines (CXCL2, CXCL3, CSF3), which are then secreted and trigger the CXCR2 receptor on white blood cells.  This is how the inflammatory cascade is “let loose” when your LDL-C is too high.  Here is a further diagram of how this works:

Diagram and legend source: LOX-1-dependent transcriptional regulation in response to oxidized LDL treatment of human aortic endothelial cells (2009)  “Model for LOX-1 functions in atherosclerosis and endothelial dysfunction. LOX-1 binding to OxLDL initiates reactive oxygen species (ROS) formation and an inflammatory response mediated in part by nuclear factor (NF)-κB and EGR1, leading to an upregulation and secretion of chemokines. The three CXC-chemokines (CXCL2, CXCL3, and IL-8) are all ligands for the same receptor, CXCR2, found on leukocytes. CXCR2 activation leads to chemotaxis and adhesion of leukocytes to endothelium. EGR1, CXCR2, and CSF3 are all implicated in animal models of atherosclerosis and represent novel molecular connections between LOX-1 and atherosclerosis. The transcription factor CREB is involved in maintaining vascular homeostasis. LOX-1 activation by OxLDL leads to a downregulation of CREB target genes and an upregulation of the CREB repressor CREM, thus providing a potential molecular mechanism of LOX-1-dependent endothelial dysfunction.”

In addition to LOX-1 mediated vascular inflammation,  oxLDL/LOX-1 internalization triggers endothelial/vascular smooth muscle cell dysfunction, apoptosis, cellular senescence, or osteoblastic differentiation. The osteoblastic differentiation of these cells is manifested as “vascular calcification”. If the oxLDL/LOX-1 internalization occurs in a monocyte, the macrophage is phenotypically transformed into the classic “foam cell”, which is the sine qua non of atherosclerosis.  Along with the other 4 “drivers” of atherosclerosis (Angiotensin II, pro-inflammatory cytokines, sheer stress, and advanced glycation end products) this oxLDL/LOX-1 pathway is considered to be the major molecular cause of the disease.

So, the first point in the story is this:  1. Too high LDL is bad because when oxidized it lets loose an inflammatory cascade that can lead to chemotaxis, adhesion, plugged arterial lumens, atherosclerosis and heart disease.

The next part of the story is simple: 2. under normal healthy conditions, LDL receptors on cells latch on to LDL particles and drag them into the cell where endosomes degrade the LDL and then the receptors pop back up to the surface of the cell and wait for another LDL particle to come along.  But if PCSK9 is present, the LDL receptor itself is destroyed.  The amazing clinical results mentioned above were due to inhibiting PCSK9.

The diagram below illustrates how PCSK9 works for elimination of LDL receptors and therefore reduction of capability to eliminate LDL.

Image source: Complementing the “Gold Standard”: Exploring PCSK9 MOA with Current Lipid Therapies  “When PCSK9 is present in that complex, what ultimately happens is that the LDL receptor is subsequently targeted via the endosome to the lysosome for degradation (situation depicted in right panel in diagram). Hence, we lose the LDL receptor; it does not recirculate back to the liver. If there is no PCSK9 that is bound to that complex, then once it is internalized, the cholesterol is released, the LDL particle is released from the receptor, and the receptor recirculates back to the surface, where it can then attach to more LDL cholesterol and clear more LDL cholesterol from the circulation degradation (situation depicted in left panel in diagram).”

The following diagram show how the final part of the story works: 3. anti-PCSK9 antibodies prevent PCSK9 from binding to LDL receptors so they are not destroyed in the lysosome.

DIAGRAM B  Image source

As already mentioned, although the PCSK9 inhibitor class of drugs have only been recently FDA approved, they have already shown the largest ACMR (all-cause mortality reduction) of any class of FDA-approved drugs ever approved in history.


These drugs are especially important for those of us who are interested in the science of aging since it has been shown that the levels of PCSK9 protein circulating in the plasma increases with aging in rodents and in humans (Tao,, J Bio Chem) (Ruscica, J Am Heart Asso).

Here is a diagram showing the relative levels of plasma PCSK9 in pre vs post menopausal women and young vs old men in a large population-based study (Diagram source: Ruscica, J Am Heart Asso).

The cause of the age-related increase in PCSK9 gene expression is due to a loss of suppression of the PCSK9 gene by the FoxO3a transcription factor.  Normally SIRT6 recruits FoxO3a to the promoter of the PCSK9 gene, but with aging, SIRT6 activity declines due to declining NAD+ levels in the cell.  As a result, there is an increase in PCSK9 gene expression with aging (Tao,, J Biol Chem). Here is a diagram illustrating this:

Note that we are here extending the story line of our NAD World blog entries detailing negative impacts of declining NAD+ LEVELS.

Reference and image source :Role of Oxidized LDL in Atherosclerosis (2015)

FoxO3a and Nrf2 are considered two of the most important transcription factors that protect mammals from aging by increasing oxidative stress resistance (Li, Ox Med Cell Long). Multiple genetic variants in the Foxo3a gene have been linked in many studies world-wide with extreme longevity (Anselmi,, Rejuvenation Research).  SIRT6 has also been shown to be a longevity gene in GWAS studies as well as in lab animals (Braidy,, Front Cell Neuroscience).  Thus it is not surprising that aging due to declines in FoxO3a/SIRT6 activity triggerw the increase in expression of the PCSK9 gene and play a major role in vascular aging.  Moreoever, statin use causes a paraxodical INCREASE in PCSK9 levels in patients with familial hypercholesterolemia, most likely due to a compensatory homeostatic feedback mechanism (Raal,, J Am Heart Asso).  The following diagram is from this study.

Another reason why PCSK9 levels increase with aging is insulin signaling. It is not surprising either that Insulin/IGF-1 signaling exacerbates this problem with age-related insulin resistance, since FoxO3a cannot enter the cell nucleus under conditions of high glucose/insulin signaling (due to the inhibition of nuclear translocation of FoxO3a by Akt).   This is why diabetes, metabolic syndrome, and insulin resistance leads to an increase in the expression of PCSK9 gene.  Here is a diagram of the relationship between insulin resistance (HOMA-IR) and insulin correlated with increases in PCSK9 expression over time

(Image source: Levenson, NMCD).

Age and diet-related increases in LDL blood levels are probably the most common clinical problem facing modern man.  Although statins reduce endogenous cholesterol synthesis, they do not directly stop the age-related increase in PCSK9 protein in the blood.  As a result, most studies have suggested that “age” is the greatest risk factor for atherosclerosis after age 50, whereas “cholesterol” is the greatest risk factor for atherosclerosis prior to age 50.  For this reason, the value of a drug that reduces the PCSK9 protein in the blood should increase with aging.  So far, this prediction has held true.  Unlike statins, where their value declines in old age (zero value by age 85), there appears to be value in reducing LDL levels even in old age, with FoxO3a and SIRT6 signaling decline.

So, in this overall story we are seeing a link-up of several “usual suspect” themes we have touched on multiple times before in this blog: insufficient expression of sirtuins and lack of NAD+, the FoxO3a transcription factor, high age-related LDL, the Insulin/IGF-1/PI3K/Akt pathway, the master trigger of inflammation NF-kB, Nrf2, reactive oxygen species, inflammatory cytokines, atherosclerosis.  With a new element which is age-related overexpression of the PCSK9 gene which kills off LDL receptors.

Cholesterol, step to the rear of the coach please

Whereas cardiologists used to consider cholesterol on this “big five” list, it is no longer considered to be as important as it once was. Instead, high blood levels of LDL is now reputed to be the #1 culprit that causes atherosclerotic disease. Since PCSK9 proteins trigger the degradation of the LDL receptor, monoclonal antibodies against PCSK9 have been successful at dropping LDL levels as low as 10 mg/dl (although no physician is recommending that you lower your LDL that much).  Getting rid of LDL reduces oxidized LDL, which dramatically reduces LOX-1 activation.  Reduced LOX-1 activation reduces endothelial cell dysfunction, apoptosis, senescence, and osteoblastic differentiation of endothelial cells (which is a major cause of vascular calcification).  It is this mechanisms that explains why PCSK9 inhibitor therapy has been shown to reduce vascular and valvular calcification (something that statins rarely do).

Despite these known (theoretical) molecular mechanisms, most cardiologists were skeptical that PCSK9 inhibitors would reduce all-cause mortality as well as statins.  However, the results of the PCSK9 inhibitor clinical trials have shattered all doubts by 10 miles!  The results on ACM reduction have been nothing short of amazing. A meta-analysis of the 24 RCTs that have been done so far (N = 10,159) show a 55% ACMR (OR = 0.45) and a 50% reduction in cardiovascular mortality (OR = 0.50). The rate of MI was also reduced by 51% (OR = 0.49). Even increases in serum creatinine kinase (CPK) was reduced (OR = 0.72). Because PCSK9 levels increase as a function of aging, it is likely that these drugs will still have beneficial effects after age 85, whereas data shows that statins probably become harmful after age 85.

F In addition to LOX-1 mediated vascular inflammation,  oxLDL/LOX-1 internalization triggers endothelial/vascular smooth muscle cell dysfunction, apoptosis, cellular senescence, or osteoblastic differentiation. The osteoblastic differentiation of these cells is manifested as “vascular calcification”. If the oxLDL/LOX-1 internalization occurs in a monocyte, the macrophage is phenotypically transformed into the classic “foam cell”, which is the sine qua non of atherosclerosis.  Along with the other 4 “drivers” of atherosclerosis (Angiotensin II, pro-inflammatory cytokines, sheer stress, and advanced glycation end products) this oxLDL/LOX-1 pathway is considered to be the major molecular cause of the disease.

DIAGRAM A above, shows how PCSK9 inhibitors work and how they are fundamentally different than statins:

ReferenceEffects of Proprotein Convertase Subtilisin/Kexin Type 9 Antibodies in Adults With Hypercholesterolemia: A Systematic Review and Meta-analysis (2015)


Costs of PCSK9 Inhibitor Therapy and Delivery methods$14,000/year in the US but cheaper overseas

This blog is designed to be a practical resource that you can use on your personal journey to improved healthspan.  It is not a substitute for your personal physician, however. For this reason, we suggest you consult with your doctor before attempting any of the interventions in this blog, including monoclonal antibodies against PCSK9.  However, your doctor may be completely unaware of this new class of drugs and your health insurance company will not pay for the drugs unless you have had an MI or a stroke AND you also have failed to control your LDL with statins (or have severe side effects from statins that cannot be treated with CoQ10).  In the US, the annual costs for PCSK9 inhibitor therapy runs about $14,000 per year so this is mow a very expensive way to try to lengthen your healthspan.  However the cost  of the same PCSK9 inhibitor drug in some other countries can be half the price found here in the US, so for those who have figured out how to do cash-based medical tourism, you can save $7,000 per year. Here are the two monoclonal antibodies against PCSK9 that are currently FDA-approved:

  • Alirocumab (Praluent) – This monoclonal antibody against the PCSK9 protein was the first to be approved by the US FDA (July, 2015). It is a twice-a-month SQ injection of 75-150 mg per dose. It has been FDA-approved for patients with heterozygous familial hypercholesterolemia or for normal patients whose cholesterol cannot be adequately controlled with diet and statins.  Patients who go on Praluent are supposed to stay on their statin, even though they may have to lower their dose to avoid statin-induced muscle pain. Side effects include nasopharyngitis (11%), injection site reactions (7%), influenza (5.7%), UTIs (4.8%), and diarrhea (4.7%).  Neurocognitive events were seen in two of the large clinical trials, but did not correlate with how low the LDL-C went.   Further studies have failed to show any neurocognitive difference between the drug and the placebo group (0.7-0.8%).  8% of patients developed anti-drug antibodies. Alirocumab costs $1200 per month or $14,350 per year in the US.  What is amazing is that PCSK9 monoclonal antibody therapy can drop your LDL-C to as low as 25 with no evidence of any major side effect.  With such dramatic reduction, vascular and valvular calcifications have been shown to be reduced.
  • Evolocumab (Repatha) – This monoclonal antibody was approved in August, 2015, shortly after Alirocumab. Like Alirocumab, it is approved for familial (genetic) hypercholesterolemia (heterozygous or homozygous) and patients who fail diet + statin therapy.  Unlike Praluent, it can be given twice-a-month dose (140 mg) or once-a-month (420 mg) SQ. Like Alirocumab, patients are supposed to remain on statins.  Most common side effects Include nasopharyngitis (11%), URI (9.3%), influenza (7.5%), back pain (6.2%), and Injection-site reactions (5.7%).  Ongoing studies are closely looking for neurocognitive side effects, but these studies are not completed.  The annual costs for Evolocumab are almost identical to those for Alirocumab.

Are PCSK9 Inhibitors Cost Effective? Answer: No

While our enthusiasm for this dramatic reduction in all-cause mortality may appear to be “exuberant”, a famous economist once said that people often suffer from “irrational exuberance”.  In the case of PCSK9 monoclonal antibodies, this is definitely true.  The “bean counters” have already figured out that these new drugs are overpriced to be cost effective (Really!….any 5th grader could tell you that!).  At $14,000 per person per year, the two PCSK9 inhibitor drugs are expected to generate $4.5 billion USD in annual sales by 2020 (Big Pharma is drowning in their saliva over this!).  If all eligible patients took these injections, it would add an approximately $150 billion annual cost to our health care system.  Only if the price of these drugs dropped to $4,536 per patient per year would the drugs start to become cost effective.  However. if you have a high LDL-C despite eating a strict cholesterol-free diet and taking your statins, taking one of these drugs could lengthen your healthspan.

ReferenceAre PCSK9 meds worth the cost? Only if Amgen, Sanofi and Regeneron slash prices by two-thirds: JAMA

Alternative to PSCK9 Inhibitor TherapyReduce Insulin signaling with diet, SIRT6 activation, fasting, and the Salvia extract, Tanshinone IIA

For those who do not have $14,000 per year to “burn”, there is a much simpler way of “shutting off” PCSK9.  As mentioned in the beginning of this section, the “longevity gene” FoxO3a functions as a suppressor of PCSK9 gene expression when the FoxO3a transcription factor can enter the cell nucleus. Unfortunately to do this, you must reduce insulin signaling, since insulin prevents FoxO3a from entering the cell nucleus (due to the Insulin/IGF-1/PI3K/Akt pathway).  Once you reduce insulin signaling, FoxO3a can enter the cell nucleus and be recruited by SIRT6.

Unfortunately, with aging, SIRT6 cannot be activated due to declining levels of NAD+ within the cell.  This can be ameliorated (at least in theory) by restoring NAD+ levels within the cell with NAD+ precursors like NR or NMN, or possibly with NAD+ given IV.  (you can look at our blog postings on the NAD World that describe the mechanisms involved.)   Specifically, however there is a fascinating phytochemical that has been isolated from the Salvia miltiorrhiza Bunge plant called Tanshinone IIA. This compound is the most pharmacologically bioactive compound found in the Salvia plan and has anti-inflammation, anti-cancer, anti-LDL-cholesterol, neuroprotective, and hypolipidemic properties.  Only recently was the molecular mechanism of the Tanshinone IIA compound elucidated by a team of researchers from Taiwan.  Below is a molecular diagram of the molecule and the reference for how it works.

There are other molecular mechanisms that regulate the PCSK9 gene besides the SIRT6-mediated FoxO3a suppression, however.  This includes binding sites for several transcription factors in the promoter region of the PCSK9 gene (SREBP-1/2, HNF1A, farnesoid X receptor, PPAR-gamma, liver X receptor, and histone nuclear factor P).  Fasting has also been shown to be a very powerful way of reducing plasma levels of PCSK9, reducing PCSK9 levels by as much as 58%, compared to fed conditions.  This is an even greater effect than the monoclonal antibodies!

Image and legend source  Effects of fasting on PCSK9 levels   “Trends in plasma LDL-c, PCSK9, and lathosterol-to-cholesterol ratio during a 48 h fast. Plasma levels of LDL-c increased modestly over the initial 32 h of the two-day fast (P = 0.004) and then stabilized. Plasma PCSK9 levels declined steadily after 8 h of fasting and reached a nadir by 36 h (P = 0.024). The decline in plasma PCSK9 occurred in tandem with a decrease in the lathosterol-to-cholesterol ratio (P = 0.091). Data (mean ± SEM) were derived from 18 healthy subjects (described in Table 1) who underwent an observed 48 h fast. PCSK9, proprotein convertase, subtilisin/kexin type 9; LDL-c, low-density lipoprotein cholesterol.”


Conclusion: The story of the strange, extracellular protein called PCSK9 has now become the focal point of a $4.5 billion/year gravy train for Big Pharma companies, due to the dramatic effect that monoclonal antibodies have of clearing this “bad” protein out of the blood.  There is no debate that the use of these monoclonal antibodies has a greater effect on reducing all-cause mortality than any other drug, diet, supplement, or lifestyle intervention at this time, with an ACM reduction of 55%.


However until the cost of these PCSK9 monoclonal antibodies drops below $4,536 per person per year, they are not cost effective.  Since the increased expression of the PCSK9 gene is due in part to the Insulin/IGF-1 signaling pathway, which prevents FoxO3a transcription factor from migrating into the cell nucleus, the most cost effective way to reduce PCSK9 expression is to reduce your insulin levels.  This can easily be done with diet and exercise.

Moreover, a phytochemical from the Salvia plant called Tanshinone IIA, and SIRT6 activation with NAD+/NR/NMN may also be ways to prevent the increase in PCSK9 gene expression which are NOT due to insulin.  The loss of SIRT6 activity due to the decline in NAD+ levels within the nucleus is a likely contributor to the age-related increase in PCSK9.

Nevertheless, because oxidized LDL is such a powerful “driver” of atherosclerosis, a dramatic reduction in PCSK9 plasma protein levels should be a major goal for lengthening lifespan.  Despite its unpopularity, fasting has been scientifically shown to be the cheapest and most effective way to reduce PCSK9. With a 48-hour fast, PCSK9 levels can be lowered by 58%, which is even more than the monoclonal antibodies do!

Mitochondria in Aging, II: Remedies

The once-popular mitochondrial free radical theory of aging proved to be too glib. Aging isn’t fundamentally about dispersed damage; rather, dispersed damage is a result when the body’s defenses stand down in old age.  Nevertheless, the mitochondria do play a role in aging, largely through signaling and apoptosis.  Antioxidants targeted to mitochondria may be an exception to the rule that antioxidants don’t prolong lifespan.  And other supplements and strategies that either promote production of new mitochondria or enhance their efficiency of operation show promise for modest lifespan extension.

Growing new mitochondria

A ketogenic diet leads to generation of new mitochondria, as do caloric restriction and exercise.  Exercise when the body is starved for sugar (low glycogen) is the most potent stimulator of new mitochondrial growth.  Exercise while fasting, or continue to exercise after you “hit your wall”.

Hormones that promote mitochondrial proliferation include thyroxin, estrogens, and glucocorticoids.  Promoting new mitochondria has a tendency simultaneously to suppress apoptosis, programmed cell death [ref].  At later ages, apoptosis of cells that are still functional tends to be a larger problem than the failure of cancerous cells to eliminate themselves by apoptosis.  In other words, suppressing apoptosis is (on balance) a good thing for anti-aging, but the downside is it can also increase risk of cancer.



Coenzyme Q-10 (aka ubiquinone) is an essential part of mitochondrial chemistry, shuttling electrons along their way to the ATP molecules that mitochondria generate as their primary energy export to the cell.  It’s often called an antioxidant, but that’s not the primary role of CoQ10.

As a supplement, it is well-established with a good reputation.  There is lots of evidence for benefits to health markers, especially athletic endurance, several aspects of heart health, and erectile dysfunction.  If you have fibromyalgia or if you are taking statins, CoQ10 is strongly indicated.  For chronic fatigue syndrome, it’s definitely worth trying.

But there’s no reason to expect it will increase your life expectancy.  Supplementing with ubiquinone increases the lifespan of worms but not mice or rats [ref, ref].

Worms that cannot make unbiquinone live 10 times as long.  Just saying…

A few years ago, ubiquinol was introduced as a more bioavailable form of ubiquinone.  It’s more expensive, but there is not clear evidence that it is more bioavailable.



Pyrroloquinoline quinone is helpful but not necessary part of mitochondrial chemistry.  Bacteria make a lot of it; plants less; mammals only tiny quantities.  Mice completely deprived of PQQ show growth deficiency, but the amount that they need is tiny compared to the quantities in PQQ supplements.

PQQ is a growth factor for bacteria, and the principal health claim for PQQ is that it can stimulate growth of new mitochondria.  The evidence is based on biochemistry and cell cultures.  In live mice, it has been shown that PQQ deficiency results in a mitochondria deficiency, but not that large quantities of PQQ lead to more mitochondria.

Bill Faloon (LEF) and Joseph Cohen (selfhacked) are big fans of PQQ, and you can read a list of benefits here.  Cohen claims PQQ helps with sleep quality and nerve growth, leading to better cognitive function.

Small quantities of PQQ can be absorbed from many plant foods, but not animal foods.  Much larger quantities come in supplement form. 100 g of tofu has just 2µg (micrograms).  Supplements are usually 5-20 mg, hundreds of times as much as you’re likely to get from a vegetarian diet.  Here is a table of PQQ concentrations in foods:


SkQ and MitoQ

These are two closely related molecules, originally synthesized in Russia in the 1970s, but it wasn’t until the 1990s that their therapeutic value was documented by two New Zealand scientists.  One end of the molecule is CoQ10 (or a version found in plants, claimed to be even more powerful as an antioxidant).  The other end of the molecule is an electric tugboat that pulls the molecule into mitochondria.

I’ve written a  detailed report three years ago.  At the time, I noted that the Russians claimed to extend lifespan of mice modestly with SkQ, and SkQ was found (also in Russian labs) to be a powerful rejuvenant for aging eyes.  The Russians sell SkQ as eye drops.  The Kiwis sell MitoQ as skin cream and also as pills.

Earlier this year, the Russian labs announced that SkQ had substantially extended lifespan of a mouse strain that was short-lived because of a mitochondrial defect.  None of the Russian claims have been reproduced in Western labs.  Three years ago, I was inclined to give the Russians the benefit of the doubt, but now I’m starting to wonder, since the New Zealand company has a laboratory arm, and they haven’t announced anything nearly so impressive.


Humanin and her sisters

Mitochondria have ringlets of their own DNA, encoding just 37 genes.  (That doesn’t mean that the mitochondria only need 37 proteins; the great majority of proteins needed by mitochondria are coded in chromosomes of the cell nucleus, and transported to the mitochondria as needed.)  Just 16 years ago, the first mitochondrial-coded protein to be discovered was named Humanin, because it was found to improve cognitive function to dementia patients, restoring some of their “humanity”.  In addition to being neuroprotective, humanin promotes insulin sensitivity.  Humannin’s action is not confined to the mitochondrion in which it was produced, but in fact it  circulates in the blood as a signal molecule.  Blood levels of humanin decline with age.


In experiments with mice, humanin injections have been shown to protect against disease.  Lifespan assays with humanin are not yet available.

To date, HN and its analogs have been demonstrated to play a role in multiple diseases including type 2 diabetes (25, 43), cardiovascular disease (CVD) (2, 3, 47), memory loss (48), amyotrophic lateral sclerosis (ALS) (49), stroke (50), and inflammation (22, 51). The mechanisms that are common to many of these age-related diseases are oxidative stress (52) and mitochondrial dysfunction (53). Mitochondria are major source of ROS, excess of which can cause oxidative damage of cellular lipids, proteins, and DNA. The accumulation of oxidative damage will result in decline of mitochondrial function, which in turn leads to enhanced ROS production (53). This vicious cycle can play a role in cellular damage, apoptosis, and cellular senescence – contributing to aging and age-related diseases. Indeed, oxidative stress is tightly linked to multiple human diseases such as Parkinson’s disease (PD) (54), AD (55), atherosclerosis (56), heart failure (57), myocardial infarction (58), chronic inflammation (59), kidney disease (60), stroke (61), cancers (62, 63), and many types of metabolic disorders (64, 65). We and others have shown that HN plays critical roles in reducing oxidative stress (6668). [2014 review]

Pinchas Cohen, MD (Dean, School of Gerontology, University of Southern California Davis, Los Angeles, California) is an expert in humanin, a protein (peptide) produced in mitochondria. Mitochondria are energy-generating organelles in cells, which have their own DNA separate from the DNA in the nucleus. The amount of DNA found in the mitochondria is much less than that found in the nucleus. As such, mitochondrial DNA contains codes for only a few proteins, humanin being one of them. Humanin was discovered by a search for factors helping to keep neurons alive in undiseased portions of the brains of Alzheimer’s disease patients.Humanin protects neurons against cell death in Alzheimer’s disease, as well as protecting against toxic chemicals and prions (toxic proteins)[ref].  Dr. Cohen’s team has shown that humanin also protects cells lining blood vessel walls, preventing atherosclerosis. In particular, they have shown that low levels of humanin in the bloodstream are associated with endothelial dysfunction of the coronary arteries (arteries of the heart).[ref] Humanin has also been shown to promote insulin sensitivity, the responsiveness of tissues to insulin. Because humanin levels decline with age, it is believed that reduced humanin contributes to the development of aging-associated diseases, including Alzheimer’s disease and type II diabetes. [Ben Best]

Personal notes: This lab near where I am visiting in Beijing is taking leadership in characterizing a group of short peptides similar in origin to humanin, and this company across the street from us is selling mitochondrial peptides.

If humanin were a patentable drug, there would be much excitement and multiple clinical trials for AD, probably leading to expansion into general anti-aging effects.



This is another short peptide of mitochondrial origin, only recently discovered and characterized.  I was alerted to its existence by a study from a USC lab that was written up here in ScienceBlog just this month (reprinted from a USC press release).  Results are new but impressive.  Mice injected with MOTS-c had more muscle mass, less fat, more strength and endurance.  MOTS-c protected their insulin sensitivity when mice were fed a high fat diet [ref].  Lifespan studies haven’t been done yet.

Like humanin, MOTS-c is manufactured inside mitochondria from a template in mitochondrial DNA, but it is exported from the cell and appears in the bloodstream as a signal molecule.  Blood levels of MOTS-c decline with age.  It is a mini protein molecule with 16 amino acids, too big to survive digestion so it can’t be taken orally.

“MOTS-c holds much potential as a target to treat metabolic syndromes by regulating muscle and fat physiology, and perhaps even extend our healthy lifespan.”[ref]

Let’s keep your eyes on this one over the next year or two.


Gutathione / NAC

I’ve never heard anyone say a bad word about glutathione.  It’s the antioxidant with no downside.  Genetic modifications that upregulate glutathione have increased lifespan in worms, flies and mice.

For a long while, it has been assumed that you can’t eat glutathione, because it doesn’t survive digestion.  Some researchers at Penn State disagree, finding impressive increases in tissue and blood levels when people were supplemented with up to 1 g per day raw glutathione.  Liposomal glutathione is an oral delivery form that gets around the digestion problem, especially when taken with methyl donors like SAMe.

The herb Sylimarin=milk thistle may increase glutathione.  For now, the precursor molecule N-Acetyl Cysteine (NAC) is the best-established supplement we have to promote glutathione.  In the one available study, supplementing with NAC greatly increased lifespan in male but not female mice.  NAC also increases lifespan in worms and flies.

N-Acetyl Cysteine



For the future, we might hope to do better.  Less than 20% of the cell’s glutathione actually makes its way to the mitochondria, where it is most needed.  There are esters of glutathione that, in theory, ought to be attracted into the mitochondria.  They have been tested in cell culture only, but are more than ripe for animal testing [ref].


Nicotinamide Riboside (NR) and other NAD+ enhancers

The chemicals NAD+ and NADH are alternative, cycled forms of an intermediate in the process by which mitochondria make energy.  Levels of NAD+/NADH decline with age.  NR is a precursor to NAD+, and it has been demonstrated (preliminary results in humans) that NR supplementation increases blood levels of NAD+.

It may be awhile before we know for sure whether this leads to better health or longer lifespan.  Niagen and Basis are heavily promoted with credible scientifists behind their products, and many early adopters offer subjective reports of short-term benefits.  There is one mouse study claiming to pull a 3% extension of lifespan out of the noise, and perhaps I am less open to the finding because the article, published prominently in Science, seems so breathless in describing benefits.



The primary role of melatonin is to regulate the body’s sleep/wake cycle.  Melatonin declines with age and the timing of our daily melatonin surge gets fuzzier and less reliable with age.sleep quality deteriorates.  Sleep quality suffers.

Melatonin is well-established in mice as a modest longevity aid, although results have been inconsistent.  12 out of 20 studies showed a lifespan increase, and the remaining 8 showed no increase or decrease.  Whether nightly supplementation affects mortality rates in humans has never been determined.

Melatonin is concentrated in mitochondria as much as 100-fold, and it may even be created there [ref], independent of the circulating melatonin that is secreted from the pineal gland at night.  One of its actions is as a mitochondrial antioxidant and scavenger of ROS.

Twenty years ago, Walter Pierpaoli promoted melatonin as a sleep aid, cancer fighting hormone that would enhance your mood and your sex life while keeping you young.  Russian labs have also been optimistic.  My take is that melatonin is a legitimate anti-aging hormone, and is especially useful for those of us whose sleep is disrupted with age.  It is widely available, cheap and safe.  Unless you’re fighting jet lag, 1 to 2 mg at night is all you need.

Also worth mentioning

Magnesium is required for manufacture of glutathione.  Selenium works along with glutathione.  Omega-3 fatty acids can promote synthesis of glutathione.  Acetyl L-carnitine transports fat fuels through the mitochondrial membrane.  Alpha-lipoic acid is part of the mitochondrial energy metabolism.

The Bottom Line

Commercial interests can make some messages louder than others, and the health news we hear is affected by what is profitable as much as by what is healthy.  Exercise is primary, but has no sales value.  Of the supplements reviewed here, NAC is the best-established for mitochondrial health and a possible effect on lifespan.  It is cheap and available.  Liposomal glutathione is certainly more expensive and possibly more effective.  Melatonin is even cheaper, and has been found to increase lifespan in multiple rodent studies, with broad benefits apart from modification of mitochondrial function.  Humanin and MOTS-c, not yet close to commercial availability, seem to be promising substances to explore for health, though not for profits.

Mitochondria in Aging, I Mechanisms and Background

A popular theory a generation back sought to trace aging to oxidative damage originating in the mitochondria.  Every cell in the body has hundreds or thousands of mitochondria, the sites of the high-energy chemistry that produces ROS as toxic waste. The hope was that by quenching the ROS, aging might be turned off. The “Mitochondrial Free Radical Theory” is built on a flawed theoretical foundation, and anti-oxidants don’t extend lifespan. Nevertheless, the mitochondria play a role in aging.  Historically, mitochondria were mediators of the first organized mechanisms of programmed death over a billion years ago, and they retain a role in processing signals that regulate lifespan.  Curiously, though a quadrillion mitochondria are dispersed through the body, they act in some ways like a single organ, sending coordinated signals that regulate metabolism and affect aging.

Mitochondria are in the cells of all plants and animals—hundreds or thousands of mini power plants in each cell.  They burn sugar to make electrochemical energy in a form the cell can use.  They are loyal and essential servants.  But it wasn’t always so.  More than a billion years ago, mitochondria came into the cell as invading bacteria.  Though they’ve long ago been domesticaed, they retain a bit of their autonomy as a relic of the past.  Mitochondria have their own DNA.  Like bacteria, mitochondrial DNA is in the form of loop, a plasmid rather than a chromosome.  Each mitochondrion keeps several copies of the plasmid.

Mitochondria retain from their distant pathological past the capacity to kill the cell.  This is an orderly process known as apoptosis=programmed cell death.  Mitochondria are not the jurors that sentence the cell to death, but only the executioners acting on external signals.

Aging of the body as a whole is centrally coordinated, though the nature and location of the clock(s) remain a major unsolved problem.  Communication about the age state of the body is carried through signal molecules in the blood, and tissues respond accordingly.  Mitochondria not only pick up on these signals, they also contribute circulating signals of their own.  Apoptosis is dialed up in old age.  Along with inflammation, it is a primary, local mode of the self-destructive process that is aging.  We lose too many cells to apoptosis, cells that are still healthy and useful, and mitochondria are the proximate cause of this loss.

Portrait by scanning electron microscope, artistically colorized


Signaling, up, down and sideways

The big picture is that mitochondria take their orders from the cell nucleus, where the vast majority of the DNA is housed.  The transcription factors that determine what mitochondrial genes are expressed are housed in the nucleus.  In addition, there is feedback, retrograde signaling, by which mitochondria communicate to the nucleus the state of their own health and of the cell’s energy mtabolism in general.  The nucleus responds with changes in transcription based on communication from the mitochondria.

A great part of the diverse benefits of caloric restriction, and perhaps of exercise, too, are thought to originate in signaling from the mitochondria.

In addition to sending and receiving signals from the cell nucleus, mitochondria talk to each other.  They coordinate extensively within a cell, and they also generate hormones that are transmitted through the bloodstream, talking to distant cells and foreign mitochondria.


Mitochondria and Cancer

Cancer cells have impaired mitochondrial metabolism.  They don’t burn sugar through the usual, high-efficiency mode that combines with the maximal amount of oxygen; rather they use fermentation—anaerobic breakdown of sugar.  Cancer cells do this even when oxygen is plentiful, despite the fact that it generates much less energy per sugar molecule.  Cancer cells are starved for energy, and they gobble up sugar at a high rate.  (PET scans are able to visualize tumors on the basis of their sugar consumption.)  Eating a very-low-carb diet is a cancer therapy.

90 years ago, a Nobelist and Big Thinker in biomedicine named Otto Warburg gave us the hypothsis that mitochondria with impaired glucose metabolism are the root cause of cancer.  We usually think of cancer as starting with mutations that lead to uncontrolled growth and proliferation, but in the Metabolic Theory of Cancer, mutations and proliferation are secondary to this change in mitochondrial chemistry.  Today, proponents of the Warburg Hypothesis are a small but enthusiastic minority, armed with facts and arguments that I have not yet found time to assess.  But I am struck by the fact that when the nucleus of a cancer cell is transplanted into a healthy cell, the healthy cell remains healthy; and when the nucleus of a healthy cell is transplanted into a cancer cell, the cell remains cancerous [ref, ref].  This seems to be prima facieevidence that the essence of cancer is not to be found in chromosomes of the nucleus.


Fewer, less efficient, and more toxic waste with age

We have fewer mitochondria as we age, and this is plausibly connected to lower muscle strength and endurance as well as energy in the organ that uses energy most intensively=the brain [ref].  The relationship is subtle enough that it is not completely nailed down, despite decades of work from true believers.  Since mitochondria mediate apoptosis, it is also plausible that loss of muscle cells and nerve cells with age (at least partially through apoptosis) is also mediated by mitochondria.

Cells that need a lot of energy have a lot of mitochondria. Heart muscle cells are packed with them.

Compounding the problem, the mitochondria that we do have become less efficient with age.  They are giving us less energy, and they are generating more reactive oxygen species (ROS).  Simultaneously, the cell is generating less of the native anti-oxidants that protect from ROS.  Glutathione, ubiquinone, and superoxide dismutase all decline with age.  This is one of the ways the body destroys itself.  Oxidative damage accumulates in old but not young people.  Oxidative damage may also contribute to telomere shortening.

Somehow, ROS generated by impaired mitochondria produce damage that accumulates, but ROS generated by exercise signal the body to ramp up the repair processes, and produce a net gain in health.  It is not clear how the two processes are distinguished.  The reason that anti-oxidants don’t work to extend lifespan is probably that they interfere with the signaling functions of ROS.

The best-documented way in which mitochondria deteriorate is that their DNA develops mutations.  I find this something of a conundrum—not that mitochondria should accumulate mutations over the course of a lifetime but that they don’t accumulate mutations from one generation to the next (in the germline).  Mitochondria proliferate clonally, without sex.  Sex shuffles genes in many combinations, so that the good genes can be separated from the mutated ones, and the latter eliminated before they get fixed into the genome.  Without sex, how do mitochondria avoid accumulating mutations over the aeons?  And since they largely do manage to avoid accumulating mutations over millions of years, why can’t they avoid accumulating mutations over the course of a few decades within a human body?


Are mutations in mitochondrial DNA a cause of aging?

Mitochondrial mutations accumulate with age.  Genetically modified mice with a defective gene for replication of mitochondrial (but not nuclear) DNA age faster and die earlier.  This has generally been taken as proof that mitochondrial mutations are a factor in aging, but it need not be so.  In fact, mitochondria function well with a high tolerance for genetic errors, and it is not clear whether levels of mitochondrial mutations in aging humans cause significant problems, or even whether mutations are related to the general decline in mitochondrial function with age.  An alternative explanation for the mito-mutator mice is that they have developmental problems already in utero, and these may lead to premature aging even without accumulation of mito mutations.

Mitochondrial mutator mice

Stem cells keep dividing and producing new functional (differentiated) cells through the life of the animal.  They seem smart enough to minimize the damage from mitochondrial mutations.  Stem cells have been observed to hold on to the best mitochondria, and pass the damaged ones off to the cells that have a limited lifetime. This helps keep the errors from proliferating, and is in the best interest of the organism as a whole.  It’s interesting that mother budding yeast cells do the opposite—they hold on to their damaged mitochondria and pass the cleanest and purest on to their daughter cells [ref].  Mammalian mothers also seem able to choose the best mitochondria to pass to their daughters, purifying the germline [ref].  In other words, though their behavior is the opposite of stem cells, both behaviors are adaptive for the long-term interest of the organism (and its progeny).

In summary, the age-related increases in oxidative damage and ROS production are relatively small and may not explain the rather severe physiological alterations occurring during aging. Consistent with this hypothesis, the absence of a clear correlation between oxidative stress and longevity [across species] also suggests that oxidative damage does not play an important role in age-related diseases (e.g., cardiovascular diseases, neurodegenerative diseases, diabetes mellitus) and aging. Experimental results from mtDNA mutator mice suggest that mtDNA mutations in somatic stem cells may drive progeroid phenotypes without increasing oxidative stress, thus indicating that mtDNA mutations that lead to a bioenergetic deficiency may drive the aging process [but this is not assured, since these mice seem to suffer substantial damage already in utero]. There is as yet no firm evidence that the overall low levels of mtDNA mutations found in mammals drive the normal aging process. One way to address this experimentally would be to generate anti-mutator animal models to determine whether decreased mtDNA mutation rates prolong their life span. [Bratic & Larsson review]


Mitochondrial evolutionary conundrum

Mitochondria reproduce clonally, like bacteria.  In fact, all the mitochondria in your body were inherited from one of your mother’s egg cells, and she got her mitochondria from your maternal grandmother, and so forth back in time—matrilineal all the way.  How is it that defects don’t accumulate in the mitochondrial genome?

As far as I know, the way in which the integrity of the mitochondrial genome is maintained remains an unsolved problem.  We do know that mutations in mitochondrial DNA increase with age in some tissues but not others [ref].  The reason you have to speak up when you talk to your grandmother is probably related to mitochondrial defects in neurons [ref].

Over the course of millions of years, mitochondria do not lose their genetic integrity, though the mitochondrial genome evolves more rapidly than the nuclear genome, and different species tend to have distinctive mitochondrial genomes.  The mystery is why detrimental mutations should accumulate over decades, but not over aeons.

To me, this is powerful evidence that there is a mechanism for managing the evolution of the mitochondrial genome.  It probably involves selection by the cell so that mitochondria that are functioning efficiently are encouraged to reproduce.  The cell acts like a human lab that is breeding tomatoes or Labrador retrievers for specific characteristics that the breeder or the cell finds most useful.  Probably there is also gene exchange among the different copies of the plasmid within a mitochondrion, and between mitochondria as they sometimes merge during the lifetime of a cell (my speculation).


What’s going on?

A theme in this blog (and in my thinking) has been that aging is not a dispersed process of locally-occurring damage, but is centrally orchestrated.  Well, mitochondria are about as far from “central” as you can get.  We have about a quadrillion of them, dispersed through every cell in the body (except red blood cells).

Mitochondria talk to each other within a single cell.  They merge and they reproduce, coordinating with one another and with the cell nucleus.  Now it appears they also send signals through the bloodstream (more next week).  Could they be acting like a single organ, dispersed through the body? Maybe.  Sensing the body’s state of energy usage and fuel sufficiency, they send signals that contribute to calculations about lifespan.

My guess is that aging is coordinated by a few biological clocks (centralized like the suprachiasmatic nucleus and the thymus or dispersed like telomeres and methylation patterns), and that mitochondria are not counted among the clocks.  But mitochondria are important intermediates.  The old story is that they generate energy and generate tissue-damaging ROS.  The new story is that they are also centers of signal transduction, probably based on their first-hand knowledge of the energy status of the body.

Building the Case that Aging is Controlled from the Brain

Last week, a new study came out fingering the hypothalamus as locus of a clock that modulates aging.  This encourages those of us who entertain the most optimistic scenarios for anti-aging medicine.  Could it be that altering the biochemistry of one tiny control center might effect global rejuvenation?  

First some background….

I believe that aging is governed by an internal biological clock, or several semi-independent and redundant clocks.  There are

  • A telomere clock, counting cell divisions on a flexible schedule, eventually producing cells with short-telomeres that poison us.
  • The thymus, crucial training ground for our white blood cells, shrinks through a lifetime.
  • An epigenetic clock alters gene expression over time in directions that give rise to self-destruction.
  • A neuroendocrine clock in the hypothalamus
  • Perhaps other clocks, yet to be identified.


A dream is to be able to reset the hands of the clock.  If we’re lucky, then changing the state of some metabolic subsystem will not just temper the rate at which we age, but actually restore the body to a younger state.  Most of the research in anti-aging medicine is still devoted to ways to engineer fixes for damage the body has allowed to accumulate; but I belong to a wild-eyed contingent that thinks the body can do its own fixing if we understand the signaling language well enough to speak the word “youth” in the body’s native biochemical tongue.

Some of these clocks are more accessible and easier to manipulate than others.  The epigenetic clock is most daunting, because it presents the spectre of a global network of signal molecules circulating in the blood, transcription factors that mutually support one another in a state of slowly-shifting homeostasis.  This system could be so complex that it might take decades to understand, and then hundreds of different signal molecules in the blood would need to be re-balanced in order to recreate homeostasis in a younger condition.  (For several years, the Mike and Irina Conboy have been looking for a small subset of molecules that might control the rest, but in a private conversation they recently told me they are less optimistic that a small number of factors controls all the rest.)

At the other end of the spectrum, the hypothalamic clock presents the most optimistic scenario.  It is tightly localized in a tiny region of the brain, and might be relatively easy to manipulate, with consequences that rejuvenate the entire body.  The hypothalamic clock hypothesis is an attractive target for research because, if correct, it will offer direct and straightforward control over the body’s metabolic age.

That aging unfolds according to an internal clock remains a controversial claim, but what everyone agrees is that the body has some way to know how old it is.  There has to be a clock for development that determines when growth surges and stops, when sex hormones turn on and, if it’s not too great a stretch, when fertility ends and menopause unfolds.

The clock that governs growth and development has yet to be elucidated—a major metabolic mystery by my lights.  The clock that we know about and (sort of) understand is the circadian day-night clock that governs sleep and waking, giving us energy at some times of the day but not others.

Is the life history clock linked to the circadian clock?  Maybe the body just counts days to tell how old it is?  This possibility was eliminated, at least for flies, using experiments with cycles of light and dark that were consistently longer or shorter than 24 hours.  Flies living with fast day-night cycles (less than 24 hours) lived shorter, as predicted; but flies living with long day-night cycles failed to have longer lifetimes,  In fact, deviation from 24 hours in either direction shorten the fly’s lifespan [2005].

But this study suggests the short-term clock and the long-term clock may be linked in a way that is less straightforward.  Melatonin may be another reason to expect a connection.  Melatonin is the body’s cue for sleep, and Russian studies have documented a role for melatonin in aging.  A third motivation comes from the fact that aging disrupts sleep cycles, and (in a downward spiral) disrupted sleep cycles are also a risk factor for mortality and diseases of old age.

Cells seem to have their own, built-in daily rhythms.  I want to say “transcriptional rhythms”, adding the idea that gene transcription is the locus of control; however, red blood cells are the counterexample—they exhibit daily cycles, even though they have no DNA to transcribe [2011].  Individual cycles are designed to be 24 hours, but they would soon drift out of phase with day and night if they weren’t centrally coordinated.  The reference clock that keeps the others in line is in the SCN, the suprachiasmatic nucleus, a handful of nerve cells in a neuroendocrine part of the brain called the hypothalamus.

Think of a million pendulums that are all tuned to swing with a period of 24 hours.  All that it takes is a tiny nudge to all these pendulums each day to keep them in phase with one another, so they are all swinging together.  The SCN provides this nudge in a smart way, based on information from the eyes (light and dark) and endocrine signals that indicate activity and sleep.  The SCN is upstream of the pineal gland, and supplies the signal that tells the pineal gland when it’s time to make melatonthematic index of scarsonatas.  The natural resonances of individual cells become entrained in a body-wide response.


What does all this have to do with aging?

Experiments in the 1980s and 90s showed that the SCN is related to annual cycles, but the relationship seems to be not as strong or as simple or as direct.  For example, squirrels in which the SCN was removed had no daily sleep-wake cycles at all, but their annual cycles of fertility and oscillations of weight were affected inconsistently, more in some animals than others.  Transplanting a SCN from young hamsters into old hamsters cut their mortality rate by more than half, and extended their life expectancies by 4 months [1998].

I have written in this column about research from the laboratory of Claudia Cavadas (U of Coimbra, near Lisbon) indicating that inflammation and inflammatory cytokines in the hypothalamus are at the headwaters of a cascade of signals that lead to whole-body aging.  They have emphasized the role of TGFß binding to ALK5 and of the neurotransmitter NPY.  We usually think of inflammation as a source of damage throughout the body, but in the hypothalamus, inflammation seems to have a role that is more insidious than this, with full-body repercussions.  Blocking inflammation in the hypothalamus is a promising anti-aging strategy.

New Paper on micro RNAs from the Hypothalamus

Along with Cavadas, Dongshen Cai (Einstein College of Medicine) has been a leader in exploring neuroendocrine control of aging that originates in the hypothalamus.  Several years ago, Cai’s group demonstrated that aging could be slowed in mice by inhibiting the inflammatory cytokine NF-kB and the related cytokine IKK-ß just in one tiny area of the brain, the hypothalamus.  “In conclusion, the hypothalamus has a programmatic role in ageing development via immune–neuroendocrine integration…”  They summarized findings from their own lab, suggesting that metabolic syndrome, glucose intolerance, weight gain and hypertension could all be exacerbated by signals from the inflamed hypothalamus.  In agreement with Cacadas, they identified GnRH (gonadotropin-releasing hormone) as one downstream target, and were able to delay aging simply by treatment with this one hormone.  IKK-ß is produced by microglial cells in the hypothalamus of old mice but not young mice.  Genetically modified IKK-ß knock-out mice developed normally but lived longer and retained youthful brain performance later in life.

In the new paper, Cai’s group identified micro-RNAs, secreted by the aging hypothalamus and circulating through the spinal fluid, that contribute to aging.  A small number of stem cells in the hypothalamus were found to keep the mouse young, in part by secreting these micro-RNAs.  Mice in which these stem cells were ablated had foreshortened life spans; old mice that were treated with implants of hypothalamic stem cells from younger mice were rejuvenated and lived longer.  A class of neuroendocrine stem cells from the third ventricle wall of the hypothalamus (nt-NSC’s) was identified as having a powerful programmatic effect on aging.  These cells are normally lost with age, and restoring these cells alone in old mice extended their life spans.

Exosomes are little packets of signal chemicals. Micro-RNAs from stem cells in the hypothalamus are collected into exosomes and shipped down through the spinal fluid.  These exosomes seem to constitute a feedback loop.  On the one hand, they are generated by the hypothalamic stem cells.  On the other hand, they play a role in keeping these same cells young, and producing more exosomes.

Life extension of about 12% was impressive given that there was just one intervention when the mice were more than 1½ years old, but of course it’s not what we would hope for if the master aging clock were reset.  For really large increases in lifespan, we will probably need to reset two or even three of the clocks at once.


The Bottom Line

The reason the body has multiple, redundant aging clocks is to assure that natural selection can’t defeat aging by throwing a single switch.  That means the clocks must be at least somewhat independent.  Nevertheless, I judge it is likely that there is some crosstalk among clocks, because that’s how biology usually works.  To effect rejuvenation, we will have to address all aging clocks, but we see some benefit from resetting even one, and expect more significant benefit from resetting two or more.

The most challenging target is the epigenetic clock,built on a homeostasis of transcription and signaling among hundreds of hormones that each affect levels of the others.  Reverse engineering this tangle will be a bear.

The idea of a centralized aging clock in the hypothalamus seems far more accessible, and is promising for the medium term.  Still, it does not suggest immediate application to remedies.  The hypothalamus is deep in the brain, and you and I might be reluctant to accept a treatment that required drilling through the skull.  A treatment based on circulating proteins and RNAs from the hypothalamus would be less invasive, but even that might have to be intravenous, and include some chemistry for penetrating the blood-brain barrier.  RNA exosomes seem to be our best opportunity

As Cavadas’s group has already pointed out, it is inflammation in the hypothalamus that is amplified by signaling to become most damaging to the entire body.  This raises the interesting question: could it be that the modest anti-aging power of NSAIDs is entirely due to their action within the brain?  In other words, maybe “inflammaging” is largely localized to the hypothalamus.

Inflammation Part 3: resolving inflammation – resolvins, protectins, maresins and lipoxins

These articles by James Watson and Vince Giuliano are not actually research, but are such in depth background that I consider must reading to understand NAD+  This one is published here

Inflammation Part 2: The Tale of Three Stress Sensors and their Interactions: 1)Inflammation, 2)Genomic Instability (p53), and 3)Oxidative stress (Nrf2)

These articles by James Watson and Vince Giuliano are not actually research, but are such in depth background that I consider must reading to understand NAD+  This one is published here

Nicotinamide Riboside supplements aid metabolic health in Mice

cells artResearchers at the Wageningen University in the Netherlands took a different approach to understand the effect of NAD+ pre-cursers and their influence on health. They presented a poster publication during the “10th World Congress on Polyphenols Applications” about an experiment  to test the effects of marginal dietary supplementation with Nicotinamide Riboside (NR) on metabolic flexibility and anti-oxidant response in mice exposed to a high fat diet. This is in particular interesting because most studies with NR have been done at higher therapeutic levels as opposed to levels more typical for daily supplementation with an aim to maintain health.

The researchers fed two groups of adult male mice with a high fat diet containing dosing of 5 or 30 mg NR/kg. They measured body weight, lean mass, fat mass and feed intake weekly. After 14 weeks the metabolic flexibility was assessed with a fasting and re-feeding challenge using indirect calorimetry. With indirect calorimetry a measurement of respiratory exchanges takes place. Oxidation of the energetic substrates by the body is associated with oxygen consumption, carbon dioxide production, and heat release specific to the nature of the energetic substrates being oxidized. In the following week (so at 15 weeks) the researchers determined the anti-oxidant response in epididymal white adipose tissue.

The results showed that in the fasting and re-feeding challenge, the delta respiratory exchange ratio (ΔRER) was higher when comparing 30 to 5 mg/kg NR dosing thus indicating an increase in metabolic flexibility. In addition the expression levels of anti-oxidant genes were enhanced in the 30 mg/kg group compared to the 5 mg/kg group. The body physiological parameters were not affected by the dietary NR.

nr nad namFrom these results it is still not possible to conclude what is the minimum level at which NR supplementation impacts the body as there are no intermediate dose test results between 5mg and 30mg / kg but it seems reasonable to conclude that NR supplementation does impact at a dosing level of  30mg / kg in mice.

Using FDA specified guidelines  we can calculate the Human Equivalent Dose (HED) for the NR diet given to the mice. Using this guideline 30mg/kg dosing in mice translates into a HED of approx. 2.4mg/kg. Or into approximately 170mg daily nicotinamide riboside dosing for a person weighing 70kg.  This is below the limit wrt to side effects and safety and less than the typical serving sizes of 250mg of supplements on the market. The result supports the view that NR supplementation may help maintain health as we age.

Here the updated overview of Nicotinamide Riboside dosing in various studies.

You can find the poster presentation summary here.