AGINGSCIENCES™ – Anti-Aging Firewalls™

I have reported on Induced pluripotent stem cells (iPSCs) in my treatise and in numerous past blog entries(ref)(ref)(ref).  I have viewed these cells as probably providing  the golden keys to closing the loop on the stem cell supply chain allowing extension of human longevity(ref)(ref)(ref).  iPS cells can be created by forcing expression of certain genes in normal somatic (body) cells taken from an individual, like skin cells.  According to most of the literature until recently such cells are pluripotent  like human embryonic stem cells (hESCs), and can be induced to differentiate into any cell type.  They seemed to offer a better alternative for stem cell therapies than use of hESCs because they are autologous, i.e. derived from the same individual they are used on and therefore not subject to immune system rejection.  

As more research on iPSCs is being done, however, evidence has been building that the iPSCs that have been created so far are second-rate in a couple of key respects and so-far unfit for use in human therapies. 

The February 2010 study Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency looks at the differentiation of iPSCs into neural cells.   “For the promise of human induced pluripotent stem cells (iPSCs) to be realized, it is necessary to ask if and how efficiently they may be differentiated to functional cells of various lineages. Here, we have directly compared the neural-differentiation capacity of human iPSCs and embryonic stem cells (ESCs). We have shown that human iPSCs use the same transcriptional network to generate neuroepithelia and functionally appropriate neuronal types over the same developmental time course as hESCs in response to the same set of morphogens; however, they do it with significantly reduced efficiency and increased variability. These results were consistent across iPSC lines and independent of the set of reprogramming transgenes used to derive iPSCs as well as the presence or absence of reprogramming transgenes in iPSCs. These findings, which show a need for improving differentiation potency of iPSCs, suggest the possibility of employing human iPSCs in pathological studies, therapeutic screening, and autologous cell transplantation.”  The study was authored by Su-Chun Zhang and colleagues.  Su-Chun Zhang has published previous studies related to creating neural cells from stem cells such as the 2009 article Differentiation of spinal motor neurons from pluripotent human stem cells.   

I do not think these findings are very surprising since iPSCs are reverted from mature cells and may have short telomeres.  In other words, though newly-created iPSC cells can differentiate into any cell type like embryonic stem cells can, unlike embryonic stem cells the iPSCs made so far are mostly old and tired cells from the viewpoint of replicative potential.  I covered this point in the blog entry Telomeres and telomerase in Induced Pluripotent stem cells – not what we thought.  That blog entry cites the March 2010 publication Spontaneous reversal of the developmental aging of normal human cells following transcriptional reprogramming.  That new study contradicts what had been the prevalent assumption that reverting a cell to iPSC status restores the expression of telomerase and telomere length to equivalent embryonic stem cell length.  This study asserts that reverting a mature cell to iPSC status does not automatically restore its telomere lengths to those found in hESCs.  “Conclusion: Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.”   

Put differently, the March 2010 study says restoring a cell to pluripotency and providing the cell with youth are two quite different things and the former does not necessarily imply the latter.  Pluripotency of an iPSC implies that, like an embryonic stem cell, the iPSC can differentiate into any somatic cell type.  Absence of youth in this case means that the restored iPSC cell could probably not lead to many generations of descendents like an embryonic stem cell could,  because its telomere lengths are short like those in old cells.   In fact, iPSC cells can be near senescent.  The February 2010 Su-Chun Zhang article does not link the observed variability and inefficiency of iPSC differentiation for generating neural cells to short telomeres, but it seems to me that the association may be a key one.   

This question of telomere lengths of iPSCs, nonetheless, appears to be somewhat controversial.  The above-cited publication directly contradicts assertions in a number of earlier publications including the 2009 publication Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells.  That report asserted “We show here that telomeres are elongated in iPS cells compared to the parental differentiated cells both when using four (Oct3/4, Sox2, Klf4, cMyc) or three (Oct3/4, Sox2, Klf4) reprogramming factors and both from young and aged individuals. We demonstrate genetically that, during reprogramming, telomere elongation is usually mediated by telomerase and that iPS telomeres acquire the epigenetic marks of ES cells, including a low density of trimethylated histones H3K9 and H4K20 and increased abundance of telomere transcripts.” 

It seems to me that it is essential to develop reliable techniques for creating iPSCs with long telomeres, perhaps by selecting mutant lines that naturally express telomerase.  That may go a long ways to correcting the problems of lack of differentiation capability and wide variability noted by Su-Chun Zhang and his colleagues.  Or, it might be necessary to address additional issues to get iPSCs to behave as well as hESCs.

At least three approaches to human longevity that have been discussed multiple times in this blog appear to be in trouble or the subject of controversy between groups of scientists: 1.  generation and use of effective and reliable induced pluripotent stem cells, 2. human telomere extension via telomerase activators, and 3. use of resveratrol and its homologs to activate SIRT1 and therefore provide health and longevity.  At best, each of these approaches faces unforeseen hurdles; at worst some of them may simply not be working out.  While I have liked these approaches and written about each from a positive viewpoint, there is a problematic or controversial side to each which I will cover in three different blog entries following this one.

How do we know if something we read about longevity is so?  This post is stimulated by the previous blog entry Another piece of DAF-16 research.  I list a few comments having to do with the creditability of longevity research reports in the general press and then comment on creditability of publications in the scientific literature: 

News reports in the popular press 

·        When the press reports discovery of a new “longevity gene,” don’t believe it until you check it out.  As in the case described in Another piece of DAF-16 research, what is being reported could just be a new study shedding incremental knowledge on a long-known and well-studied gene.

·        The same is true of other reports in the popular press relating to “longevity breakthroughs.”  In recent months, I have come across literally hundreds of articles with titles like “new fountain- of-youth substance discovered” that relate to telomerase.  Most report on minor incremental pieces of research.  Few point out that telomerase was discovered in 1985(ref) and has been intensely studied since. 

·        Recognize that most such reports are written by intelligent well-meaning writers who do not have in depth scientific backgrounds.  Typically, they will pick up on a press report issued by a university or a literature abstract that describes current research but provides no background. 

Reports in the published scientific literature 

·        Most basic discoveries relating to health or longevity are not one-time events but are reported in a developing stream of studies by different researchers located in different centers over a period of years.  Important results are invariably confirmed in multiple publications and the stream becomes a little river.

·        Once in a while, an important concept may not be picked up for years.  For example, the telomere shortening hypothesis and the possibility of telomerase were first proposed by Alexey Olovnikov in 1973(ref), but little attention was paid to it until the 1990s.  Eventually, telomerase was rediscovered and explained by Greider, Blackburn and Stoszak in 1985 and became part of a current rapidly-running river of research.

·        I tend not to trust therapies or health or longevity substances researched, developed, publicized and sold by a lone practitioner or single company, especially if they are based on a “scientific secret” or proprietary non-published data.

·        Once in a while some reported results are controversial with different researchers publishing contradictory results.  In such cases my commitment is to keep digging deeper. If I cannot come up with a conclusion, I will say so.

·        Scientific truth are not absolute, They are relative to the culture and knowledge of the time.  So, even streams of seemingly consistent reports can turn out to be incorrect.  For many decades, based on reputable published studies the medical establishment sternly warned people of the possible toxic consequences of taking more than 400IU of vitamin D a day.  Nowadays the medical establishment has finally acknowledged the important benefits of vitamin D and doses of 2,000IU per day and up are routinely recommended. 

·        In particular, care has to be paid to “what is so” when very large amounts of money are involved as in the case of blockbuster drugs.  Many lucrative money-maker drugs have remained on the market for years before being pulled off for safety or lack-of-efficacy reasons.  See the list here.  In the case of almost all drugs eventually pulled from the market, there were positive previously-published research results, in many cases written by consultant medical people quietly paid off by the drug companies involved(ref)(ref).  Reporting of research paid for by pharmaceutical companies tends to be systematically biased towards favoring the sponsor’s products(ref). “Conclusion Systematic bias favours products which are made by the company funding the research. Explanations include the selection of an inappropriate comparator to the product being investigated and publication bias.” 

It is often very difficult to spot biased research publications ghost-written by doctors paid off by pharmaceutical companies.  When I see a highly positive research review of a new drug, one of my initial reactions is to be a little suspicious.   In writing this blog I try to keep my reporting as unbiased as possible citing multiple sources over long time frames and avoiding reporting where there is a strong commercial interest involved coupled with scant knowledge.  But I am not sure I have always been successful.  For a period, for example, I was possibly too captivated by the idea of telomere extension via astragaloside IV or cycloastragenol as a key anti-aging technology.  This morning, a reader raised a question in a blog comment: do resveratrol and resveratrol homologs really activate SIRT1 or is that mostly hype promoted by Sirtris Pharmaceuticals and the scientists who made many millions of dollars from the acquisition of Sirtris by GlaxoSmithKline(ref)?  This is an issue I will be looking into. 

Some of the popular media touted new April 2010 research as heralding the discovery of a new gene(e.g., AOL news(ref)), but what the new piece of research does is only add another perspective to a long-developing story on how the DAF-16 gene contributes to longevity in nematode worms, and possibly – only possibly – in higher organisms like us. 

I have mentioned the DAF-16 gene in previous blog entries.  See FOXO genes and protecting stem cells — What does resveratrol do?, Calorie restriction research roundup – Part II, and my comment MicroRNAs in cancers and aging, and back-to-the-nematode.  It is an evolutionary-conserved gene belonging to what is called the forkhead family involved in the insulin/IGF-1 pathway and the response to calorie restriction and possibly to taking resveratrol.  Most of the research on DAF-16 the gene has been on members of the Caenorhabditis  family of nematodes, but since the related pathways seem to be evolutionary-conserved, have been thought possibly to apply to us as well. 

Background

For over 22 years it has been known that mutations in certain genes can drastically extend the lives of nematodes.  For example, the 1988 publication A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility reports “age-1(hx546) is a recessive mutant allele in Caenorhabditis elegans that results in an increase in mean life span averaging 40% and in maximal life span averaging 60% at 20 degrees; at 25 degrees age-1(hx546) averages a 65% increase in mean life span (25.3 days vs. 15.0 days) and a 110% increase in maximum life span (46.2 days vs. 22.0 days for wild-type hermaphrodites).”  It was known back then that the IGF1 insulin-like signaling pathway was involved and that two other key genes involved on both longevity and reproduction of caenorhabditis elegans were DAF-2 and DAF-16(ref).  So, forget the idea that DAF-16 is “a newly discovered gene.”

Identification of the potential life-expanding role of DAF-16 in C. elegans is evidenced in the 1993 paper A C. elegans mutant that lives twice as long as wild type: “We have found that mutations in the gene daf-2 can cause fertile, active, adult Caenorhabditis elegans hermaphrodites to live more than twice as long as wild type. This lifespan extension, the largest yet reported in any organism, requires the activity of a second gene, daf-16. — daf-2 and daf-16 provide entry points into understanding how lifespan can be extended.” Also, DAF-16 is discussed in the 1997 paper The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans.   The 2006 review article Worming pathways to and from DAF-16/FOXO summarizes the knowledge as of that time: “Caenorhabditis elegans, the insulin/IGF-1 signaling pathway controls many biological processes such as life span, fat storage, dauer diapause, reproduction and stress response . This pathway is comprised of many genes including the insulin/IGF-1 receptor (DAF-2) that signals through a conserved PI 3-kinase/AKT pathway and ultimately down-regulates DAF-16, a forkhead transcription factor (FOXO). DAF-16 also receives input from several other pathways that regulate life span such as the germline and the JNK pathway –. JNK regulates lifespan in by modulating nuclear translocation of forkhead transcription factor/DAF-16. Therefore, DAF-16 integrates signals from multiple pathways and regulates its downstream target genes to control diverse processes. Here, we discuss the signals to and from DAF-16, with a focus on life span regulation.” So, DAF-16 has long been known to be involved in lifespan regulation in caenorhabditis elegans. 

The new research 

The this-week publication of interest is Phenotypic Covariance of Longevity, Immunity and Stress Resistance in the Caenorhabditis Nematodes.  Basically, the research looked at four different members of the Caenorhabditis family of nematodes that have widely different levels of expression of DAF-16 and widely different disease resistance and longevities.  Higher levels of expression of DAF-16 were found to be correlated with increased disease resistance and longevity. “It is known that lifespan varies significantly among the Caenorhabditis species but, although DAF-16 signaling is highly conserved, it is unclear whether this phenotypic linkage occurs in other species. Here we investigate this phenotypic covariance by comparing longevity, stress resistance and immunity in four Caenorhabditis species.  — We show using phenotypic analysis of DAF-16 influenced phenotypes that among four closely related Caenorhabditis nematodes, the gonochoristic species (Caenorhabditis remanei and Caenorhabditis brenneri) have diverged significantly with a longer lifespan, improved stress resistance and higher immunity than the hermaphroditic species (C. elegans and Caenorhabditis briggsae). Interestingly, we also observe significant differences in expression levels between the daf-16 homologues in these species using Real-Time PCR, which positively correlate with the observed phenotypes. Finally, we provide additional evidence in support of a role for DAF-16 in regulating phenotypic coupling by using a combination of wildtype isolates, constitutively active daf-16 mutants and bioinformatic analysis.”

Going on, “The gonochoristic species display a significantly longer lifespan (p<0.0001) and more robust immune and stress response (p<0.0001, thermal stress; p<0.01, heavy metal stress; p<0.0001, pathogenic stress) than the hermaphroditic species. Our data suggests that divergence in DAF-16 mediated phenotypes may underlie many of the differences observed between these four species of Caenorhabditis nematodes. These findings are further supported by the correlative higher daf-16 expression levels among the gonochoristic species and significantly higher lifespan, immunity and stress tolerance in the constitutively active daf-16 hermaphroditic mutants(ref).”

So, an interspecies comparison among closely related nematodes establishes a correlation between expression level of DAF-16 and disease resistance/lifespan.  It was already known that “In the nematode Caenorhabditis elegans, these phenotypes are molecularly linked such that activation of the forkhead transcription factor DAF-16 both extends lifespan and simultaneously increases immunity and stress resistance(ref).”  The new research is interesting but, given what was already known, hardly earth shaking or even surprising. 

Implications for humans of the new research are not completely clear.  It appears that “sir-2.1 and daf-16 have both overlapping and distinct functions in regulation of C. elegans life span(ref).” But I have had difficulty finding any research directly related to DAF-16 in mice or rats, let alone in humans.  Will resveratrol activate DAF-16 in humans?  Probably not.  A 2008 publication reports “Treatment of C. elegans with the small molecule resveratrol, however, extends life span in a manner fully dependent upon sir-2.1, but independent of daf–16.”  So, it looks like it is going to be a while before implications in humans are known.  First, we will have to hear about implications in mice.

This blog entry is intended to clarify what aging, life extension and anti-aging are about.  It suggests a new way of looking at the theories of aging in my treatise ANTI-AGING FIREWALLS THE SCIENCE AND TECHNOLOGY OF LONGEVITY.

It is easiest to start out with distinguishing between life extension and postponing aging (or anti-aging), which are not quite the same things.  Aging is the most difficult of these matters to define and I will get to it a bit later.

First of all, life-extension approaches are not sure things; they negatively affect the probability of death of a member of a defined population.  Even if you could halt and reverse all causes of aging, the next day you could get run over and killed by a bus.  A lot of things will extend the average lifespan of a population, including quality sewage systems, unpolluted air, and living where there is a law-abiding citizenry.  Included in this list can be pursuance of personal choices such as exercising daily, eating a Mediterranean diet and taking certain supplements.  However, not all things that extend average lifespans also postpone aging.  For example requiring people to wear seatbelts in cars and observe speed limits can extend average lifespans but not affect aging which I see to be a biological process.

In this blog, I have mainly been concerned with life extension approaches that involve biological processes and that slow, stop or reverse aging.  So, the next question is “What is aging?”  Aging starts with birth (actually before birth) and lasts through life.  The part of it I have mainly been concerned with is post-maturational aging, aging that happens after an organism reaches reproductive age.   This later-stage aging is characterized by a number of degenerative processes that accelerate with advancing age, and these are the degenerative processes described by the 14 theories of aging in my treatise and an additional 7 “candidate” theories of aging:

1.     Oxidative Damage

2.     Cell DNA Damage

3.     Mitochondrial Damage

4.     Tissue Glycation

5.     Lipofuscin Accumulation

6.     Chronic Inflammation

7.     Immune System Compromise

8.     Neurological Degeneration

9.     Declines in Hormone Levels

10.   Susceptibility to Cancers

11.   Susceptibility to Cardiovascular Disease

12.   Telomere Shortening and Damage

13.  Programmed Epigenomic Changes

14.  Stem Cell Supply Chain Breakdown

15.   Incorrect protein folding

16.   Accumulation of progerin

17.   Gene mutations leading to hellicase abnormalities

18.   Increasing mTOR signaling

19.   Declining hypoxic response 

20.  Epigenomic changes in DNA methylation and histone acetylation 

21.   Micronutrient triage with aging 

As pointed out in the treatise and many blog discussions, many of these “theories of aging” are highly interdependent, existing in feedback loops where one promotes another, or are results of one or more common underlying processes.  In fact, it is possible to add additional theories of aging in the same spirit such as

22.    Metabolic degradation affecting the expression of sirtuins(ref)(ref), and

23.     Decline in effectiveness of autophagy(ref), and, probably many more yet.

A few key points are:

·        These are not so much theories of aging as they are corollaries of aging or processes that go on during aging. They are all processes that, once one of them is started, it can then can affect multiple body systems and unchain rapid aging in each of them.

·        There are many more corollaries of aging everyone is familiar with such as bone loss, grey hair, loss of hair, wrinkled skin, far-sightedness, loss of teeth, eyesight, hearing, and balance, etc.  While these are not usually seen as theories of aging because they each seem to involve only one system, this is not strictly true in all cases.  Hair stem cells, for example, can differentiate into epidermal and other kinds of cells(ref) and the same is true of stem cells living in tooth pulp(ref).  Bone marrow houses important mesenchymal and haemopoietic stem cells needed for renewal of cells in the blood and other parts of the body.  So, loss of hair or bone or even natural teeth could affect body renewal elsewhere.

.·        It might be possible to extend expected lifespan by interventions that address only one or several of these processes, just as it is possible to do so by diligently wearing seatbelts in a car.  Wearing eyeglasses and hearing aids may extend the lives of those who wear them by preventing accidents.  So, life-extension does not need to address fundamental aging.  

·        Since body systems and the theories of aging are so inter-related, a biologically-oriented antiaging intervention addressed to one theory, such as taking antioxidants(ref), is likely to affect aging with respect to several other theories as well, such as tissue glycation, inflammation and susceptibility to cancers.  The actions in my anti-aging lifestyle regimen affect aging with respect to several of the theories. The same is true of almost all of the supplements in the supplement regimen.  For this reason I call them anti-aging interventions though individually they may not slow or halt aging according to all the corollary theories.

·        The life extension interventions of most interest to me are the ones that slow or halt aging from the viewpoint of most of the 23 theories of aging listed.   In that category appear to be systematic exercise, weight management, and most of the other interventions suggested in my lifestyle regimen. 

·        On the supplement side, to the extent that resveratrol can activate SIRT1 producing the well-studied longevity results of calorie restriction(ref)(ref)(ref), it too affects most of the theories of aging.  Some others of the supplements might do that as well.
 

 What is aging?

This is a question I have ducked up to this point: What is aging?  Actually, we don’t know for sure.  We know all the corollaries of aging as listed, but we are not sure if any of these or any other as-yet unknown process is basic to all of the others.  The later could be the case, or it simply could be that we will never know aging except as through its corollaries.  More or less the same holds for anti-aging interventions.  If we could find an anti-aging intervention that halts or reverses aging according to all of the listed corollaries of aging, then the mechanisms of that intervention would go a long way towards telling us what aging is.  As I have mentioned above activation of SIRT1 via taking resveratrol probably comes as close to being a full anti-aging intervention as we have now.

Some of the 23 theories of aging listed are without doubt more fundamental than others as underlying drivers of aging.  That is, some of the theories are sufficient to explain what goes on according to some of the other theories of aging.   My favorites at the moment are Epigenomic changes in DNA methylation and histone acetylation and Stem Cell Supply Chain Breakdown with Telomere Shortening and Damage being in third place.  Since I have discussed each of these extensively elsewhere I comment only briefly on them here.  You can follow the hyperlinks to find out more about them. 

Epigenomic changes in DNA methylation and histone acetylation is actually a special case of Programmed Epigenomic Changes.  It is a global theory relating to changes that accumulate over a lifetime and that are capable of regulating all genes.  Anti-aging interventions could involve many specialized approaches to protein or histone demethylation or deacetylation.  It is interesting that SIRT1 mentioned above works through being a powerful deacetylase.  To see past blog posts related in some respect to this theory of aging start out with Feb 2009 blog entry Epigenetics, Epigenomics and Aging.  To see more just enter the phrase “epigenomic changes” into the search box to the left of this main text.  

Stem Cell Supply Chain Breakdown is a favorite theory of mine, and not just because because I have formulated it myself.  It says that we are charged up with a supply of pluripotent stem cells at conception (cells capable of differentiating into any body cell type), and these differentiate into our normal body cells (somatic cells) as well as various troves of adult stem cells which throughout life differentiate to make new replacement somatic cells.  The process runs down when the adult stem cells get old and are not replaced, so old senescent and near-senescent somatic cells are not replaced and aging results.  Anti-aging interventions according to this theory could include introduction of embryonic stem cells (ESCs) or “closing the loop” by reverting a few cells taking from a person to induced pluripotent stem cell (iPSC) status(ref)(ref).  It too is a global theory that could explain everything.  To see multiple past blog posts on this theory of aging just enter the word “supply chain” into the search box to the left of this main post.  

Telomere Shortening and Damage is the theory all over the news since the last Nobel prizes were given out, but it has fallen to third place in my listing because 1.  It is not clear to me that telomere shortening is the critical factor limiting human lifespans.  In a healthy young human if telomeres get too short in a cell and apoptotic processes are working well, the cell dies and is replaced by a new cell that is differentiated from an adult stem cell.  The same is not true for adult stem cells because there are no pluripotent cells around to differentiate to replace them, so we are back to the previous theory of aging, and 2.  Despite all the commercial hoop-la about supplements that induce “telomerase activation,” there is no published experimental evidence that people, monkeys, rats, mice or even worms live longer when they are subject to telomerase activation.  And we know for sure that there are a lot of other interventions that make worms, mice and rats live a lot longer than normal. To see multiple past blog posts related to telomeres and telomerase activation just enter the word “telomerase” into the search box to the left of this main text.  

I note that the second and third of these theories could in fact be highly complementary.  Adult stem cells could age and beome depleted because of shortening of their telomeres. If controlled telomerase expression could be activated in adult stem cells, in theory at least it might take a lot longer before they become depleted.

I expect that in another year it will be possible to refine or update the above observations in a significant way.  I could never have written the above when I first drafted my treatise in May 2008.  At that point I had identified only the first dozen of the above 23 theories/corollaries of aging.

It was disturbing to some readers when I characterized niacinamide as a pro-aging substance in the March 24 blog post  SIRT1, mTOR, NF-kappaB and resveratrol, as it was disturbing to me when I first came to that realization years ago.  In response to several blog comments I promised some readers to further research niacinamide and niacin for both health benefits and possible pro-aging hazards, and this blog post reports on that research. I will report on a number of research studies and finally I will give my opinions on how it all comes together and I will even disclose my past secret affair with niacin.

Niacin and Niacinimide

“Nicotinamide, also known as niacinamide and nicotinic acid amide, is the amide of nicotinic acid (vitamin B3/ niacin). Nicotinamide is a water-soluble vitamin and is part of the vitamin B group. Nicotinic acid, also known as niacin, is converted to nicotinamide in vivo, and though the two are identical in their vitamin functions, nicotinamide does not have the same pharmacologic and toxic effects of niacin, which occur incidental to niacin’s conversion. Thus nicotinamide does not reduce cholesterol or cause flushing,^[1] although nicotinamide may be toxic to the liver at doses exceeding 3 g/day for adults.^[2] In cells, niacin is incorporated into nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), although the pathways for nicotinamide and nicotinic acid are very similar. NAD+ and NADP+ are coenzymes in a wide variety of enzymatic oxidation-reduction reactions.^[3] (ref)” 

Continuing, “Nicotinamide adenine dinucleotide (NAD) and its relative nicotinamide adenine dinucleotide phosphate (NADP) are two of the most important coenzymes in the cell. NADP is simply NAD with a third phosphate group attached – (ref).  — NAD plays several key roles in metabolism.  It “participates in many redox reactions in cells, including in glycolysis and most of those in the citric acid cycle of cellular respiration(ref).”  And, as I have discussed previously, SIRT1 requires NAD for its actions(ref)(ref). 

General information on dietary sources of niacin/niacinamide and problems associated with deficiency of vitamin B3 can be found here and here.   “Niacin can be found in nuts, dairy products, lean meats, poultry, fish, and eggs. Some niacin is also supplied by legumes and enriched breads and cereals. The best dietary sources of vitamin B3 are found in beets, brewer’s yeast, beef liver, beef kidney, pork, turkey, chicken, veal, fish, salmon, swordfish, tuna, sunflower seeds, and peanuts. The body can synthesize niacin from the essential amino acid tryptophan, but the synthesis is extremely slow; 60 mg of tryptophan are required to make one milligram of niacin. For this reason, eating lots of tryptophan is not an adequate substitute for consuming niacin. As serotonin synthesis is reliant on tryptophan availability, inadequate dietary intake of vitamin B3 may also therefore lead to depression. The liver is the main storage area for this vitamin and absorption of vitamin B3 takes place in the intestines(ref).”  

Further,  “Niacin works closely with vitamin B1, vitamin B2, vitamin B6, pantothenic acid, and biotin to break the carbohydrates, fats, and proteins in food .  — Vitamin B3 is essential in the metabolism of carbohydrates (to produce energy), fats, and proteins. It also aids in the production of hydrochloric acid, needed for proper digestion. Additionally, vitamin B3 facilitates the body’s ability to eliminate toxins.” —  Vitamin B3 is required by the body for digestive processes, activating enzymes which nourish the brain, regulating blood pressure and regulating cholesterol levels.”  “Niacin is a water-soluble vitamin that participates in more than 50 metabolic functions, all of which are important in the release of energy from carbohydrates. Because of its pivotal role in so many metabolic functions, niacin is vital in supplying energy to, and maintaining the integrity of, all body cells. Niacin also assists in antioxidant and detoxification functions, and the production of sex and adrenal hormones. Vitamin B3 (niacin, niacinamide, nicotinic acid) lowers cholesterol by preventing its buildup in the liver and arteries. Niacin moves fat from tissues for fat metabolism, burning it for energy. It promotes healthy skin, the health of the myelin sheath (the protective covering of the spinal nerves), and good digestion, where it is also vital for the production of hydrochloric (stomach) acid. It is an aid in protecting the pancreas, and is necessary for the health of all tissue cells(ref).”

The government-recommended daily allowance (RDA) for niacin/niacinamide is 16 mg a day for men, 14 mg for women.  The 95^th percentile intake for both food and supplements is estimated to be 40-70 mg for both food and supplements(ref).  Pharmacological doses of either form of B3 can range from 500mg to 2gm daily.  There is a possibility of liver endangerment at the level of 3gms or more daily.

The case for niacinamide promoting health and longevity

B3 has been used now for over 40 years, often in large doses as a drug(ref).  A major application has been control blood cholesterol levels.  According to a 1986 publication: “The Coronary Drug Project was conducted between 1966 and 1975 to assess the long-term efficacy and safety of five lipid-influencing drugs in 8,341 men aged 30 to 64 years with electrocardiogram-documented previous myocardial infarction. The two estrogen regimens and dextrothyroxine were discontinued early because of adverse effects. No evidence of efficacy was found for the clofibrate treatment. Niacin treatment showed modest benefit in decreasing definite nonfatal recurrent myocardial infarction but did not decrease total mortality. With a mean follow-up of 15 years, nearly 9 years after termination of the trial, mortality from all causes in each of the drug groups, except for niacin, was similar to that in the placebo group. Mortality in the niacin group was 11% lower than in the placebo group (52.0 versus 58.2%; p = 0.0004). This late benefit of niacin, occurring after discontinuation of the drug, may be a result of a translation into a mortality benefit over subsequent years of the early favorable effect of niacin in decreasing nonfatal reinfarction or a result of the cholesterol-lowering effect of niacin, or both.”  Later it has become popular to combine niacin with a statin to control blood lipids.  A 2009 study concludes: “ Combined use of extended-release niacin with atorvastatin was superior to atorvastatin monotherapy alone in lipid profile regulation. Combination therapy with niacin ER and atorvastatin was well tolerated and safe in patients with coronary artery disease.” 

Several researchers think that pathways activated by niacin/niacinamide could offer hope for development of new drug treatments for several diseases, diabetes being prime among them,  but further understanding of the complex metabolic pathways involved is a prerequisite.   

The 2008 review article NAD+ and vitamin B3: from metabolism to therapies espouses this viewpoint.  The 2008 publication Triple play: promoting neurovascular longevity with nicotinamide, WNT, and erythropoietin in diabetes mellitus, states “Here we discuss the novel application of nicotinamide, Wnt signaling, and erythropoietin that modulate cellular oxidative stress and offer significant promise for the prevention of diabetic complications in the nervous and vascular systems. Essential to this process is the precise focus upon diverse as well as common cellular pathways governed by nicotinamide.” 

The 2010 publication Diabetes Mellitus: Channeling Care through Cellular Discovery comes to essentially the same conclusion “For these reasons, innovative strategies are necessary for the implementation of new treatments for DM that are generated through the further understanding of cellular pathways that govern the pathological consequences of DM. In particular, both the precursor for the coenzyme beta-nicotinamide adenine dinucleotide (NAD(+)), nicotinamide, and the growth factor erythropoietin offer novel platforms for drug discovery that involve cellular metabolic homeostasis and inflammatory cell control.” 

Health and potential life-extending properties of niacin/niacinamide 

“More recently, the health benefits of niacin have been shown to be far more extensive than previously appreciated — Numerous reports indicate niacin might help prevent atherosclerosis, diabetes and hypercholesterolemia. Niacin is effective in assisting burn-wound recovery, and in the prevention of cataracts and skin cancer. Unfortunately, the molecular basis for most of these health benefits remains unclear(ref).”

The 2006 e-publication Nicotinamide extends replicative lifespan of human cells reports “We found that an ongoing application of nicotinamide to normal human fibroblasts not only attenuated expression of the aging phenotype but also increased their replicative lifespan, causing a greater than 1.6-fold increase in the number of population doublings. Although nicotinamide by itself does not act as an antioxidant, the cells cultured in the presence of nicotinamide exhibited reduced levels of reactive oxygen species (ROS) and oxidative damage products associated with cellular senescence, and a decelerated telomere shortening rate without a detectable increase in telomerase activity. Furthermore, in the treated cells growing beyond the original Hayflick limit, the levels of p53, p21WAF1, and phospho-Rb proteins were similar to those in actively proliferating cells. The nicotinamide treatment caused a decrease in ATP levels, which was stably maintained until the delayed senescence point. Nicotinamide-treated cells also maintained high mitochondrial membrane potential but a lower respiration rate and superoxide anion level. Taken together, in contrast to its demonstrated pro-aging effect in yeast, nicotinamide extends the lifespan of human fibroblasts, possibly through reduction in mitochondrial activity and ROS production. 

Observation 2:  The research literature related to therapeutic use of niacin/niacinamide is at first confusing expressing different viewpoints that have emerged during different periods of time and representing different ways of looking at the effects of substance. Some articles cite reasons why large doses of niacin may promote longevity, and other articles cite reasons why such use may shorten life.

Observation 3:  For about 40 years large doses of niacin have been used for several therapeutic purposes.  Many physicians currently prescribe large doses of niacin for lipid control and other purposes. Mild benefits seem to exist in some areas like raising HDL cholesterol.  The long-term consequences of repeatedly taking large doses are unknown.

Observation 4:  Large scale niacin dosage profoundly affects multiple genes through multiple pathways producing both wanted and unwanted results.  While many researchers are excited by the possibilities of niacin-related therapies for a variety of conditions, the one thing they agree on is a need for further understanding of the pathways involved.

Observation 5:  The sirtuin-related pathways involving the niacin metabolites NAD and NADP are among those related to niacin most intensely studied in recent years.  Large doses of niacin/niacinamide inhibit the expression of SIRT1 and therefore prevent the health and longevity benefits associated with expression of SIRT1.   

Niacin and my personal regimen I have been taking niacin included in a B-complex “B-50” pill taken twice daily, for a total of 100 mg daily.  And added to this is niacin/niacinamide included in my breakfast cereals and other food, perhaps another 40mg.  So, with food I have probably been ingesting  140 mg a day of B3.  This is considerably over the minimum USRDA of 16mg and is possibly excessive for my optimal health.  I am changing my personal regimen and my suggested anti-aging Supplement Regimen to include only one B-50 a day and am looking into different B-complex supplements.  And  clearly, I do not plan to take larger doses of either niacin or niacinamide.

Personal note on my affair with niacin

In the spirit of disclosure, I need to own up to the fact that I once had a torrid affair with niacin.   And I really mean torrid because the stuff gives you hot flushes.  In the late 1970s there were some psychiatric and medical practitioners prescribing megadoses of niacin for all kinds of conditions, including chronic hypoglycemia and what later has come to be called bipolar disease.  It was a period where megavitamin therapy and orthomolecular medicine seemed to be the latest and best things for health.  Proponents of this approach like Dr. Allan Cott were respected people to be paid attention to.   I came under the influence of a New Hampshire medical practitioner who tested me and several of my sons for hypoglycemia, “evaluated” our mental states, and soon the whole bunch of us were taking megadoses of niacin, 1,000-1,500 mg a day.  This at first may have actually benefitted one of my sons who had been having problems with his schoolwork.  Reading some of the orthomolecular medicine literature of the day, I thought niacin was wonderful stuff.  After about three years, however, two things happened that resulted in the affair with niacin ending.  The first is that the son mentioned started taking larger and larger doses after he entered college, hoping it would help him cope with harder and harder schoolwork.  He ended up being seriously ill with a very inflamed liver and had to be hospitalized.  The diagnosis was overdose on Niacin.  The second thing is that we learned that the New Hampshire practitioner was not really a medical doctor as he had represented himself to be.  He was a fake.  We stopped taking megadoses of niacin.  In essence looking back, I think the spirit and intent of orthomolecular medicine was and continues to be a good thing, but that the scientific knowledge required to allow effective orthomolecular therapy to become a systematic practice is only now emerging.

Please see the medical disclaimer for this blog.

In the recent blog entry SIRT1, mTOR, NF-kappaB and resveratrol, I pointed out how “three different theories of longevity seem to be collapsing into one: 1. suppression of mTOR signaling, 2. activation of SIRT1, and 3. inhibition of expression of NF-kappaB.  Activating SIRT1 does all of these things, and this seems to be accomplishable to some extent by taking resveratrol supplements.”  Amplifying on that blog post here, I point out three additional theories of longevity that can be added to the list: 4. hypoxic signaling, 5. autophagy and 6. hormesis.   SIRT1 can activate the pathways of each of these and can in turn be activated by resveratrol

Hypoxic signaling

One my early blog entries was Another longevity-related biochemical pathway – the hypoxic response and I have listed The Hypoxic Response in my treatise as a fifth additional candidate theory of aging. “Another cross-species pathway has been discovered that allows interventions to lengthen life in primitive organisms, C. elegans nematode worms in this case.  The pathway is related to the hypoxic response, how cells respond to protect themselves when there is insufficient oxygen.  It turns out that if the hypoxic response can be turned on when normal oxygen is present, nematodes live significantly longer.  A recent research report indicates that this was experimentally accomplished by breeding nematodes that could not produce the protein VHL-1which destroys another protein called HIF which keeps the hypoxic response turned off when oxygen is present.  Also, it appears that the cells in such long-lived nematodes are relatively free of lipofuscin and toxic age-related protein aggregations such as seen in Alzheimer’s, Huntington’s and other age-related diseases(ref). — As of yet, however, just how HIF works downstream to extend longevity is still unclear.  The hypoxic response appears to operate in higher animals as well, including humans.”

Quoting the June 2009 publication Regulation of Hypoxia-Inducible Factor 2alpha Signaling by the Stress-Responsive Deacetylase Sirtuin 1:  “To survive in hostile environments, organisms activate stress-responsive transcriptional regulators that coordinately increase production of protective factors. Hypoxia changes cellular metabolism and thus activates redox-sensitive as well as oxygen-dependent signal transducers. We demonstrate that Sirtuin 1 (Sirt1), a redox-sensing deacetylase, selectively stimulates activity of the transcription factor hypoxia-inducible factor 2 alpha (HIF-2 ) during hypoxia. The effect of Sirt1 on HIF-2 required direct interaction of the proteins and intact deacetylase activity of Sirt1. Select lysine residues in HIF-2 that are acetylated during hypoxia confer repression of Sirt1 augmentation by small-molecule inhibitors. In cultured cells and mice, decreasing or increasing Sirt1 activity or levels affected expression of the HIF-2 target gene erythropoietin accordingly. Thus, Sirt1 promotes HIF-2 signaling during hypoxia and likely other environmental stresses.”

Autophagy

“In cell biology, autophagy, or autophagocytosis, is a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery. It is a tightly-regulated process that plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products. It is a major mechanism by which a starving cell reallocates nutrients from unnecessary processes to more-essential processes(ref).”  Efficiency of autophagy tends to decline with aging and increasing effective autophagy is seen as a possible anti-aging intervention. According to the 2009 publication Regulation of the aging process by autophagy “During aging, the efficiency of autophagic degradation declines and intracellular waste products accumulate. In Caenorhabditis elegans, there is clear evidence that lifespan is linked to the capacity to regulate autophagy. Recent studies have revealed that the same signaling factors regulate both aging and autophagocytosis, thus highlighting the role of autophagy in the regulation of aging and age-related degenerative diseases.”

The September 2009 publication SIRT1: Regulation of longevity via autophagy reports: “Recent studies have emphasized the importance of SIRT1, a mammalian homolog of Sir2 longevity factor, in the regulation of metabolism, cellular survival, and organismal lifespan. The signaling network interacting with SIRT1 continues to expand as does the number of functions known to be regulated by SIRT1. Autophagy is also an emerging field in longevity studies. Autophagocytosis is a housekeeping mechanism cleaning cells from aberrant and dysfunctional molecules and organelles. The extension of lifespan has been linked to the efficient maintenance of autophagic degradation, a process which declines during aging. Interestingly, recent observations have demonstrated that SIRT1 regulates the formation of autophagic vacuoles, either directly or indirectly through a downstream signaling network. — The interactions of SIRT1 with the FoxO and p53 signaling can also regulate both the autophagic degradation and lifespan extension emphasizing the key role of autophagy in the regulation of lifespan.”

Hormesis

My blog posting Hormesis and age retardation describes hormesis as a process of “challenging cells and body systems by mild stress resulting in them becoming stronger and resistant to aging(ref).  The stress can be physical, chemical and even possibly psychological.”  Exercise is an example.  That blog entry reviews the science behind hormesis and some of its demonstrable anti-aging effects.  Also, see my blog entry Stress and longevity for further discussion of how moderate stresses confer longevity.

Calorie restriction activating SIRT1 is a prime example of hormesis.  The article Sirt1 Regulates Aging and Resistance to Oxidative Stress in the Heart.  “– moderate expression of Sirt1 induces resistance to oxidative stress and apoptosis. These results suggest that Sirt1 could retard aging and confer stress resistance to the heart in vivo, but these beneficial effects can be observed only at low to moderate doses (up to 7.5-fold) of Sirt1.”  And SIRT1 is important for cellular response to other forms of genotoxic stress(ref).  The heat shock response is a well-studied example of hormesis, involving heat shock proteins.  The 2009 publication Stress-inducible regulation of heat shock factor 1 by the deacetylase SIRT1 states “Activation of the deacetylase and longevity factor SIRT1 prolonged HSF1 binding to the heat shock promoter Hsp70 by maintaining HSF1 in a deacetylated, DNA-binding competent state. Conversely, down-regulation of SIRT1 accelerated the attenuation of the heat shock response (HSR) and release of HSF1 from its cognate promoter elements. These results provide a mechanistic basis for the requirement of HSF1 in the regulation of life span and establish a role for SIRT1 in protein homeostasis and the HSR.”

So, when researchers like Leonard Guarante or David Sinclair talk about the sirtuin SIRT1 affecting multiple longevity-related pathways, there is a lot of emerging science behind what they are saying.  And there is a lot more to say about SIRT1 than I have covered so far.   Up to this point I have talked about how six of the 21 theories and candidate theories of aging/longevity described in my treatise are implicated.  But SIRT1 also links to several more of the aging theories, links I will discuss in future blog entries.

A few days ago I visited Leonard Guarante, Director of the Glenn Laboratory for the Science of Aging at MIT and pioneer in the investigation of sirtuins and their longevity properties.  The lead line of the Laboratory’s web site is “We work on mechanisms of aging so that people may lead healthier lives,” an intention that generates a lot of empathy in me.  We talked about some of the current investigations going on in the laboratory and some of Leonard’s views on aging science.  I cover high points of our discussion here, in the processes reviewing some of Leonard’s and the laboratory’s past achievements.  I also cite selected literature references pertinent to the science involved. 

Leonard started investigating aging genes in yeast back in 1991 and his 2003 book Ageless Quest: One Scientist’s Search for Genes That Prolong Youth presents a personal and very readable view of his investigations up to the time of its writing.  His studied the roles of sirtuins in yeast complexes and among his first major contributions over the years was identifying that the genetic pathways activated by the sirtuin SIR2 are the same ones activated by calorie restriction, an evolutionary-conserved pathway known to be capable of reliably conveying longevity across a number of species.  The 1999 publication co-authored by Guarante The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms has been cited by hundreds of subsequent publications and helped to prime the pump leading to what is now a steady river of publications related to sirtuins.  Leonard told me that a year or so ago, one new article relating to sirtuins  appeared just about every day and now about two such articles are appearing daily.  (I would love to cover those here but the task would clearly be impossible.)

Guarante’s 2004 publication The Sir2 family of protein deacetylases tells the story of actions of sirtuins in different organisms.  “We summarize the current knowledge of the Sir2 homologs from different organisms, and finally we discuss the role of Sir2 in caloric restriction and aging.”  This article is cited by 87 others in Pubmed Central.  A short video in which Guarante emphasizes how he believes the benefits of calorie restriction can be realized through activating sirtuins can be found here.  For discussions of pathways involved in calorie restriction and its roles in cancers, see my December 2009 blog entries Calorie restriction research roundup – Part I and Calorie restriction research roundup – Part II. 

The Glenn lab started to focus on sirtuin proteins about a dozen years ago.  Currently, about half of the activity in the lab is focused on the mammalian gene and sirtuin SIRT1, and the rest of the research relates to the other 6 members of the mammalian sirtuin family. SIRT1 is a mammalian homolog gene corresponding to the SIR2 yeast gene.  The working hypothesis with respect to longevity has been that the SIRT genes mediate the pathways of longevity related to calorie restriction, and that better understanding of the actions of the sirtuins could lead to practical interventions that postpone aging.   

The same interventions, it turns out, are likely to be highly useful for prevention or management of many late-onset diseases and diseases that flare with advancing age such as diabetes, Parkinson’s, Huntington’s  and Alzheimer’s.  In fact, Guarante points out that delaying aging is the same as delaying onset of the major diseases of aging, and I heartily agree with him.

About sirtuins, “Sir2 is active as an NAD+-dependent deacetylase, which is broadly conserved from bacteria to higher eukaryotes(ref).”  NAD+ dependency means essential involvement in metabolism.  The discovery of NAD dependency of sirtuins was in Guarante’s lab. Being a deacetylase (or an HDAC) means a capability for transcriptional silencing of gene expression.  See the blog entry Histone acetylase and deacetylase inhibitors for an explanation. “In mammalian cells, Sir2 proteins also deacetylate non-histone proteins such as the p53 tumour suppressor protein, alpha-tubulin and forkhead transcription factors to mediate diverse biological processes including metabolism, cell motility and cancer(ref).”

Guarante has very recently co-authored a review Ten years of NAD-dependent SIR2 family deacetylases: implications for metabolic diseases, a March 10, 2010 e-publication ahead of print. “Since the discovery of NAD-dependent deacetylase activity of the silent information regulator-2 (SIR2) family (‘sirtuins’), many exciting connections between protein deacetylation and energy metabolism have been revealed. The importance of sirtuins in the regulation of many fundamental biological responses to various nutritional and environmental stimuli has been firmly established. Sirtuins have also emerged as critical regulators for aging and longevity in model organisms. Their absolute requirement of NAD has revived an enthusiasm in the study of mammalian biosynthesis of NAD. Sirtuin-targeted pharmaceutical and nutriceutical interventions against age-associated diseases are also on the horizon. This review summarizes the recent progress in sirtuin research (particularly in mammalian sirtuin biology) and re-evaluates the connection between sirtuins, metabolism, and age-associated diseases (e.g., type-2 diabetes) to set a basis for the next ten years of sirtuin research. Copyright © 2010 Elsevier Ltd. All rights reserved.” 

Earlier papers of relevance authored or co-authored by Guarante include Genetics and the specificity of the aging process (2003), Calorie restriction extends life span by lowering the level of NADH (2004), Mammalian SIRT1 represses forkhead transcription factors (2004), Calorie restriction and SIR2 genes—Towards a mechanism (2005), and Calorie restriction–the SIR2 connection (2005).

The Glenn lab works basically with mice, either mice where a gene such as SIRT1 is knocked out in selected tissues or in mice where an extra copy of the gene has been added to the genome, super-sirtuin mice.  In typical past experiments, mice might be challenged, say by feeding them with high-calorie high-fat diets to attempt to induce diabetes in them.  Or a mouse might be genetically modified both to increase susceptibility to Alzheimer’s disease and to express increased amounts of SIRT1.   

Another area of research being pursued in Guarante’s lab involves the roles of sirtuins in the brain.  Guarante thinks sirtuins play important roles in brain aging and repair. In the 2009 publication Neuronal SIRT1 regulates endocrine and behavioral responses to calorie restriction, Guarante and his colleagues reported “Since the somatotropic axis is controlled by the brain, we created mice lacking Sirt1 specifically in the brain and examined the impacts of this manipulation on somatotropic signaling and the CR response. These mutant mice displayed defects in somatotropic signaling when fed ad libitum, and defects in the endocrine and behavioral responses to CR. We conclude that Sirt1 in the brain is a link between somatotropic signaling and CR in mammals.”    In a late 2008 publication by some of Guarante’s counterpart colleagues at Harvard they reported “Using embryonic stem cells, we show that mammalian Sir2, SIRT1, represses repetitive DNA and a functionally diverse set of genes across the mouse genome. In response to DNA damage, SIRT1 dissociates from these loci and relocalizes to DNA breaks to promote repair, resulting in transcriptional changes that parallel those in the aging mouse brain. Increased SIRT1 expression promotes survival in a mouse model of genomic instability and suppresses age-dependent transcriptional changes. Thus, DNA damage-induced redistribution of SIRT1 and other chromatin-modifying proteins may be a conserved mechanism of aging in eukaryotes.”

Among the few interventions that demonstrably extend lifespans across multiple species besides calorie restriction are 1. inhibition of the mTOR pathway, 2. the activation of sirtuins such as via calorie restriction or substances such as resveratrol, and 3. Inhibition of the expression of a cell nuclear factor, NF-kappaB. For a long time these pathways were thought to be independent.  However, recent research reviewed here shows that these aging and longevity-related  pathways are very closely related.   

Background 

The ability to extend lives of organisms from yeast cells to mammals via inhibition of the mTOR (mammalian target of rapamycin) pathway is discussed in my previous blog entries Longevity genes, mTOR and lifespan, Viva mTOR! Caveat mTOR!, and More mTOR links to aging theories.  In my treatise I discuss Aberrant mTOR as one of the Additional Candidate Theories of Aging. Among other actions, Rapamycin fed late in life extends lifespan in mice(ref). 

The mammalian sirtuin SIRT1 activates the same pathway that conveys longevity via calorie restriction, and SIRT1 in turn can be activated by resveratrol and other substances being developed by Sirtris Pharmaceuticals.  I have a long post on this subject in the works based on a recent visit to the Glenn Laboratory for the Science of Aging at MIT and its key leader Leonard Guarante.  A current review of research on the sirtuins over the last 10 years can be found here and discussions of calorie restriction can be found in my December 2009 blog entries Calorie restriction research roundup – Part I and Calorie restriction research roundup – Part II.   

Both laboratory and small-animal studies strongly implicate expression of NF-kappaB in aging and suggest anti-aging interventions based on the inhibition of NF-kappaB.   In my Anti-Aging Firewalls treatise the role of NF-kappaB in aging is discussed under the Programmed epigenomic changes theory of aging.  Also, see the blog posts Updates on NF-kappaB and A further update on NF-kappaB.    

The links between SIRT1 activation, mTOR signaling suppression, and inhibition of NF-kappaB   

A 2007 publication linked the two pathways in the yeast Saccharomyces cerevisiae: MSN2 and MSN4 Link Calorie Restriction and TOR to Sirtuin-Mediated Lifespan Extension in Saccharomyces cerevisiae.  “Here we show that TOR inhibition extends lifespan by the same mechanism as CR: by increasing Sir2p activity and stabilizing the rDNA locus. Further, we show that rDNA stabilization and lifespan extension by both CR and TOR signaling is due to the relocalization of the transcription factors Msn2p and Msn4p from the cytoplasm to the nucleus, where they increase expression of the nicotinamidase gene PNC1. These findings suggest that TOR and sirtuins may be part of the same longevity pathway in higher organisms, and that they may promote genomic stability during aging.”   It was already believed that SIRT1 activation extends lifespan by the same mechanism as CR(see the video).  But, it was yet to be established that a clear link exists in mammals between SIRT1 and mTOR signaling. 

A 2007 doctoral thesis revealed more about the link: Regulation Of Translation And Transcription By Sirt1: Potential Novel Mechanisms For Regulating Stress Response And Aging.  “Both SIRT1 and the target of rapamycin (TOR) are involved in age related diseases and lifespan. We demonstrate for the first time that these two pathways are interconnected. We show that SIRT1 null mouse embryonic fibroblasts (MEFs) have larger cell morphology and upregulated mTOR signaling. Furthermore, SIRT1 activator reduces, whereas SIRT1 inhibitor nicotinamide activates the mTOR pathway.  Rapamycin is effective in inhibiting mTOR activity in both SIRT1 positive and deficient cells. Finally, we show that SIRT1 physically associates with TSC2 in HeLa cells. These observations demonstrate that SIRT1 negatively regulates mTOR pathway upstream of mTOR complex-1 (TORC1), potentially, by regulating the TSC1/2 complex.” 

The same dissertation relates the expression of SIR1 to the suppression of gene activation by NF-kappaB.  “TLE1 is co-repressor for several transcriptional factors including NF-κB. We demonstrate that SIRT1 and TLE1 repress NF-κB activity and that the catalytic activity of SIRT1 may not be critical for this. Using knock-out cell lines, we further demonstrate that both SIRT1 and TLE1 are required for the down-regulation of NF-κB activity. Our results suggest that the interaction between SIRT1 and TLE1 is important for mediating repression of NF-κB activity, potentially through a deacetyalse independent mechanism.”   In the blog post A further update on NF-kappaB, I mention the role of histone deacetylation in preventing the expression of NF-kappaB.  “it involves coiling up the DNA in the neighborhood of genes so that those genes are not accessible for activation by the NF-kappaB. This appears to be the main mechanism used by curcumin, resveratrol and other dietary polyphenols for inhibition of gene activation by NF-kappaB(ref).”   Not surprisingly, SIRT1 is a powerful deacetylase and is activated by resveratrol.

The 2010 publication SIRT1 Negatively Regulates the Mammalian Target of Rapamycin is co-authored by Hiyaa Singhee Ghosh, the author of the aforementioned dissertation, and rounds out our understanding of the linkages even more completely.  “We demonstrate that SIRT1 deficiency results in elevated mTOR signaling, which is not abolished by stress conditions. The SIRT1 activator resveratrol reduces, whereas SIRT1 inhibitor nicotinamide enhances mTOR activity in a SIRT1 dependent manner. Furthermore, we demonstrate that SIRT1 interacts with TSC2, a component of the mTOR inhibitory-complex upstream to mTORC1, and regulates mTOR signaling in a TSC2 dependent manner. These results demonstrate that SIRT1 negatively regulates mTOR signaling potentially through the TSC1/2 complex.”  This publication is rich in detail and I suggest it as a good read for those of you interested in digging further.  Among the observations in this publication are:

·        “Resveratrol suppressed mTOR signaling regardless of stress or growth conditions, suggesting that inducing the catalytic activity of SIRT1 negatively regulates mTOR signaling,”

·        “Both SIRT1 and mTOR have been linked to age-associated diseases with SIRT1 activation having a protective effect, whereas inhibition of mTOR conferring a beneficial effect. For example, SIRT1 activation confers a therapeutic effect in type 2 diabetes, obesity and neurodegenerative diseases such as Alzheimer’s and amyotrophic lateral sclerosis, whereas inhibition of mTOR is protective against cardiovascular and neurological diseases, diet-induced obesity and cancer [31], [32], [33], [34], [35], [36], [37], [38]. Autophagy, a mechanism important in regulating stress response and aging is negatively regulated by mTOR [39], [40], whereas SIRT1 has been reported to activate autophagy by deacetylating several essential components of the autophagy machinery [41], ”

·        “The inverse relationship between the roles of SIRT1 and mTOR in aging-associated diseases and lifespan extension suggests a functional interrelationship between these two proteins. Our results demonstrate that SIRT1 and mTOR signaling pathways are indeed interconnected in a way that promotes stress sensing pro-survival signals, where the regulation of mTOR is mediated potentially through an interaction of SIRT1 with the TSC1-TSC2 complex,” and

·        “Resveratrol has been reported to affect insulin signaling through SIRT1 independent pathways. Consistent with these reports, our data demonstrated that at lower doses, resvetratrol regulated the mTOR pathway in a SIRT1 dependent manner. However, at higher doses, reveratrol likely activated SIRT1 independent pathways in parallel, to inhibit mTOR activity.”

Summary

So, three different theories of longevity seem to be collapsing into one: suppression of mTOR signaling, activation of SIRT1, and inhibition of expression of NF-kappaB.  Activating SIRT1 does all of these things, and this seems to be accomplishable to some extent by taking resveratrol supplements.  As time goes on, even more powerful activators of SIRT1 are likely to become available.

Why not nicotinamide? 

As a side to this discussion, I have often been asked why I do not include nicotinamide, one of the two principal forms of the B-complex vitamin niacin, in my anti-aging supplement regimen.  In fact, nicotinamide was recommended by my physician for control of cholesterol levels but I don’t take it and effectively control my cholesterol in other ways.  The answer is simple: nicotinamide is a powerful inhibitor of SIRT1 and its anti-aging activities, not only capable of negating any benefits from taking resveratrol but also stopping natural expression of SIRT1 in the body.  As stated above “The SIRT1 activator resveratrol reduces, whereas SIRT1 inhibitor nicotinamide enhances mTOR activity in a SIRT1 dependent manner(ref).”  That is, nicotinamide is a pro-aging substance so I avoid it.

Mechanisms for getting stuff into and out of cells are of great importance.  A new item of research came to my attention related to substance-trafficking that goes on within cells and across cell membrane barriers.  It has relevancy in terms of several disease processes and aging.  I review the research in the context of the fascinating cell biology involved.   

Background

We humans may have around 100 trillion eukaryotic cells, complex entities consisting of many components and constantly engaged in many internal activities.   Some of these activities involve transportation of substances either within cells or from the cell’s outer membrane surface into the cell or visa-a-versa.  For those of you not familiar with cell biology I need to identify a number of cell entities that are important for understanding how substance-trafficking in cells works.  I will try to keep things as simple as possible for the purpose of this discussion.  Vesicles are little bubbles of fluid in cells surrounded by lipid bilayers that serve a number of functions including molecular cargo transportation.  Among other things, they can transport needed substances into a cell or unwanted ones out.  “Vesicles store, transport, or digest cellular products and waste. The membrane enclosing the vesicle is similar to that of the plasma membrane, and vesicles can fuse with the plasma membrane to release their contents outside of the cell. Vesicles can also fuse with other organelles within the cell.”  “ — an endosome is a membrane bound compartment inside eukaryotic cells. It is a compartment of the endocytic membrane transport pathway from the plasma membrane to the lysosome. Molecules internalized from the plasma membrane can follow this pathway all the way to lysosomes for degradation, or they can be recycled back to the plasma membrane. Molecules are also transported to endosomes from the Golgi and either continue to lysosomes or recycle back to the Golgi. — Early Endosomes Consist of a dynamic tubular-vesicular network (vesicles up to 1 µm in diameter with connected tubules of approx. 50 nm diameter). Markers include RAB5 and RAB4, Transferrin and its receptor and EEA1(ref).“  “Lysosomes are spherical organelles that contain enzymes (acid hydrolases). They break up food so it is easier to digest. — Lysosomes digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria(ref).”  Finally, “the Gogli apparatus (also Golgi body)^[1] is an organelle found in most eukaryotic cells.  The primary function of the Golgi apparatus is to process and package macromolecules, such as proteins and lipids, after their synthesis and before they make their way to their destination; it is particularly important in the processing of proteins for secretion. The Golgi apparatus forms a part of the cellular endomembrane system(ref).”  OK, enough biological distinctions for now.

The new research

The February 2010 paper The Connecdenn DENN Domain: A GEF for Rab35 Mediating Cargo-Specific Exit from Early Endosomes deals with signaling that tells vesicles how fast they have to move when they are transporting substances to and from the surfaces of cells.  According to ScienceDaily (Mar. 18, 2010) “Defects in this trafficking pathway can have severe consequences, leading to numerous diseases such as high cholesterol, neuropathies, sterility and complications in immune response.– Dr. Peter McPherson and Dr. Brigitte Ritter and their colleagues have discovered how a molecule called Rab35, which acts as a switch is turned on in order to activate the fast-track recycling pathway — in which cargo that needs to be recycled back to the surface of the cell is rapidly selected and transported. — “The cells that make up our bodies are like a busy city,” says Dr. McPherson, neuroscientist at The Neuro and the co-principal investigator for the study. “The cell surface is defined by a membrane that separates its interior from the external world, like the walls or borders of a city. Within this environment, there are simultaneous trafficking pathways that transport vital nutrients, receptors and other components required for cells to function, within cargo vehicles called ‘vesicles.’ Like traffic in a city, these ‘cargo’ vesicles travel at different speeds to numerous destinations within the cell with different purposes. For example, the receptors on the cell surface that bind to cholesterol are on the fast track pathway, so that once they deliver the cholesterol inside the cell, they move back to the surface quickly so that they can pick up some more. It is therefore crucial to understand the controls and switching mechanisms of trafficking inside cells, as this system is of vital importance to the proper functioning of the body.”  

The ScienceDaily article continues “The Rab35 molecule is the trafficking switch for the fast-track or high-priority recycling pathway signaling the quick return of cargo to the cell surface membrane. It is known that Rab35 exists in two forms, ‘on’ (GTP- bound) or off (GDP- bound). When Rab35 is turned ‘on’, it allows the cargo to go back up to the cell surface. What Dr. McPherson and Dr. Ritter and colleagues have discovered is the switch that turns Rab35 on. — “In this study we identified that a particular region of the vesicle-bound protein connecden, called the DENN domain, is the ‘finger’ that flips the switch,” says Dr. Ritter. “The DENN domain connects with the Rab35 molecule and through enzymatic activity converts Rab35 from the inactive to the active form, in essence, turning on the switch.”  You can find articles relevant to the DENN domain here.  According to the new publication the DENN domain is a lipid-binding module with enzymatic guanine nucleotide exchange factor (GEF) activity for Rab35, and Rab35 controls cargo-specific recycling from early endosomes.  “The DENN domain is an evolutionarily ancient protein module. Mutations in the DENN domain cause developmental defects in plants and human diseases, yet the function of this common module is unknown. We now demonstrate that the connecdenn/DENND1A DENN domain functions as a guanine nucleotide exchange factor (GEF) for Rab35 to regulate endosomal membrane trafficking. Loss of Rab35 activity causes an enlargement of early endosomes and inhibits MHC class I recycling. Moreover, it prevents early endosomal recruitment of EHD1, a common component of tubules involved in endosomal cargo recycling. Our data reveal an enzymatic activity for a DENN domain and demonstrate that distinct Rab GTPases can recruit a common protein machinery to various sites within the endosomal network to establish cargo-selective recycling pathways.” 

A second February 2010 relevant e-publication ahead of print is The connecdenn family: Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery.  ‘We recently identified connecdenn (DENND1A), which contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a common and evolutionarily ancient protein module. Through its DENN domain, connecdenn functions enzymatically as guanine-nucleotide exchange factor (GEF) for Rab35. Here we identify two additional connecdenn family members and demonstrate that all connecdenns function as Rab35 GEFs, albeit with different levels of activity. The DENN domain of connecdenn 1 and 2 binds Rab35 whereas connecdenn 3 does not, indicating that Rab35 binding and activation are separable functions. Through their highly divergent C-termini, each of the connecdenns binds to clathrin and to the clathrin adaptor AP-2. Interestingly, all three connecdenns use different mechanisms to bind AP-2.”  Clathrin is a protein which plays a major role in the formation of coated vesicles. 

The ScienceDaily article points to the importance of the new results in helping us understand healthy cell cargo transportation:  “If the finger or the switch itself is mutated or missing, cargo can’t recycle, which has dire consequences,” adds Dr. McPherson. “For example a very important cargo transported by this specific fast track recycling pathway, controlled by Rab35 is the MHC class I receptor involved in the immune system response. If a cell becomes infected by a virus, the MHC receptor is loaded with fragments of the virus that have infected the inside of a cell. The MHC receptor needs to be taken back to the cell surface quickly so that so that it can act as a signpost indicating to circulating immune cells that this particular cell has been infected by a virus and needs to be destroyed, preventing viral infection to other cells.”  If the cargo trafficking system can’t work well the immune system won’t be able to do its job well and the result will be disease and premature aging.   

So, some diseases and aging processes are consequent to problems associated with cell substance-trafficking.  New insights being developed related to the control mechanisms for the cells’ trafficking system are likely to prove very useful.