This post is about a race between two exponentially-accelerating complexes of processes which I will refer to here as death-stalker and life-prolonger.   I am very concerned about how this race will go in the coming few years, for my life will depend on that.

Death-stalker

The death-stalker complex includes all those biological changes that exponentially raise the probability of death with advancing age, making sure that every human being is gone by 123.  The death-stalker complex is evolutionarily determined and up to this point we see no clear way around it.  Death-stalker makes no exceptions.  Death-stalker is complicated and thorough, involving age-related changes in the expression of thousands of genes.  Each of the 14 major theories of aging and six candidate theories of aging laid out in my treatise is an aspect of death-stalker.  Death-stalker works through many mechanisms to kill older people.  Cancers, cardiovascular diseases, neurological and muscular degeneration, deadly falls, loss of hearing, eyesight, memory and balance – all are aspects of death-stalker.  Sooner or later he gets everybody and he will surely get me eventually.  At my age of 80, death-stalker is normally kicking in with full force, each year drastically raising the probability that I will contract a deadly cancer, cardiovascular problem, a debilitating fall or something else that will soon kill me.  So, I am very impatient.  Death-stalker is near my door.

My game with death-stalker is to play to live two, three or more times longer than the normal maximum lifespan of 123.  This entails not defeating him but forcing him to bend the rules he has had for humans during our entire history.  If I can win this concession from death-stalker, it will only be possible with the assistance of life-prolonger.

Life-prolonger

On the other side of the scale is the life-prolonger complex, an amorphous collection of contexts, situations, developments and matters which increase the probability of my life and the lives of others becoming ever-longer.  Included in the life-prolonger complex are:

a.     The contexts of the possibility of eliminating many diseases and radical extensions of lifespans, contexts which affect individual behavior and   which give rise to research and activities directed towards making those possibilities real,

b.     Health-supporting changes in the basic behavior patterns in large population segments, an example being great reduction in smoking and, hopefully in the near future, reduction in obesity,

c.       Improvements in the physical, public health, medical and commercial environments, including extensive health education programs, improved nutrition and sanitation, inoculation programs, a massive health establishment, the popularity of health clubs, and the availability and sale of health supplements. 

d.     Massive government, foundation and NGO investments in health care and health care research.

Health care in the US today represents roughly 18% of GDP.

In the May 2009 blog entry Social ethics of longevity I argued that social evolution requires that people live longer – and is in fact leading to longer and longer life spans.  The above kinds of changes along with a possible evolution in the human epigenome have been leading to a rise in life expectancy in advanced societies every year.  See the blog posts Average US life expectancy up 73 days in one year and  Ever-increasing longevity– is epigenomics involved? 

These developments may well help me get to over 100, but not to 235 which has been my age target for a number of years now.  However, there are other life-prolonger factors which exhibit exponential acceleration in growth and are likely to lead to flowering of new anti aging technology, as outlined in my blog post Factors that drive Giuliano’s Law.  These include improvements in life sciences R&D technology and technological infrastructure, increase in scientific knowledge related to longevity drawing from multiple disciplines, evolution in personal behavior and individuals assuming direct responsibility for both life extension and health.

Because I am so aware of the death-stalker complex and because I monitor anti-aging science developments on a daily basis, positive changes in the life-prolonger complex seem to be progressing only at a slow creep while the death-stalker process is proceeding at its usual inexorable rate. Further, it is easy for me to lose sight of who is working on life-prolonger for me, where and how.  And how effectively? The good folk in my HMO are helping a lot in the short term, but they have no notion of how to get me over the 123 year hump.  It seems that unless life-prolonger speeds up a lot, I am not going to make it. 

Viewed over the 20 month period since I generated the first online version of my treatise ANTI-AGING FIREWALLS THE SCIENCE AND TECHNOLOGY OF LONGEVITY, however, a tremendous amount has happened in the life-prolonger complex qualifying as exponentially-accelerating progress.  A single measure that I report on in this blog is genome sequencing, particularly the cost and time for sequencing of entire human or disease genomes.

I have reported before on gene testing, in May 2009, Consumer Genomics and Individual DNA testing. Genetic testing is quite different from genome sequencing.  The former involves scanning a few dozen, hundreds or even thousands of genes for variations.  Genetic testing is typically done to check for the probable efficacy of a drug, to predict susceptibilities to certain cancers, or to provide consumers with personal gene information using a service like 23 and me.   Genome sequencing is far more comprehensive, looking at the entire genome of an individual consisting of about 3 billion base pairs of DNA.  It requires 46 separate chromosome sequences in order to represent the complete genome of a human individual.  Again, the objective is to identify evolutionary and mutational differences from what is expected in a normal human genome like small deletions and insertions, SNPs, multiple copies of genes, interchromosomal translocation events, mutations, gene mislocations, etc.  Here are some benchmark points:

·        For over 30 years there has been continuous improvement in the technology of sequencing, lowering of its cost and improvement of its accuracy.  There have been multiple generations of DNA sequencing technology(ref).

·        Between 1994 and 2004 the cost of sequencing dropped a hundredfold.

·        The cost for sequencing the genome of a single individual in 2004 was about $10 million dollars(ref).  At that time the goal was to reduce the cost to $100,000 in 5 years(ref).  The goal was exceeded, the cost in mid-2009 being around $50,000.

·        Today the cost of sequencing the genome of a single individual has dropped to the $6,000 range and is expected to drop to $1,000 in less than another year.

From the latest issue of Gen: “GEN News Highlights — Life Technologies reports that it is $5,000 away from reaching the hallowed ground of the $1,000 genome. The company has introduced the Applied Biosystems SOLiD™ 4 Sequencing System, which generates up to 100 gigabases of mappable sequence data per run at a cost of $6,000 per genome. Illumina would be another contender in this race, with its HiSeq2000, generating data at $10,000 per genome.”

·        With a continuation of the present trend I expect costs of sequencing an entire human genome to come down to around $100 by 2015, making whole-genome sequencing a routine health-supporting process for everyone with health care.

·        The same economics apply to sequencing the entire genomes of cancer cells and other organisms, making available vast databases of comparative genomic information.

Having everyone’s genome sequenced and on-file will serve the cause of longevity in multiple ways.  Included in these are creation of immense databases that will assist:

·        in clarifying the functions of genes and gene regulation throughout the genome,

·     Understanding variations in the DNA sequences among individuals  and determining what they mean. Understanding small differences may help predict a person’s risk of particular diseases and response to certain medications. For example, see the discussion in the blog post CETP gene longevity variants,

·     Developing treatment protocols related to drug selections, therapy selections for certain cancers and other medical conditions that differentially depend on a patient’s genomic profile,

·     Understanding the 3-dimensional structures of proteins and identifying their functions (see the blog post Protein origami and aging),

·     Exploring how DNA and proteins interact with one another and with the environment to create complex living systems (See the blog post The new omics and longevity research)

·     Understanding the epigenomic factors that are involved in aging and possible anti-aging epigenomic interventions, and

·     Using this knowledge to develop and apply genome-based strategies for lifestyle interventions for longevity and the early detection, diagnosis, and treatment of diseases.  (The blog post Genes discussed or mentioned in this blog provides links to a number of relevant discussions in this blog.)

Researching the above discussion led me to an example of who is out there working on life-prolonger for me, namely the good folk at Life Technologies, the ones out to bring the cost of whole-genome scanning down to $1,000 this year.  From the Life Technologies website “Life Technologies (NASDAQ: LIFE) is a global biotechnology tools company dedicated to improving the human condition. — Our systems, consumables and services enable researchers to accelerate scientific exploration, driving to discoveries and developments that make life even better. — Life Technologies customers do their work across the biological spectrum, working to advance personalized medicine, regenerative science, molecular diagnostics, agricultural and environmental research, and 21st century forensics. The company had sales of more than $3 billion, employs approximately 9,500 people, has a presence in more than 100 countries, and possesses a rapidly growing intellectual property estate of approximately 3,600 patents and exclusive licenses. Life Technologies was created by the combination of Invitrogen Corporation and Applied Biosystems Inc.” 

Wow, they are part of life-prolonger even if I never met any of them, and they are getting breakthrough results.There are hundreds of more biotech companies out there working on various aspects of life-prolonger for me, representing hundreds of billions of dollars in economic activity and utilizing incredible computer and brainpower on my behalf.  Add in the university and hospital researchers, the government labs, the pharma company labs.  The results being produced in many cases represent exponential change, not just linear change.  That’s life-prolonger, getting stronger and meaner every day.  Watch out death-stalker, you are going to have to share the stage when it comes to calling the life-and-death shots.  And, death-stalker, I do recognize that it is more comfortable for most people to call you by your usual name, aging.

A review article published in the February, 9, 2010, issue of the Journal of the American College of Cardiology points out that a number of herbal remedies may become dangerous when their use is combined with taking certain cardiovascular drugs.  How to view this fact depends on your perspective.  One perspective, that of the mainline cardiology profession, places the onus on the herbal supplements.  It says that if your doctor puts you on a life-saving drug, you should avoid herbal supplements that combine dangerously with that drug.  The minority perspective, that of the supplement industry, points out that the supplements of concern are generally a lot safer than the drugs of concern.  For example, if you are taking the drug warfarin (also known as Coumadin), a classical rat poison, you certainly do have to be careful about what you combine it with.  Stay away from garlic, ginkgo biloba, ginger, alfalfa, saw palmetto, green tea, bilberry, fenugreek, ginseng, chondroitin sulfate or vitamin k.  Warfarin kills rats by excessively thinning their blood and those herbal or vitamin substances tend to either potentiate or inhibit warfarin’s blood thinning.  The green tea is only evil when you are already taking the rat poison.

The article Use of Herbal Products and Potential Interactions in Patients With Cardiovascular Diseases starts out “More than 15 million people in the U.S. consume herbal remedies or high-dose vitamins. The number of visits to providers of complementary and alternative medicine exceeds those to primary care physicians, for annual out-of-pocket costs of $30 billion. Use of herbal products forms the bulk of treatments, particularly by elderly people who also consume multiple prescription medications for comorbid conditions, which increases the risk of adverse herb-drug-disease interactions. — In this review, we highlight commonly used herbs and their interactions with cardiovascular drugs. We also discuss health-related issues of herbal products and suggest ways to improve their safety to better protect the public from untoward effects.” 

Drug-herb interactions should be taken seriously for they can be matters of life and death.   According to an accompanying Feb 1 2010 ACC news release entitled As Use of Herbal Remedies Soars, Patients Taking These and Cardiovascular Medications May be at Heightened Risk of Dangerous, Potentially Life-Threatening Interactions “Many people have a false sense of security about these herbal products because they are seen as ‘natural,’” Arshad Jahangir, M.D., Professor of Medicine and Consultant Cardiologist, Mayo Clinic Arizona, — “But ‘natural’ doesn’t always mean they are safe. Every compound we consume has some effect on the body, which is, in essence, why people are taking these products to begin with.” — In addition to their direct effects on body function, these herbs can interact with medications used to treat heart disease, either reducing their effectiveness or increasing their potency, which may lead to bleeding or a greater risk for serious cardiac arrhythmias.  — “We can see the effect of some of these herb-drug interactions—some of which can be life-threatening—on tests for blood clotting, liver enzymes and, with some medications, on electrocardiogram,” Dr. Jahangir said.  — According to the report, a major concern is that patients do not readily disclose their use of herbal remedies, and healthcare providers may not routinely ask about such use. In addition, because these herbs are regarded as food products, they are not subject to the same scrutiny and regulation as traditional medications.”

Other examples of drug-herb interactions exist besides those involving blood thinning.  “For instance, St. John’s wort, which is often taken to treat depression and anxiety, affects how the body absorbs dozens of prescription medications and may diminish the efficacy of statins and beta-blockers, a class of drugs used to treat high blood pressure and heart-rhythm disorders. — Even grapefruit juice, which people often drink for weight loss and heart health, can increase the blood concentration of statins, raising the risk of liver damage and muscle pain, the report notes(ref).”

Any reader of a vampire novel knows that acquiring the blood of a young person is the secret of a vampire’s eternal youth.  In fact, the essence of being a vampire is a constant quest for such acquisition.  According to a news stories that broke today, it seems like scientific knowledge is finally catching up. 

It is also common knowledge among us longevity-science types that somatic stem cells are subject to senescence and that, with aging, these stem cells progressively lose their capability to reproduce and differentiate. (See the discussion in my treatise related to the Stem Cell Supply Chain Breakdown theory of aging.)  “Buildup of levels of Ink4a/P16 associated with aging slows down the rate of differentiation of adult stem cells.” Further, age-related loss of capability to reproduce and differentiate has to do with what is going on in the niches in which stem cells live.  “Our results reveal that aged differentiated niches dominantly inhibit the expression of Oct4 in hESCs and Myf-5 in activated satellite cells, and reduce proliferation and myogenic differentiation of both embryonic and tissue-specific adult stem cells (ASCs). Therefore, despite their general neoorganogenesis potential, the ability of hESCs, and the more differentiated myogenic ASCs to contribute to tissue repair in the old will be greatly restricted due to the conserved inhibitory influence of aged differentiated niches(ref).”  Along with this decline in stem cell renewal capability comes a breakdown in the efficacy of the stem cell supply chain, aging and death.  According to a January 30 2010 news item appearing in Science Daily “A team of Howard Hughes Medical Institute (HHMI) researchers has found that in old mice, a several-week exposure to the blood of young mice causes their bone marrow stem cells to act “young” again.”  Dracula, why are you acting bored? 

The publication related to the new research is Systemic signals regulate ageing and rejuvenation of blood stem cell niches and appeared in the January 28 issue of Nature. “Ageing in multicellular organisms typically involves a progressive decline in cell replacement and repair processes, resulting in several physiological deficiencies, including inefficient muscle repair, reduced bone mass, and dysregulation of blood formation (haematopoiesis). Although defects in tissue-resident stem cells clearly contribute to these phenotypes, it is unclear to what extent they reflect stem cell intrinsic alterations or age-related changes in the stem cell supportive microenvironment, or niche. Here, using complementary in vivo and in vitro heterochronic models, we show that age-associated changes in stem cell supportive niche cells deregulate normal haematopoiesis by causing haematopoietic stem cell dysfunction. Furthermore, we find that age-dependent defects in niche cells are systemically regulated and can be reversed by exposure to a young circulation or by neutralization of the conserved longevity regulator, insulin-like growth factor-1, in the marrow microenvironment. Together, these results show a new and critical role for local and systemic factors in signaling age-related haematopoietic decline, and highlight a new model in which blood-borne factors in aged animals act through local niche cells to induce age-dependent disruption of stem cell function.”

This does not sound much like vampire talk.  Some of the press reports about the work are more lucid if not lurid.  According to the Science Daily writeup “Hematopoietic stem cells give rise to all the cells of the blood system, including immune cells and red blood cells. As animals age, these stem cells become more numerous, but less effective at regenerating the blood system, Wagers says. That translates into a less effective immune system and a greater susceptibility to disease. — To see if younger blood could reverse the sluggishness of aging blood cells, the researchers began by surgically joining the bloodstreams of pairs of mice that were of different ages, but nearly clones of one another.” (Hmmn, joining bloodstreams?  That does sound rather vampire-like.)  “Each mouse carried distinctive genetic markers so that researchers could differentiate between its cells and those of its partner. The technique, called parabiosis, enables researchers to test the long-term effects of one animal’s blood on the tissues and organs of the other. “It’s the only model that really allows us to come close to mimicking an in vivo systemic environment,” Wagers (Amy Wagners, the lead investigator) said. “There is a constant exposure to any cell or soluble factor that circulates, at close to physiologic levels.”  — After several weeks of sharing their blood systems with young mice, the hematopoietic stem cells of the older mice changed markedly. Exposure to a younger animal’s blood somehow pushed the older animal’s hematopoietic stem cells back to a more youthful state, in which they were fewer in number but recovered nearly all of their blood-cell-generating capacity. When transplanted into mice whose own blood-producing cells had been eliminated by radiation, the “rejuvenated” stem cells repopulated the blood with a mixture of cell types similar to that generated by transplanted young stem cells. No such changes occurred in the young mice in these pairings, or among age-matched pairs of animals.”

There is significantly more to the recent research findings, and that is that IGF-1 expression in osteoblasts present in the haematopoietic stem cell bone marrow niches is responsible for the decline in vitality and differentiation capabilities of haematopoietic stem cells in older mice, and neutralizing the IGF-1 in the bone marrow also restores the vitality and differentiation capabilities of these stem cells.

In more detail, ” Wagers and her team haven’t yet discovered the blood-borne factor that triggers this apparent restoration of youthfulness in aged hematopoietic stem cells. But they did find two important clues to how it transmits its effects.  — First, they found evidence that this factor works via bone-forming cells known as osteoblasts, which also are present in bone marrow and help regulate hematopoietic stem cells. When old animals were exposed to young blood, their osteoblasts reverted to more youthful numbers. They also behaved more like younger osteoblasts in their interactions with hematopoietic stem cells. Hematopoietic stem cells grown in cultures with these “rejuvenated” osteoblasts regained the blood-cell-generating capacity characteristic of youthful stem cells. For osteoblasts, the opposite was also true: the bone-forming cells of young animals- from humans as well as mice — showed signs of aging when they were exposed to blood from an older animal. — The team also found that the insulin-like growth factor 1 (IGF-1) hormone appears to be necessary to maintain these stem-cell-regulating osteoblasts in an aged state. When they blocked IGF-1 activity in osteoblast cells in culture or in bone marrow, aged osteoblasts reverted to a “younger” state, and could pass that rejuvenation effect on to hematopoietic stem cells. Blocking IGF-1 activity in the bloodstream of mice didn’t have the same effect, which suggests that IGF-1 acts specifically through osteoblasts. — Oddly enough, IGF-1 is best known for its growth-promoting and potentially anti-aging effects in other tissues, including muscles and bones. “Our findings highlight the fact that IGF-1 signaling is complex and depends in part on the tissue involved,” said Wagers(ref).”

Getting back to vampires, the results of the new study suggests a cure for their centuries-old thirst for blood, since it suggests that blocking the effects of IGF-1 in bone marrow osteoblasts could have the same rejuvenating affect as the blood of a young person.  Of course this would have to be validated by a clinical trial.  Can you imagine a drug company setting up such a trial for vampires where half the participants take a drug that blocks bone marrow IGF-1 and the control group participants go out and hunt human victims and drink their blood in normal vampire fashion? 

Seriously, there just could be some longevity benefit to selective blocking of IGF-1 in osteoblasts.  More must be learned about this possibility.  And the mouse results must be validated in humans.

Do you remember the Monopoly card that says “Go to jail.  Go directly to jail.  Do not pass Go, Do not collect $200?”  Well, imagine that there is a cell reprogramming card that says, say when you land on skin cell, “Go to nerve cell, go directly to nerve cell.  Do not pass iPSC status. Do not collect pluripotent reprogramming factors.”  Very recent research shows that that card exists, and its existence is giving us a broad new perspective on epigenetic regulation of cell fates.  By modifying epigenetic factors, it appears that one type of body cell can be changed, very likely, into any other type of body cell, directly and without a need for reversion into induced pluripotent stem cell (iPSC) status. And the process is efficient.  This post reviews background on cell reprogramming, the new research in context, and speculates on the implications.

Background on cell reprogramming

Research on reprogramming cells from one type to another goes back to the 1980s, long before the first iPSC was produced.  The first work in this area involved fusing two different kinds of cells together to form heterokaryons.  A hetrokaryon is “A cell with two separate nuclei formed by the experimental fusion of two genetically different cells(ref).”  A 1986 publication reports Rapid reprogramming of globin gene expression in transient heterokaryons, where “Interspecific heterokaryons were formed by fusing adult mouse erythroleukemia (MEL) cells and human embryonic/fetal erythroid (K562) cells with each other, or with a variety of mouse and human nonerythroid cell types.”  A series of other publications based on studies of heterokaryons followed.  A 1993 publication Reversibility of the differentiated state in somatic cells reported “Analysis of de novo gene activation in multinucleated heterokaryons has shown that the differentiated state, although stable, is not irreversible, and can be reprogrammed in the presence of appropriate combinations of trans-acting regulatory molecules.”

A 1999 publication Use of somatic cell fusion to reprogram globin genes reports “Experiments with heterokaryons demonstrate that the reprogramming is due to trans-acting factors that are developmental-stage-specific. These results suggest the feasibility of using fusisome-carried sets of nuclear factors to reprogram somatic cells.”  A relatively recent January 2009 study Nuclear reprogramming in heterokaryons is rapid, extensive, and bidirectional reports “Here, we show that hundreds of genes are activated or repressed within hours of fusion of human keratinocytes and mouse muscle cells in heterokaryons, and extensive changes are observed within 4 days.”

Another thread of research related to cell reprogramming was cloning.  Dolly, the world’s most famous sheep, was cloned in 1996.  “The production of Dolly showed that genes in the nucleus of such a mature differentiated somatic cell are still capable of reverting back to an embryonic totipotent state, creating a cell that can then go on to develop into any part of an animal(ref).^[11]”

Another chain of studies in the mid 2000s related to cell reprogramming involved the impact of microenvironment on cell fate.  It was found that when cloned liver stem cells were placed into a cardiac microenvironment, they transformed themselves to acquire a cardiac phenotype and function(ref)(ref)(ref). “Collectively, these results support the conclusion that these adult-derived liver stem cells respond to signals generated in a cardiac microenvironment ex vivo acquiring a cardiomyocyte phenotype and function(ref).”

The hetrokaryon studies, the cloning work and the studies related to the effect of microenvironment indicate that cells of one kind can be directly reprogrammed into cells of another kind and that there is some kind of molecular signaling process involved.  The big more-recent cell reprogramming news of course was the ability to revert any cell to embryonic stem cell-like pluripotency, the creation of induced pluripotent stem cells (iPSCs) starting in 2006.  The first comprehensive discussion of iPSCs in this blog was the March 2009 post Rebooting cells and longevity, and iPSCs have been mentioned or discussed in many subsequent blog posts.  The first studies described the use of four transcription factor proteins to create iPSCs: Oct4, Sox2, Klf4, and c-Myc.  Much progress in creating iPSCs in the last year including use of other transcription factor combinations, safer less-oncogenic vectors for insertion of the transcription factors, induction of stem cell expression without using transgenes, and, most recently, the use of vitamin C to improve the efficiency of reprogramming(ref)(ref). 

The 2008 publication Reprogramming of somatic cell identity summarized the situation as of the time “Nuclear transfer and cell-fusion experiments demonstrate that the epigenetic signature directing a cell identity can be erased and modified into that of another cell type. Furthermore, in the case of cloning, differentiated cells can be reprogrammed back to pluripotency to support the reexpression of all developmental programs. Recent breakthroughs highlight the importance of transcription factors as well as epigenetic modifiers in the establishment, maintenance, and rewiring of cell identity.” 

Nonetheless, the excitement about iPSCs led many researchers to forget or ignore the earlier research on cell reprogramming and assume that if one wants to start with, say, skin cells and end up with nerve (or heart or liver) cells, just about the only practical approach is a two-step one: 1.  Take some skin cells and revert them to being iPSCs, an inefficient process even when using vitamin C, and then  2.  Somehow convince those iPSCs to progressively differentiate to become nerve cells, possibly a quite tricky thing to do in-vivo.  The new research finding suggests that with the right transcription factors it might be possible to start out with any kind of cell and end up with any other kind of cell without going through an intermediate stage. 

Direct cell reprogramming

2008 saw the publication of a breakthrough study In vivo reprogramming of adult pancreatic exocrine cells to beta cells.  “Here, using a strategy of re-expressing key developmental regulators in vivo, we identify a specific combination of three transcription factors (Ngn3 (also known as Neurog3) Pdx1 and Mafa) that reprograms differentiated pancreatic exocrine cells in adult mice into cells that closely resemble -cells. The induced -cells are indistinguishable from endogenous islet -cells in size, shape and ultrastructure. They express genes essential for -cell function and can ameliorate hyperglycaemia by remodeling local vasculature and secreting insulin. This study provides an example of cellular reprogramming using defined factors in an adult organ and suggests a general paradigm for directing cell reprogramming without reversion to a pluripotent stem cell state.”

The new January 2010 research study report Direct conversion of fibroblasts to functional neurons by defined factors reports “Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses.”

The transcription factors used in both studies are different than those used to create iPSCs and the cell-type conversion process is much more efficient than that of reverting cells to iPSC status.  The 2008 study was exciting because it described direct cell reprogramming in-vivo in a way that addresses a disease, albeit in a mouse model.  Regarding the 2010 study, the “neurons could integrate into pre-existing neural networks and form independent synapses with each other.  — This system bypasses production of tumorigenic pluripotent cells, a main barrier to using iPSCs in regenerative medicine, and may provide a platform for more efficient disease modeling and drug discovery(ref).” “They also tested the procedure on skin cells from the tails of adult mice. They found that about 20 percent of the former skin cells transformed into neural cells in less than a week. That may not, at first, sound like a quick change, but it is vast improvement over iPS cells, which can take weeks. What’s more, the iPS process is very inefficient: Usually only about 1 to 2 percent of the original cells become pluripotent(ref).”

Implications include:

·        hESCs, iPSCs and other stem cell types are likely to turn out to be extremely important, but are not the only games-in-town for producing desired cells where and as needed.

·        Cloning taught us that all body cells encompass the same genes and that any one cell encompasses the possibilities in all other cells.  The differences among cells are ones of epigenetic gene expression.  The latest research indicates it may be possible freely to change one cell type to another via introducing highly specific transcription factors.

·        It may turn out to be practical to convert many cell types to other cell types in-vitro, in-vivo or both, allowing the development of many new regenerative medicine applications.  The challenge is discovering the transcription factors and other epigenetic modifiers needed and how to introduce them so as safely get a desired result.

·        The new work probably makes addressing cell senescence even more critical.  I suspect that transforming an old-near-senescent skin cell into a nerve cell will produce an old near-senescent nerve cell unless issues like telomere lengths are also addressed.

·        The new work is likely to contribute to an acceleration in research relating to the discovery and isolation of gene transcription factors(ref), micro-RNAs(ref), HATs and HDACs(ref), DNA demethylases(ref) and other gene regulatory factors.  Unraveling all of those may well take decades. 

I am not worried about running out of work here.

In the blog entry The stem cell supply chain – closing the loop for very long lives, I have suggested that it might be possible to re-introduce fully pluripotent stem cells into the body so as to close the loop in the stem cell supply chain and enable much longer lives. This post reports progress towards that point, again a topic related to Vitamin C suggested by reader jeg3.

Background

Recapitulating the essence of the stem cell supply chain concept: “Stem Cell Supply Chain Breakdown is the newest theory of aging described in my treatise and the one I am currently most excited about.  According to a simplified model of this theory a newly-conceived human embryo consists of pluripotent stem cells (Type A), ones that can potentially divide into any body cells.  With growth, these proliferate and, in a remarkably articulated manner, progressively differentiate into multipotent stem cells (Type B), progenitor cells (Type C), mature body somatic cells (Type D), and many eventually become senescent cells (Type E).”  — ‘According to the best current understanding of stem cells this is an open-loop once-through process.  The above list is in order of increasing cell-type specificity and decreasing cell-type potency to differentiate into other cell types.  Starting at conception and throughout life, all cells on this list except the senescent ones will selectively reproduce and possibly differentiate into cells of types further down in the list.  The state of the body in terms of makeup of cell types continues to change through life and the process goes inexplicably from start (conception) leading to end (death’).  The stem cells themselves are subject to replicative senescence.   Early in life, Type A cells tend to vanish.  With aging, pools of type B and type C cells become exhausted and are less capable of differentiation to renew the supply of Type D cells.  The stem cell supply chain slows down and ceases to function well.  There are fewer healthy Type D cells and more Type E cells, and disease and death soon follow.

The blog entry The stem cell supply chain – closing the loop for very long lives suggests an approach that could conceivably transform the stem cell supply chain from being a once-through process to being a continuous open-loop process.   “There is a possibility of keeping the stem cell supply chain active indefinitely.  The key idea is to use induced Pluripotent Stem cells (iPSCs) which are fully pluripotent and equivalent to embryonic stem cells(ref)(ref)(ref) as feedstock Type A cells in adults to make the stem cell supply chain as a continuous loop process instead of a once-through process.”

I assume the reader is generally familiar with iPSCs and the general approaches to reprogramming cells to iPSC status.  See, for example, the blog posts Rebooting cells and longevity, Update on induced pluripotent stem cells and “Footprint-free” iPSCs – and a crazy wager offer.

This blog post reports research progress on creating iPSCs which may eventually lead to closing the loop.  An old dear friend seems to be involved, Vitamin C.  The study Vitamin C Enhances the Generation of Mouse and Human Induced Pluripotent Stem Cells was timed as a Christmas present and published December 24 2009.  Some of the points related to the new study are:

1.     The approaches to reverting cells to iPSC status have been remarkably inefficient.  “Soon after the exciting discovery of a method to transform human skin cells into stem cells in 2007 came the frustration of actually trying to make a sufficient amount of these induced pluripotent stem (iPSC) cells.  The process is so inefficient that scientists typically only get 0.01 percent of a sample of human skin, or fibroblast, cells to form iPS cell colonies after they infect fibroblasts with the retroviruses used to induce pluripotency(ref).”  The new study report indicates  “However, the low efficiency of iPSC generation is a significant handicap for mechanistic studies and high throughput screening, and also makes bona fide colony isolation time consuming and costly. The efficiency of alkaline phosphatase-positive (AP⁺) colony formation with the four Yamanaka’s factors (Sox2, Klf4, Oct4, c-Myc; SKOM) in mouse fibroblasts is about 1% of the starting population, but only around 1 in 10 of those colonies is sufficiently reprogrammed to be chimera competent – -. ”  This iPSC reprogramming inefficiency has been noted by others as well(ref)(ref).

2.     It is hard to revert old or near-senescent cells to iPSC status given age-related upregulation of tumor suppressor genes. “While our work was in progress, six independent laboratories identified cell senescence as a roadblock for reprogramming (Hong et al. 2009) — “Functional analyses of these genes demonstrate that the p53-p21 pathway serves as a barrier not only in tumorigenicity, but also in iPS cell generation,”  Kawamura et al., 2009, Li et al. 2009 “In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming by activation of p53 (encoded by Trp53) and p21 (encoded by Cdkn1a); whereas, in human fibroblasts, INK4a is more important than ARF. Furthermore, organismal ageing upregulates the Ink4/Arf locus and, accordingly, reprogramming is less efficient in cells from old organisms,” Marión et al. 2009 “These observations indicate that during reprogramming cells increase their intolerance to different types of DNA damage and that p53 is critical in preventing the generation of human and mouse pluripotent cells from suboptimal parental cells,”  Utikal et al., 2009, Zhao et al., 2008).”  This has led to significant interest in finding compounds that “alleviate cell senescence without increasing the risk of mutations.”  The researchers set out testing antioxidants for this purpose.  The one that worked was vitamin C.

3.    The main finding of the study is that vitamin C can markedly improve the efficiency of the reprogramming process for both mouse and human cells. “We show here that vitamin C, a common nutrient vital to human health, enhances the reprogramming of somatic cells to pluripotent stem cells. By adding Vc to the culture medium, we can now obtain high-quality iPSCs from mouse and human cells routinely.”   Exactly how vitamin C works to achieve this end is not clear.  Other tested antioxidants appeared not to have an effect.  It is highly possible that epigenetic factors are involved.  “Besides reducing p53, Vc accelerates transcriptome changes during reprogramming and allows the conversion of pre-iPSCs to iPSCs. The extent to which these observations relate to cell senescence is unclear, and it is possible that Vc is acting in other ways as well. For example, it could accelerate stochastic events during reprogramming, perhaps by promoting epigenetic modifications that allow further changes to proceed. In this regard, Vc is a cofactor in reactions driven by dioxygenases including collagen prolyl hydroxylases, HIF (hypoxia-inducible factor) prolyl hydroxylases, and histone demethylases (Shi, 2007), and it is interesting to consider that Vc might influence reprogramming by increasing the activity of these enzymes. Histone demethylases are important for development and modulate the expression of the ESC master transcription factor Nanog (Cloos et al., 2008), so it is possible that Vc allows the reprogramming to run more smoothly by facilitating histone demethylation.” 

The new finding can result in increased productivity in creating iPSCs.  However, there is still a way to go before the “closing the stem cell supply chain loop” hypothesis can be tested.  If iPSCs are created outside the body from a person’s tissue, safe ways must be found to introduce them back into the body so they will go about replenishing stocks of Type B and Type C stem cells without creating problems such as tumors or teratomas.

Oh Spirit of Linus Pauling Great Father of Vitamin C, are you listening?  A few days ago I wrote a blog entry Surprise!  Just when we thought we knew everything about vitamin C, pointing to new research indicating that vitamin C could be a cure for Werner’s Syndrome.  It looks like regularly taking vitamin C does a myriad of other things besides serving as a good antioxidant, things like preventing DNA damage induced by renovascular hypertension, and helping to control obesity.  This week’s new finding relates to the usefulness of Vitamin C in creating iPSCs, possibly an important finding for regenerative medicine.

We have new powerful frameworks for looking at old familiar substances like vitamin C, frameworks like epigenetics, proteomics, telomere science and cell cycle molecular biology, and these frameworks are telling us things about vitamin C that Linus may have intuited but could not have put into words.  Because most of the needed words did not exist in his time.

The  responses to my blog posts tell me that many of you readers out there join me in being telomerase life-extension aficionados.  In case you don’t already know about it, you might want to have a look at the Sierra Sciences website.  Sierra Sciences is a small biotech research company (30 scientists), completely devoted to discovering new activators of the telomerase gene to serve the cause of human longevity.  The company was founded by Bill Andrews, one of the discoverers of the telomerase gene back when he was in charge of the molecular biology research group at Geron.

Going to the Sierra Sciences website you are greeted by a video presentation featuring Bill Andrews.  Going on to the home page you are greeted with the slogan “Cure Aging of Die Trying,” which I can personally identify with.  “Sierra Sciences, LLC is a company devoted to finding ways to extend our healthspans and lifespans beyond the theoretical maximum of 125 years.” 

Basically, the company is screening substances, searching for telomerase inducers. The home page reports: “As of January 20, 2010: We have screened 189,264 compounds — We have found 555 telomerase inducers — These represent 34 distinct drug families — Most potent compound = 6% of goal — Check back frequently for updates! — We are screening 4,000 compounds per week”. 

The website features a few fairly current video presentations relating to telomerase as well as older ones.   Finally, the company is seeking the involvement of others:  “Sierra Sciences is seeking individuals passionate about finding the cure for aging to get involved in the company at levels of grant funding, strategy, and management. — We are looking for individuals who will be interested in giving us their input on strategies and helping us to take whatever steps necessary to achieve this cure within our lifetimes.”

This blog is now a year old and represents an accumulation of 232 posts and 270 comments.  My favorite thing seems to be reporting recent research findings in context, providing discussion and a network of citations for understanding how newly-reported research fits in to what is already known.  This has required much thinking on my part and many ideas that are original to me have appeared here.  But there also have been two streams of brand-new original thinking that have appeared in this blog, ideas that did not exist “out there” before they appeared here.  The purpose of this post is to highlight those two streams and point to the posts that contain them.

Giuliano’s Law

The first stream relates to Giuliano’s Law, which is “Starting now, every seven years will see the emergence of practical age-extension interventions (ones that have a potential of leading to extraordinary longevity) that double the power of the interventions available at the start of the 7 year period.  That is, on an average basis, the practical anti-aging interventions available at the end of a seven-year period will enable twice the number of years of life extension than did the interventions available at the start of the period.  Life extension is measured in years of life expectancy beyond those actuarially predicted for a given population.

This law and some of the rationale for it was laid out in the March 2009 post Giuliano’s Law: Prospects for breaking through the 122 year human age limit.  It is an analog of Moore’s law for the power of computers and is valid for many of the same reasons.  The post Factors that drive Giuliano’s Law goes into those reasons in detail, a positive feedback loop  of interaction exists between societal need, market, marketing channels and economics, changes in user expectations, market vehicles, user applications, marketing channels, advancement in the relevant basic science, advancement in technology, advancement in manufacturing, and entrepreneurial environment. 

I argue that the juggernaut defined by these interacting factors is already immense, growing mightily in power and on the road just as surely as the computer revolution was on the road in 1958.  The same advances that further health and medicine will further the cause of longevity.  Anti-aging science is not some arcane discipline off to the side.  It is a natural byproduct of the life sciences revolution that is well underway.  

The blog post More on Giuliano’s Law; calculating my longevity prospects is a more personal one, looking at my own life expectancy assuming Giuliano’s law is correct under three scenarios: Case 1:  I discontinue my anti-aging firewalls program and go about living a normal life.  Case 2: I continue pursuing my existing anti-aging firewall program keeping it exactly as it is now and Case 3: I continue to follow all the relevant threads of anti-aging research, to update the Anti-Aging Firewalls Treatise weekly or more as I have been doing, and periodically update the firewalls and firewall program to reflect this emerging new knowledge.  Further, I incorporate new science-based anti-aging substances and procedures into the firewall program as they become available. As you might guess, the Case 3 projection is that I have a good shot at breaking the 123 years maximum age barrier.  I hope that I am right!

The stem cell supply chain theory of aging

After generating a number of blog posts related to stem cells and stem cell differentiation I started to see a whole new viewpoint on aging connected with stem cells.  I first laid this viewpoint out in my September 2009 blog post An emerging new view of aging – the stem cell supply chain.  The idea is that there is a hierarchy of stem cells in human bodies ranging from pluripotent embryonic-like stem cells at the top to specialized progenitor cells just above ordinary somatic cells, with senescent cells at the very bottom of the heap.  In a healthy living organism, a supply chain is in constant operation.  Cells at one level are replenished by differentiation of cells at a higher level.  In aging the pools of stem cells at the higher levels become exhausted, cell regeneration via differentiation of stem cells is compromised, and sickness and death follow. 

The blog entry The stem cell supply chain – closing the loop for very long lives suggests an approach that could conceivably transform the stem cell supply chain from being a once-through process to being a continuous loop.  The idea is to generate autologous induced pluripotent stem cells in order to keep the stem cell supply chain operating indefinitely.  I have created a large number of posts about stem cells and the stem cell supply chain is an underlying concept of several of them.

A follow-up posting will comment on how my views of aging and anti-aging interventions have evolved since starting this blog.

It seems like scarcely a day goes by now without new telomerase research news items showing up in the popular press, the latest having to do with fish oil.  I mention this news here but my purpose is to make a few broader points:

1.      Taking a number of popular supplements in the anti-aging firewalls Supplement Regimen like Vitamin E, fish oils, Vitamin D3 and resveratrol can lead to telomeres being longer than they otherwise might be, possibly because they induce the production of telomerase, possibly for other reasons.  As such, these supplements are quite possibly life-extending.

2.     Despite the popular conception, telomere lengths do not uniformly get shorter with advancing age.  Sometimes they get longer over substantial periods of time.  Nobody is quite sure of how or why.

3.     Many of the same supplements that lead to longer telomeres in healthy people seem to have the capacity to turn off telomerase and shorten telomeres in cancer cells and help kill them.

Fish Oil and longer telomeres

Yesterday’s news is based on a January 20 publication in JAMA: Association of marine omega-3 fatty acid levels with telomeric aging in patients with coronary heart disease.  “Context  Increased dietary intake of marine omega-3 fatty acids is associated with prolonged survival in patients with coronary heart disease. However, the mechanisms underlying this protective effect are poorly understood. ^— Objective  To investigate the association of omega-3 fatty acid blood levels with temporal changes in telomere length, an emerging marker of biological age. ^— Design, Setting, and Participants  Prospective cohort study of 608 ambulatory outpatients in California with stable coronary artery disease recruited from the Heart and Soul Study between September 2000 and December 2002 and followed up to January 2009 (median, 6.0 years; range, 5.0-8.1 years). ^— Main Outcome Measures  We measured leukocyte telomere length at baseline and again after 5 years of follow-up. — Results  Individuals in the lowest quartile of DHA+EPA experienced the fastest rate of telomere shortening (0.13 telomere-to-single-copy gene ratio [T/S] units over 5 years; 95% confidence interval [CI], 0.09-0.17), whereas those in the highest quartile experienced the slowest rate of telomere shortening (0.05 T/S units over 5 years; 95% CI, 0.02-0.08; P < .001 for linear trend across quartiles). –. Each 1-SD increase in DHA+EPA levels was associated with a 32% reduction in the odds of telomere shortening (adjusted odds ratio, 0.68; 95% CI, 0.47-0.98). ^— Conclusion  Among this cohort of patients with coronary artery disease, there was an inverse relationship between baseline blood levels of marine omega-3 fatty acids and the rate of telomere shortening over 5 years.”

The temptation is to conclude that “taking fish oils leads to less telomere length shortening,” but that is not what the study says.  The conclusions of this study are based on levels of DHA and EPA measured at baseline and do not take possible supplementation during the study period into account.  Further, the study population was a very special one, people with coronary artery disease.  Another temptation is to conclude that fish oil leads to the expression of telomerase, but this conclusion is also not directly supported.  The study does not say why the rate of telomere shortening was less in those with higher baseline levels of the fish oils.  Nontheless, the popular press has yielded to these temptations with news story titles like Is Fish Oil the Elixir of Life? And  Stay young by eating fish oil, say scientists. I chalk this up to a general hunger in the population for anti-aging news.  And now, after Blackburn, Greider and Szostak  have received a Nobel prize for work on telomeres and telomerase, it is almost household news that longer telomeres are associated with longevity and are better for health.

Natural telomere lengthening with age

The report on omega-3 fish oils and telomeres was preceded two weeks ago by another PLoS ONE report based on data for the same 608 individuals in the Heart and Soul Study Telomere length trajectory and its determinants in persons with coronary artery disease: longitudinal findings from the heart and soul study.  “METHODOLOGY/PRINCIPAL FINDINGS: In a prospective cohort study of 608 individuals with stable coronary artery disease, we measured leukocyte telomere length at baseline, and again after five years of follow-up. We used multivariable linear and logistic regression models to identify the independent predictors of leukocyte telomere trajectory. Baseline and follow-up telomere lengths were normally distributed. Mean telomere length decreased by 42 base pairs per year (p<0.001). Three distinct telomere trajectories were observed: shortening in 45%, maintenance in 32%, and lengthening in 23% of participants. The most powerful predictor of telomere shortening was baseline telomere length (OR per SD increase = 7.6; 95% CI 5.5, 10.6). Other independent predictors of telomere shortening were age (OR per 10 years = 1.6; 95% CI 1.3, 2.1), male sex (OR = 2.4; 95% CI 1.3, 4.7), and waist-to-hip ratio (OR per 0.1 increase = 1.4; 95% CI 1.0, 2.0). CONCLUSIONS/SIGNIFICANCE: Leukocyte telomere length may increase as well as decrease in persons with coronary artery disease. Telomere length trajectory is powerfully influenced by baseline telomere length, possibly suggesting negative feedback regulation. Age, male sex, and abdominal obesity independently predict telomere shortening.”

Note that this is not the first study to show average telomere length increasing for a substantial part of the study population over a substantial period of time. According to a large Swedish study, a third of the population experienced telomere lengthening over 9 to 11 year intervals(ref).

Fish Oil, other supplements and turning off telomerase in cancers

According to the 2005 report Polyunsaturated fatty acids inhibit telomerase activity in DLD-1 human colorectal adenocarcinoma cells: a dual mechanism approach “We investigated the inhibitory effect of various fatty acids on telomerase, with particular emphasis on those with antitumor properties, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).  — In contrast, cis-unsaturated fatty acids significantly inhibited the enzyme, and the inhibitory potency was elevated with an increase in the number of double bonds. Accordingly, polyunsaturated fatty acids (PUFAs), like EPA and DHA, appeared to be powerful telomerase inhibitors. — Culturing DLD-1 cells with either EPA or DHA resulted in a remarkable decrease in telomerase activity. EPA and DHA inhibited telomerase by down-regulating human telomerase reverse transcriptase (hTERT) and c-myc expression via protein kinase C inhibition. These results indicate that PUFAs can directly inhibit the enzymatic activity of telomerase as well as modulate the telomerase at the transcriptional level.” 

So there we have it.  The same DHA and EPA fish oils that seem to be correlated with longer telomeres in the recent population study also clobber telomerase in a cancer cell line.  This property seems to be shared by several other popular supplements as well. 

Alpha-tocopherol (Vitamin E) seems to repress age-related telomere shortening(ref). Yet, the 2007 study Vitamin E suppresses telomerase activity in ovarian cancer cells concludes “Our data suggest that, by suppressing telomerase activity, Vitamin E may be an important protective agent against ovarian cancer cell growth as well as a potentially effective therapeutic adjuvant.”  

Another supplement that is both associated with longer telomere lengths and that inhibits telomerase expression in cancer cells is Vitamin D3.   The 2007 paper Higher serum vitamin D concentrations are associated with longer leukocyte telomere length in women reports ”Serum vitamin D concentrations were measured in 2160 women aged 18–79 y (mean age: 49.4)  — Serum vitamin D concentrations were positively associated with LTL (longer telomere length) (r = 0.07, P = 0.0010), and this relation persisted after adjustment for age (r = 0.09, P < 0.0001) and other covariates (age, season of vitamin D measurement, menopausal status, use of hormone replacement therapy, and physical activity; P for trend across tertiles = 0.003). The difference in LTL between the highest and lowest tertiles of vitamin D was 107 base pairs (P = 0.0009), which is equivalent to 5.0 y of telomeric aging.” Yet, D3  is another substance that can help inhibit the expression of telomerase in cancer cells as pointed out in the 2003 publication Combination treatment with 1alpha,25-dihydroxyvitamin D3 and 9-cis-retinoic acid directly inhibits human telomerase reverse transcriptase transcription in prostate cancer cells.  Also, see Induction of Ovarian Cancer Cell Apoptosis by 1,25-Dihydroxyvitamin D3 through the Down-regulation of Telomerase.

Resveratrol is another supplement substance that seems to have a dual personality, on the one hand associated with enhancing telomerase activity in healthy cells(ref)(ref) and on the other hand inhibiting expression of telomerase in cancer cells(ref)(ref).   

The active ingredient in green tea EGCG appears to be yet another dietary substance with the dual personality characteristic.  Drinking ample quantities of green tea appears to slow down age-dependent telomere shortening on the one hand(ref),  and EGCG represses telomerase expression in cancer cells(ref)(ref).  I am sure the same point can be made for other substances in the anti-aging supplement regimen.

Telomerase regulation is in fact a very complex process(ref).   As I have put it in my treatise “These results suggests to me that telomere shortening is a complex process involving a balance of shortening due to cell division, lengthening due to natural telomerase expression and perhaps cell replacement due to differentiation of stem cells. And these in turn are affected by many lifestyle and dietary factors and moderated by cell-signaling feedback loops.” 

Yet, it could well be the case that management of telomere length is our best hope for realizing extraordinary longevity in the nearer future.  The 12^th theory of aging in my treatise Telomere Shortening and Damage forwards the hypothesis that longer telomere lengths are likely to be correlated with longer lifespans and that keeping one’s telomeres as long as possible through expression of telomerase is vital for health and longevity. Telomeres and telomerase are among my favorite subjects for treatment in this blog.  Among the many relevant blog postings are the recent postings Exercise, telomerase and telomeres, Timely telomerase tidbits, Breakthrough telomere research finding, and Telomere and telomerase writings. And, as time proceeds, I expect there will be more.

In researching the previous blog post Changing the threshold for neuromuscular fatigue in the young and old, carnosine or beta-alanine supplementation, I discovered a fascinating set of relationships among the substances mentioned in the title of this post and promised to report further on them.  I do that here, requiring a review of some of the basic biochemistry involved.  Although I am not clear of all the implications involved, I flag a few of these in the areas of pain management, synapse development and learning, maintaining mental balance, sleep and mental acuity.   

GABA

GABA (gamma-Aminobutyric acid) “is the chief inhibitory neurotransmitter in the mammalian central nervous system. It plays a role in regulating neuronal excitability throughout the nervous system. In humans, GABA is also directly responsible for the regulation of muscle tone.^[1] (ref)”  The operation of GABA is complex.  “In vertebrates, GABA acts at inhibitory synapses in the brain by binding to specific transmembrane receptors in the plasma membrane of both pre- and postsynaptic neuronal processes. This binding causes the opening of ion channels to allow the flow of either negatively charged chloride ions into the cell or positively charged potassium ions out of the cell. This action results in a negative change in the transmembrane potential, usually causing hyperpolarization. Two general classes of GABA receptor are known: GABA_A in which the receptor is part of a ligand-gated ion channel complex, and GABA_B metabotropic receptors, which are G protein-coupled receptors that open or close ion channels via intermediaries (G proteins).  GABA_A receptors are chloride channels, that is, when activated by GABA, they allow the flow of chloride ions across the membrane of the cell (ref).” “GABA_B receptors (GABA_BR) are metabotropic transmembrane receptors for gamma-aminobutyric acid (GABA) that are linked via G-proteins to potassium channels.^[1] These receptors are found in the central and peripheral autonomic nervous system^[2](ref). 

Carnosine, homocarnosine, anserine and beta-alanine

Carnosine and homocarnosine are closely related dipeptide substances, both found in substantial quantities in the mammalian brain and muscles, and they are similar also to anserine found in bird muscles and brains as well as humans.  L-Carnosine is a dipeptide composed of the two amino acids L-histidine and beta-alanine.  And Homocarnosine is a dipeptide composed of the amino acids L-histidine and GABA.  The chemical structures of the two substances are remarkably similar; you can see them diagrammed here.  (Unfortunately, the way this blog software is set up it is hard for me to include diagrams here).  This little article L-Carnosine and Related Histamine-Derived Molecules comments further on the three substances. “Carnosine and homocarnosine are both produced by the same ATP-driven enzyme, carnosine synthetase, and both molecules exhibit very similar properties. The concentration of homocarnosine in the human brain, however, is about 100 times that of carnosine. It is manufactured by glial cells (oligodendrocytes) except in the olfactory bulb, where it is synthesized by neurons. The highest brain homocarnosine concentrations are found in the substantia nigra, dentate gyrus and olfactory bulb as well as in the cerebrospinal fluid.”

So, going back to the discussion of the previous blog entry, Changing the threshold for neuromuscular fatigue in the young and old, carnosine or beta-alanine supplementation, chemically, beta-alanine is one of the dipeptide components of l-carnosine, the wonderful stuff discussed in the blog entry The curious case of l-carnosine.  Homocarnosine, on the other hand, molecularly substitutes GABA for beta-alanine. 

The three dipeptides Carnosine, homocarnosine, and anserine, have a number of biological properties in common.  All appear to be antioxidants(ref).  All three are protective against peroxyl radical-mediated Cu,Zn-superoxide dismutase modification(ref).  Both carnosine and homocarnosine can detoxify the highly reactive aldehyde acrolein(ref).  Of particular note, all three are inhibitors of GABA metabolism.  That is, they lead to higher levels of GABA in the brain.  This was pointed out in a 1978 publication Homocarnosine, carnosine and anserine on uptake and metabolism of GABA in different subcellular fractions of rat brain.  “L-Carnosine, L-homocarnosine and L-anserine are inhibitors of GABA metabolism. They show differential action on GABA-transaminase from synaptosomes compared to the extrasynaptosomal enzyme.” A 2004 publication also identifies beta-alanine as a GABA uptake inhibitor. 

Much of the research literature on these substances was published prior to 2000. The more-recent literature has since been scanty and scattered as indicated in the 2005 title Carnosine and homocarnosine, the forgotten, enigmatic peptides of the brain.  

Carnosine and homocarnosine are degraded in the body by carnosinase, “An enzyme that hydrolyzes carnosine (amino-acyl-l-histidine) and other dipeptides containing l-histidine into their constituent amino acids(ref).”  Activity of carnosinase tends to increase with age, leading to lower levels of carnosine in older people, however a very recent publication suggests that the presence of homocarnosine tends to inhibit the degradation of carnosine by carnosinase.  “Activity of carnosinase (CN1), the only dipeptidase with substrate specificity for carnosine or homocarnosine, varies greatly between individuals but increases clearly and significantly with age. — Further, CN1 activity was dose dependently inhibited by homocarnosine. — Homocarnosine inhibits carnosine degradation and high homocarnosine concentrations in cerebrospinal fluid (CSF) may explain the lower carnosine degradation in CSF compared to serum. Because CN1 is implicated in the susceptibility for diabetic nephropathy (DN), our findings may have clinical implications for the treatment of diabetic patients with a high risk to develop DN. Homocarnosine treatment can be expected to reduce CN1 activity toward carnosine, resulting in higher carnosine levels.”

Further background information on carnosine, homocarnosine, anserine and other related nerve and muscle histidine can be found in the online monograph Carnosine and Oxidative Stress in Cells and Tissues. This monograph also describes several pathways through which these substances can be created as metabolic products of each other.

A few of the key points for the purpose of this discussion are:

·        While carnosine can play key health and longevity-supporting roles, it or beta-alanine are far from the only games in town.  Carnosine acts in synergy with homocarnosine and its levels are controlled by carnosinase. 

·        Although exogenous supplementation is possible with one or several of these dipeptides, they are created and broken down in the body in complex ways.  Homocarnosine can be formed when GABA replaces the beta-alanine component in carnosine.  It appears that carnosine and beta-alanine release is stimulated by glutamatergic receptors, at least in cultured rat oligodendrocytes(ref).  

·        Supplementation with carnosine and/or beta-alanine may be valuable for athletes and older people as described in the blog entry Changing the threshold for neuromuscular fatigue in the young and old, carnosine or beta-alanine supplementation.

·        Carnosine, beta-alanine and homocarnosine increase levels of GABA. 

Gabapentin

I have discussed the drug gabapentin in the blog entry Spinal cord injury pain – a personal story and a new paradigm. As pointed out there it has been both a very popular as well as controversial drug used as an antic-convulsant, for control of neuropathic pain and, off-label, for a number of other psychiatric and medical conditions.  Gabapentin, like carnosine, beta-alanine and homocarnosine, is a GABA agonist that increases GABA levels in the brain(ref)(ref).  Further, gabapentin increases brain levels of homocarnosine(ref)(ref).  The studies cited were on patients or on tissues from patients prone to seizures, but I would wager that at least some increases in GABA and homocarnosine levels due to taking gabapentin would apply in general.

In my blog entry on neuropathic pain, I highlighted the possible role of gabapentin in quieting pathological pain due to over-excited microglia.  A July 2009 e-publication suggests that carnosine and N-acetyl carnosine might possibly be able to accomplish a similar result.  “Chronic inflammation and oxidative stress have been implicated in the pathogenesis of neurodegenerative diseases. A growing body of research focuses on the role of microglia, the primary immune cells in the brain, in modulating brain inflammation and oxidative stress. One of the most abundant antioxidants in the brain, particularly in glia, is the dipeptide carnosine, beta-alanyl-L-histidine. — The aim of the present study was to examine the role of carnosine and N-acetyl carnosine in the regulation of lipopolysaccharide (LPS)-induced microglial inflammation and oxidative damage. –. The data shows that both carnosine and N-acetyl carnosine significantly attenuated the LPS-induced nitric oxide synthesis and the expression of inducible nitric oxide synthase by 60% and 70%, respectively.  — we demonstrated a direct interaction of N-acetyl carnosine with nitric oxide. LPS-induced TNFalpha secretion and carbonyl formation were also significantly attenuated by both compounds. N-acetyl carnosine was more potent than carnosine in inhibiting the release of the inflammatory and oxidative stress mediators. These observations suggest the presence of a novel regulatory pathway through which carnosine and N-acetyl carnosine inhibit the synthesis of microglial inflammatory and oxidative stress mediators, and thus may prove to play a role in brain inflammation.”

Having been on gabapentin for three months now has contributed to vanishing my neuropathic pain due to a spinal injury and keeps me sleeping soundly.  See my blog entry Spinal cord injury pain – a personal story and a new paradigm. 

However, there are a few things I don’t like about gabapentin, one being that it often leaves me sleepy in the mornings making it difficult to concentrate. More seriously, I recently discovered something that bothers me a lot: gabapentin inhibits neuron synapse formation and therefore probably impairs new learning.  This was reported in an October 2009 study Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. “We have previously identified thrombospondin as an astrocyte-secreted protein that promotes central nervous system (CNS) synaptogenesis. Here, we identify the neuronal thrombospondin receptor involved in CNS synapse formation as alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin.“  As explained in the LA Times Booster Shots: “Stanford University researchers examined the interaction between neurons and brain cells called astrocytes. Previous studies showed that a protein that astrocytes secrete, thrombospondin, is critical to the formation of the brain’s circuitry. In the study, researchers found that thrombospondin binds to a receptor, called alpha2delta-1, on the outer membrane of neurons. In a study in mice, they showed that the neurons that lacked alpha2delta-1 could not form synapses in response to the presence of thrombospondin. — Alpha2delta-1 is the receptor for gabapentin. That has been known, although scientists did not understand how gabapentin worked. But the new research revealed that when gabapentin was given to mice, it prevented thrombospondin from binding to the receptor, thus stopping the synapse formation.  While gabapentin, which is sold under the trade name Neurontin, does not dissolve pre-existing synapses, it prevents the formation of new ones. That’s why the medication may be dangerous if given to pregnant women or young children, the authors said. The majority of the brain’s synapses are formed in uteri and early childhood.”  We now know that synapse formation goes on throughout life, and I don’t like the idea of it being stopped in me. 

Some of the questions I am left with are:

·        Is seizure control associated with taking gabapentin due to higher levels of brain homocarnosine or GABA, or due to some other effect of the drug?

·        Can l-carnosine or N-acetyl carnosine achieve some of the pain control and other benefits attributed to gabapentin?

·        What if any of the general health benefits of supplementation by l-carnosine are also achieved by supplementation with beta-alanine, by taking gabapentin?  Is gabapentin a “longevity drug?”

·        What are the actual implications on adult learning of gabapentin inhibiting new synapse formation?

·        What are the differential effects of supplementation with l-carnosine, supplementation with beta-alanine, supplementation with GABA or taking the drug gabapentin on brain neurons, in CNS glial cells and in muscle tissues? 

My current personal plans are 1. to stay on 500mg of l-carnosine twice daily, 2.   To phase off of gabapentin as soon as possible consistent with my neuropathic pain not returning; pursuant to this,I have just phased down from 900mg a day to 600mg, and 3. Of course to keep alert to any new research developments that might affect these decisions.

First of all, my thanks to reader Jeg3 who put me onto this topic via a comment to the blog post Exercise, telomerase and telomeres.  It seems that both younger people who participate in strenuous sports and old folks who are in danger because of loss of muscle strength can benefit considerably from increasing the carnosine levels in their muscles.  And this can be accomplished to some extent by eating meat, supplementation with l-carnosine or supplementation with beta-alanine.  This blog post reviews the research in this area and steps towards muscle strengthening that can be taken by both athletes and older folks like me. 

I am also planning a follow-up blog post that looks at a fascinating set of similarities and relationships in behavior of beta-alanine, l-carnosine and gabapentin in terms of actions on GABA receptors in nerves and glia.  This post will relate these substances to topics like pain management, maintaining mental balance, sleep and mental acuity. 

I fell in love with l-carnosine over ten years ago when I learned how it could delay or reverse cellular senescence.  It can triple the replicative lifespan of fibroblasts in culture.  For an introduction to this fascinating substance, see the blog post The curious case of l-carnosine.  L-carnosine has long been part of my personal supplement regimen and is in my suggested anti-aging Supplement Regimen.

Beta-alanine and carnosine

The research of relevance to this blog entry has to do with supplementation to increase muscle carnosine levels in two populations: those who participate in high-intensity exercise like long-distance running, and the elderly. The studies I cite are mostly concerned with beta-alanine supplementation, though I am not completely convinced that this is the best approach to building up carnosine levels in muscles.  Beta alanine “is a naturally occurring beta amino acid, — is a component of the naturally occurring peptides carnosine and anserine and also of pantothenic acid (vitamin B₅) which itself is a component of coenzyme A. Under normal conditions, β-alanine is metabolized into acetic acid. — β-Alanine is the rate-limiting precursor of carnosine, which is to say carnosine levels are limited by the amount of available β-alanine(ref).” 

Another source indicates “The greatest natural dietary sources of beta-alanine are believed to be obtained through ingesting the beta-alanine containing dipeptides: carnosine, anserine and balenine, rather than directly ingesting beta-alanine. These dipeptides are found in protein rich foods such as chicken, beef, pork and fish. It is predominantly through ingesting the dipeptide carnosine that we ingest most of our beta-alanine, as the two other dipeptides are not found nearly as plentiful in our typical coniferous diet. However, obtaining beta-alanine through these dipeptides is not the only way, as our bodies can synthesize it in the liver from the catabolism of pyrimidine nucleotides which are broken down into uracil and thymine and then metabolized into beta-alanine and B-aminoisobutyrate.” 

Simply put, in the body both carnosine and beta alanine create each other and the presence of one leads the body to create the other.  Beta alanine has been a very popular sports and body-building supplement but carnosine itself is just now emerging to be known as a sports supplement(ref). 

A 2009 study looks at how long carnosine stays in muscles, once its level has been built up by supplements, Carnosine loading and washout in human skeletal muscles. “The oral ingestion of β-alanine, the rate-limiting precursor in carnosine synthesis, has been shown to elevate the muscle carnosine content both in trained and untrained humans.– The β-alanine supplementation significantly increased the carnosine content in soleus by 39%, in tibialis by 27%, and in gastrocnemius by 23% and declined postsupplementation at a rate of 2–4%/wk. Average muscle carnosine remained increased compared with baseline at 3 wk of washout (only one-third of the supplementation-induced increase had disappeared) and returned to baseline values within 9 wk at group level. — It can be concluded that carnosine is a stable compound in human skeletal muscle, confirming the absence of carnosinase in myocytes. The present study shows that washout periods for crossover designs in supplementation studies for muscle metabolites may sometimes require months rather than weeks.” It is remarkably persistent stuff!

Beta alanine supplementation for endurance athletes

The 2007 publication β-Alanine supplementation augments muscle carnosine content and attenuates fatigue during repeated isokinetic contraction bouts in trained sprinters states “The ingestion of β-alanine, the rate-limiting precursor of carnosine, has been shown to elevate the muscle carnosine content. We aimed to investigate, using proton magnetic resonance spectroscopy (proton MRS), whether oral supplementation with β-alanine during 4 wk would elevate the calf muscle carnosine content and affect exercise performance in 400-m sprint-trained competitive athletes. Fifteen male athletes participated in a placebo-controlled, double-blind study and were supplemented orally for 4 wk with either 4.8 g/day β-alanine or placebo. — In conclusion, 1) proton MRS can be used to noninvasively quantify human muscle carnosine content; 2) muscle carnosine is increased by oral β-alanine supplementation in sprint-trained athletes; 3) carnosine loading slightly but significantly attenuated fatigue in repeated bouts of exhaustive dynamic contractions; and 4) the increase in muscle carnosine did not improve isometric endurance or 400-m race time.”

The 2007 study Influence of beta-alanine supplementation on skeletal muscle carnosine concentrations and high intensity cycling capacity concludes: “Muscle carnosine was significantly increased by +58.8% and +80.1% after 4 and 10 wks beta-alanine supplementation. Carnosine, initially 1.71 times higher in type IIa fibres, increased equally in both type I and IIa fibres. No increase was seen in control subjects. Taurine was unchanged by 10 wks of supplementation. 4 wks beta-alanine supplementation resulted in a significant increase in TWD (total work done) (+13.0%); with a further +3.2% increase at 10 wks. TWD was unchanged at 4 and 10 wks in the control subjects. The increase in TWD with supplementation followed the increase in muscle carnosine.” 

The purpose of the 2009 study Effects of beta-alanine supplementation and high-intensity interval training on endurance performance and body composition in men; a double-blind trial “was to evaluate the effects of combining beta-alanine supplementation with high-intensity interval training (HIIT) on endurance performance and aerobic metabolism in recreationally active college-aged men. — CONCLUSION: The use of HIIT to induce significant aerobic improvements is effective and efficient. Chronic BA supplementation may further enhance HIIT, improving endurance performance and lean body mass.”

The 2006 study Effects of twenty-eight days of beta-alanine and creatine monohydrate supplementation on the physical working capacity at neuromuscular fatigue threshold looked at non-athletes. “ — findings suggested that b-Ala supplementation may delay the onset of neuromuscular fatigue. Furthermore, there appeared to be no additive or unique effects of CrM vs. b-Ala alone on PWCFT (neuromuscular threshold fatigue test).”

Beta-alanine supplementation for the elderly

Of course there is much to the conventional wisdom that exercise is an important part of any anti-aging program for the elderly as well as for the young(ref).   But what about muscular carnosine?

The 2008 study The effect of beta-alanine supplementation on neuromuscular fatigue in elderly (55-92 Years): a double-blind randomized study looked at a small sample of elderly people. In the introduction, the article makes a compelling argument for strengthening the carnosine concentrations in muscles of older people: “Carnosine (beta-alanyl-L-histidine), a dipeptide is an efficient hydrogen ion (H⁺) buffer over the physiological pH range [1,2]. In muscle, where its concentration is highest, carnosine makes an important contribution to the maintenance of intracellular pH, which is vital for normal muscle function during intense exercise [1]. While the dipeptide is found in both Type I and Type II muscle, its concentration is highest in Type II muscle. Studies in humans and rats have demonstrated an inverse relationship between age and muscle carnosine content [3,4]. Sarcopenia, the loss in muscle mass with age, is associated with significant reductions in strength, power, and the ability to resist fatigue in elderly men and women [5,6]. Significant decreases in skeletal muscle and decline in muscle function are clearly evident after the age of fifty [5,7]. Deterioration of motor coordination, as a result of losses in strength and/or fatigue, is related to an increase in the frequency of falls [6,8] which repeatedly lead to injury and even deaths among the elderly [9].”  — “Twenty-six elderly men and women (Table 1) from independent-living communities in South Florida volunteered to participate in the study. None of the participants had any previous history of BA supplementation and maintained their regular activity and dietary patterns throughout the study”

The study looked at Pre- to post-test values for physical working capacity (PWC) at fatigue threshold (PWC_FT) for BA and PL groups.  A significant difference was found.  “Data from this study suggest that ninety days of BA supplementation may increase physical working capacity in elderly men and women. These findings may be clinically significant, as a decrease in functional capacity to perform daily living tasks has been associated with an increase in mortality [18], primarily due to increased risk of falls [9]. Further, deVries et al. [13] and Alexander et al. [8] have suggested that falls may be related to fatigue-induced deterioration of motor coordination. Thus, an improved resistance to fatigue, as reported in this study, may be important to consider when working with a similar population.  — The results of this study suggest that ninety days of BA supplementation may have significantly increased intramuscular carnosine resulting in a 28.5% increase in PWCFT due to a greater H+ buffering capacity.” This study also contains an excellent set of hyperlinked references relating to muscular fatigue,  muscular carnosine and age-related muscle functioning. 

I surmise that changing the fatigue threshold for exercise via raising muscle carnosine levels would also change the point where telomere shortening due to exercise over-stress might occur.  However, none of the papers regarding muscular carnosine and beta-alanine supplementation shed direct light on this issue, an issue raised in the blog entry Exercise, telomerase and telomeres. 

The new-to-me citations listed above leave me impressed with how important carnosine augmentation in muscles might be for health and functionality in the elderly.  On the other hand I am not sure at this point that beta-alanine supplementation is the best way to go. 

Supplementation: beta-alanine vs. l-carnosine

Studies in the literature about augmenting muscular carnosine seem to be mostly about supplementation using beta-alanine.  I am not sure the reasons for that including possibly a) the sport-medicine oriented researchers have always thought in terms of using beta-alanine instead of directly taking carnosine, b) the research was motivated or influenced by commercial makers of beta-alanine supplements, no doubt large money-makers, or c) beta-alanine is in fact the best approach to augmenting muscular carnosine.

As mentioned earlier, muscle levels of carnosine can also be raised by eating meat or by directly taking carnosine supplements.  I have had difficulty finding any study that systematically compares the efficacy of these approaches vs beta-alanine supplementation.  I have seen claims on body-building sites that beta-alanine is possibly less expensive and more bioavailable  than directly taking carnosine. 

One such site recommends taking a combination of beta-alanine and Histidine.  On the other hand, the 2006 report The absorption of orally supplied beta-alanine and its effect on muscle carnosine synthesis in human vastus lateralis states “Dietary supplementation with I) 3.2 and II) 6.4 g . d(-1) beta-alanine (as multiple doses of 400 or 800 mg) or III) L-carnosine (isomolar to II) for 4 w resulted in significant increases in muscle carnosine estimated at 42.1, 64.2 and 65.8%.”

I also found a seemingly-credible blog entry relative to beta-alanine and carnosine: “Anti-crosslinking properties of carnosine: significance of histidine. — Hobart LJ, Seibel I, Yeargans GS, Seidler NW. Department of Biochemistry, University of Health Sciences, 1750 Independence Avenue, Kansas City, MO 64106-1453, USA.  Carnosine, a histidine-containing dipeptide, is a potential treatment for Alzheimer’s disease. There is evidence that carnosine prevents oxidation and glycation, both of which contribute to the crosslinking of proteins; and protein crosslinking promotes beta-amyloid plaque formation.  It was previously shown that carnosine has anti-crosslinking activity, but it is not known which of the chemical constituents are responsible. We tested the individual amino acids in carnosine (beta-alanine, histidine) as well as modified forms of histidine (alpha-acetyl-histidine, 1-methyl-histidine) and methylated carnosine (anserine) using glycation-induced crosslinking of cytosolic aspartate aminotransferase as our model. beta-Alanine showed anti-crosslinking activity but less than that of carnosine, suggesting that the beta-amino group is required in preventing protein crosslinking. Interestingly, histidine, which has both alpha-amino and imidazolium groups, was more effective than carnosine.  Acetylation of histidine’s alpha-amino group or methylation of its imidazolium group abolished anti-crosslinking activity. Furthermore, methylation of carnosine’s imidazolium group decreased its anti-crosslinking activity. The results suggest that histidine is the representative structure for an anti-crosslinking agent, containing the necessary functional groups for optimal protection against crosslinking agents. We propose that the imidazolium group of histidine or carnosine may stabilize adducts formed at the primary amino group.” 

At this time I will not stop taking l-carnosine as a supplement and substitute beta-alanine in its place because of the multiple demonstrated benefits of l-carnosine that are independent of its effects in muscle tissues(ref)(ref).   I am open, however, to the question of whether adding beta-alanine to my regimen could be useful or would be redundant, and will be on the lookout for further research on this issue. I am planning a follow-up blog entry that will go deeper into the biochemical actions of carnosine, beta-alanine and gabapentin insofar as they impact on GABA receptors in nerve cells and glia.