Studies of Caloric Restriction, Resveratrol and SIRT1 Gene Regulation of Intermediary Metabolism and Mitochondrial Biogenesis Demonstrate a Requisite

‘Metabotype’ Continuum from Cellular Rejuvenation to Aging to Cancer, Independent of Cancer Cell Growth Factors, Suppressors or Immortalizers. Gregory S. Bambeck, Ph.D. Abstract
In the early to mid twentieth century Otto Warburg hypothesized that cancer cells could be characterized by dramatically elevated glycolysis and mitochondrial respiratory deficiency locked in a relationship he called ‘aerobic glycolysis’. The respiratory deficiency claim was proven false and cancer cell biochemistry shifted its studies toward oncogenes, cell growth factors and their cascades, cancer cell growth suppressor systems, apoptosis, telomeric immortalization, cell recognition and adhesion mechanisms etc. Any global hypothesis devoted to mitochondrial inefficiency or dysfunction related to the anabolic and catabolic control requirements to cancer cell function, such as those proposed by G. Bambeck were refused funding or publication, and in fact, were quietly chaperoned out of the halls of science. Now, some 30 and some 50 years later, the pariahs may have been proven to be visionaries. Glycolytic blocking and retrafficking agents, such as dichloroacetate etc. can either kill or ‘renormalize cancer cells. Caloric restriction, the only known mechanism for extending life well beyond its normal span, in everything from roundworms to primates, is now known to rejuvenate aging cells and down regulate cancer cell initiation and growth by turning on a suite of 745, or more, genes that renormalize glycolysis and initiate phagocytosis of inefficient mitochondria concomitant with biogenesis of efficient, new mitochondria. Resveratrol turns on the exact same suite(s) of genes, and by every measure, has the same cytological impact. The ‘metabotype’ continuum

from juvenile cell, to aging cell, to cancer cell, make even more sense in an evolutionary context. FORWARD This is a general review article which is presented as a narrative. It is not intended for peer review, in part, for reasons contained in the abstract and the body of the text. That does not mean that this work is unimportant. In fact, it might be very important, because it connects together lines of research that converge on medical implications of great magnitude. It is written by a PhD scientist with over thirty fruitful years of scientific research under his belt, who hopes that he may have the time, later, to write a more formal article. This information should be gotten out there informally and quickly, rather than not to be gotten out at all. There is no bibliography or references in this work, but at the end of this text, a short list of search engine words and suggested readings are provided, so that more than enough bibliography is provided to see ‘the big picture’ presented by the hypothesis contained, herein. This article is restricted to the basic catabolic and anabolic changes in intermediary metabolism and how they relate to juvenile, adult and cancer cells, because this area of cancer research has been left fallow. Other very important areas of research, such as the aforementioned cell growth factors etc., have tens of thousands of articles that may be referred to. This article is devoted to an area of research that has been relegated to a backwater, in particular, in regards to cancer cell intermediary metabolics. If it had not been for anti-aging and life extension research results, stumbling through the metabolic ‘back door’, so to speak, some thirty years after a modified Warburg hypothesis, coupled with some new cancer metabolism blockers, the important connections could not have been made. Throughout history, serendipities and/or convergences have come together to form holistic emergent systems with more than just notable impact. Hopefully, this one of

those times. Lastly, this article is written a la Scientific American in that it is, hopefully designed to be understood by the educated lay person, while not short changing the serious scientist, too much. Professional jargon will be held to a minimum. It is my hope that a more in depth review won’t be necessary, if enough interest ensues. It would certainly be far more gratifying if interested parties would take up the discussion, or even initiate lines of investigation that might knit the outlines of these semi-integrated patches into a more complete fabric of either enhanced, or wholly new understanding. So, here we go, more or less ‘off the cuff’. Earlier Times Approximately 96% of a cell’s organic biomass consists of carbohydrates, fats, nucleotides and proteins. All four of these components can be either burned as fuel to power the cell, or as construction materials to replace defective parts in a quiescent cell or to build a new cell, as in the case of a dividing cell. In a normal, healthy quiescent cell, there is a balance between energy production and repair rates, so that the cell is said to be in homeostasis, or balance. In a normal, healthy dividing cell, growth factors set up a cascade of directives that order the cell to both increase its energy output and to import raw materials and build those raw materials into the components of a new cell. In short, nucleotides become RNA and DNA, amino acids become proteins, sugars become complex carbohydrates and fatty acids become lipids in a process called anabolism, while these same four starting materials can, alternatively, be burned as fuel to create energy, in a process called catabolism. The anabolic intermediate products can further assemble, by anabolism, into molecular assemblies, such as ribosomes, enzyme complexes etc., and, even further, into organelles such as mitochondria, lysosomes, a cell nucleus etc. Eventually, a whole new cell is formed. Cancer cells do this to, but as we shall see later, they don’t do it the same metabolic way as normally dividing cells. Instead, they do it more like in an extended metabolic version

of aged cells. Although, what has been presented so far, is a gross oversimplification, suffice it to say, that by the middle of the twentieth century, a pretty sophisticated outline of many hundreds of the molecular trafficking pathways of the catabolic and anabolic interplay of organic molecular transitions, were mapped out. Wall posters in classrooms demonstrated these processes in a fashion analogous to watching automotive freeway traffic flow from an aerial view over a large city. Many discoveries have been made by observing disruptions in the flow, just as auto accidents cause clogs on off-ramps, onramps and thru-ways on the freeway system. Otto Warburg, in charge of a large research organization, and a highly respected molecular researcher of his day, being the early to mid twentieth century, thought he saw an intermediary metabolic perturbation both unique and specific to all cancer cells. More precisely, he hypothesized that this perturbation was critically confined to the catabolic, or fuel burning side of metabolism, but also set in motion an anabolic shift as a consequence. Even more specifically, he targeted the anomaly to the burning of a single fuel, glucose, to the relative exclusion of other fuels. Glucose is the primary, but, by no means, only fuel that is burned by cells of the body, and it is burned in two complex systems, called glycolysis and the Krebs cycle. Glycolysis obtains energy from glucose and other sugars by an anaerobic (non-oxygen utilizing) mechanism to produce ATP, an energy carrying molecule. The Krebs cycle, contained in an organelle called the mitochondrion, burns the glycolytic end product, pyruvate, utilizing an aerobic (oxygen consuming) mechanism to produce ATP. The burning with oxygen process sequentially strips hydrogens from the glycolytic end product, converting NAD to NADH2 and then couples the NAD hydrogens to oxygen to form water, while releasing the sugar carbons to form carbon dioxide. The formation of water is a stepwise process in which the energy of the hydrogens and their electrons are rejoined in a stepwise process called electron transport,

while the energy of the process is captured by converting ADP to ATP, while H and O form water, in a process called oxidative phosphorylation. Anabolism is the opposite process in that NAD hydrogens and ATP energy are utilized to build cell components. Thus the two swing molecules in the process are ATP and NAD as they switch back and forth between their low and high energy forms and their oxidation and reduction forms, respectively. In a healthy homeostatic cell, many fuels are used, and about 5% of the ATP is produced by glycolysis, while about 95% of the ATP is produced by the mitochondria. Anabolism and catabolism are in a steady state balance with mitochondria producing this ATP energy at about 99% efficiency. In a healthy dividing cell, the entire energy production system is upregulated to make ATP energy and NADH reducing power for the anabolic requirements to make a new cell. Otto Warburg notice an uniqueness in the catabolism of cancer cells, in that glycolysis was considerably elevated, that respiration was depressed and that mitochondria in cancer cells appeared small, malformed or disorganized. Furthermore, he proposed that, unlike fetal or other normally dividing cells, the cancer cell was irreversibly ‘stuck’ in this metabolic phenotype. Although the glycolytic part of his hypothesis was never refuted, the respiratory defect notion was struck down in a furious debate in 19551956. Mitochondrial respiratory deficiency, although found in many tumor types, was not found in all tumor types, and was not considered required as a fundamental requisite of the cancer cell condition. As mentioned before, Warburg was a big name during his time. He made and broke many scientific careers, and was known for having a bit of an irascible nature. Well, the bigger they are, the harder they fall. Many of Warburg’s detractors became journal editors, reviewers and laboratory directors. Woe be it to anyone positing any form of mitochondrial defect/cancer hypothesis, even twenty five years later. After the Warburg hypothesis rejection, cancer research shifted away from metabolic studies toward oncoviruses, oncogenes, cell growth factors and

their cascades, cell growth suppressor systems, apoptosis mechanisms, telomeric immortalization, cell recognition and adhesion systems etc. These studies have had a huge impact upon our understanding of the normal, to cancer cell, transformation process. It has become obvious that evolution has provided numerous impediments to lethal carcinogenesis in its attempts to keep cell division under control. There is also no doubt that these new areas of research would have opened up regardless of the outcome of the Warburg hypothesis. However, it is also true that investigations of mitochondrial interactions in the intermediary metabolic interplay in the cancer cell, would not have all but dried up, as it most certainly did. In 1975, some twenty years after the Warburg hoopla, a very politically naïve graduate student, named G. Bambeck became fascinated with mitochondria, in a Kent State University laboratory, that happened to have a mouse lymphoblastic lymphoma model. He isolated mitochondria from many mouse tissues and noted that the lymphoma mitochondria had uniquely low ADP:O ratios. This means that these mitochondria were producing abnormally low amounts of ATP energy per oxygen consumed. This could mean that either ATP was being uncoupled from oxygen consumption via the respiratory chain, that reduced NAD was either being decoupled from oxygen or being exported from mitochondria in abnormally high rates, via some kind of chemiosmotic shuttle, for anabolic purposes, or in some combination, thereof, by some unknown mechanism(s). Thus, he began a literature search and, among other things, he ran into the Warburg debacle. But, being socially unsophisticated, he pressed on, and he pressed on because he found something very interesting, so interesting, in fact, that he virtually abandoned his wet lab (hands on) research for a mental form of research. In mental research, one reviews the work of other researchers in the hopes of ‘an unique synthesis of thought’, or, in other words an overlooked ‘big picture’, that, in this case, might also be suitable for a PhD dissertation.

First, G. Bambeck found that there were a lot of cancer cell mitochondria vs. normal cell mitochondria papers out there, a number of them showing that Warburg’s respiratory defect was not there, in more than just a few cases. What he did find, in every cases where the data was taken, that net mitochondrial ATP production on a per mitochondrion and/or, more importantly, per cell basis, was significantly lowered compared to normal dividing cells. He further noted that mitochondrial net ATP synthesis shortfall could be due to low mitochondrial numbers per cell, inefficient electron transport, increased NAD/NADH shuttle export of reducing power, inefficient coupling of ATP formation to hydrogen chemiosmotic potential, a shift in the ATP synthetase to ATPase equilibrium dynamics or some fundamental metabolic Km shift in the chemical pathway linking glycolytic end products to the Krebs cycle. Anything that would block the connection between the NAD/NADH redox coupling to ATP formation, could tip the balance of carbon flow to anabolism. Most importantly, these mitochondrial shortfalls of ATP production could change the glycolytically produced to mitochondrially produced ATP production ratio by over 1000%. He proposed that this ratio differential forced fuel dependency upon glucose while simultaneously forcing other metabolites, reducing power and ATP energy flux toward anabolism. In the light of modern discoveries, to be discussed later, one could say that such a system pre-adapts the cell, metabolically, for a more uncontrolled growth format when not necessarily signaled by a growth factor: a precancerous state or hyperplasic non-dividing growth condition, as one might have it. If carcinogenesis is a multi-step process, why not have a metabolic process, or a progression toward a metabolic state that predicates the terminal process when suitable mutations and their associated stimuli arrive? G. Bambeck optimistically presented his conclusions to the scientific community with the expected dismissive results. Even though his dissertation was of a somewhat heretical nature, he received his PhD because the logic was essentially sound. But with nowhere to publish and nowhere to work on

his findings, he evaporated from obscurity to nothingness in the annals of cancer research. Instead, he plied his trade in medical diagnostics and research tool technologies. The tools of 1975-1980 were not available to address a rationale for the collapse of mitochondrial efficiency in cells. For one, there seemed to be too many ways for it to happen. Gene switches and gene switching systems were just in their early days of initial elucidation. There were simply too many unknowns and alternatives. G. Bambeck took a stab at the problem, and notioned that reactive oxygen species (ROS), called free radicals at the time, might be peppering the mitochondrial and nuclear genome, causing a sequential randomizing of the mitochondrion and manifesting itself as a progressive decline in fuel burning efficiency. Paraphrasing, he said that, ‘by whatever means, the per cell mitochondrial ATP production deficiency is there’. We now know this to be true, and not just in cancer cells, but in aging cells as well. Its most severe manifestation appears to occur in the cancer cell. The anaerobic to aerobic ratio of ATP production appears to increasingly exacerbate as cells age and become transformed. Cell heat production and adaptation to hypoxic conditions are hallmarks of glycolytically adapted cells with poorly coupled ATP formation, as is witnessed by the limited success with hyperthermic, hypobaric and lactic acid export blocking cancer therapies. G. Bambeck hypothesized that more specific blocking agents to glycolytic and mitochondrial systems, might have greater efficacy, either for instituting differential kill or cancer cell renormalization. The now known facts that glycolytic blocking agents can kill or that fetal pyruvate kinase blocking agents can renormalize cancer cells to a non-anabolic and non growing state, and that conditions and agents that create efficient aerobic catabolism via mitochondrial biogenesis, reduce cancer incidence and increase cell rejuvenation, show a more mechanistic support for the concept. However, much of these data come from research areas originally perceived as only tangentially related, or not at all related, to cancer. Toward Present Times

From the 1980’s to the present, there has been a lot of work with ROS and their mutagenic and aging effects on cells. Basically, ROS are highly reactive oxygen species containing an unshared electron which allows the ROS to react with just about any available organic molecule, including DNA, RNA, proteins etc. Naturally, ROS are mutagenic and, therefore, carcinogenic, akin to ionizing radiation, causing the highly specific and organized genome and its protein products to become more randomized, or nonsensical. Because they are immersed in an oxygen atmosphere, either directly or by blood delivery, body cells produce ROS and utilize free radical scavenger molecules to mop up these nemeses. Anti-oxidants, such as vitamin C, vitamin E, bioflavinoids etc. are such free radical scavengers, and experiments with elevated doses of anti-oxidants and their effects upon cancer induction and aging rates are rife. In general, the results are positive, helping organisms to more closely achieve their well nourished natural life expectancy potential, but not beyond that potential. The mitochondrion produces more ROS than any other part of the cell because it is the seat of oxidative fuel burning, where the electron cascade of the respiratory chain is used couple the energy of the Krebs cycle intermediate hydrogen electrons to ATP formation, then, ultimately to oxygen, to yield water. To protect itself from its own internally produced ROS, the mitochondrion utilizes endemic anti-oxidants and a special enzyme called SOD to mop up ROS. Each mitochondrion has several copies of its own DNA, and when ordered to do so, mitochondria can multiply, similar to cells. But unlike cells, mitochondria can do something amazing. Via a mostly unresolved process, mitochondrial biogenesis (to be distinguished from simple division), results in new efficient mitochondria arising from old inefficient mitochondria. Cell nuclear genomes pass on the mistakes that their DNA repair systems fail to detect or faultily repair, as do mitochondria, when simply dividing, but not when undergoing biogenesis. Perhaps it is because a single cell has only one nuclear genome (or at most, two half genomes), but as many as over a thousand mitochondrial genomes that might be coupled to some selection

process, maybe based upon some kind of selection for fidelity of consensus sequence. It seems obvious that there must be some kind selective renewal system, because mitochondria pass from generation to generation via oogenesis, on average, without a single point mutation. Without such a process, it would not take many generations for eggs to contain a gibberish of mitochondrial sequences, rendering the following of the matrilineal line to a virtual impossibility. The fact that we can follow the matrilineal line through thousands of generations, supports this notion. I envision something like a polytene chromosomal sequence comparator mechanism of some kind, or perhaps, a highly protected and sequestered mitochondrial ‘mother genome’ somewhere in the cell. Evidence for real biogenesis mounts. In adult body cells, mitochondria progressively degenerate into inefficient couplers of hydrogen, electrons and oxygen to ATP and water production. And not just one thing, but a host of things go wrong with mitochondria as adult cells age, with one of the hottest causal suspects being ROS mediated mutagenesis. Concomitant with reduced mitochondrial efficiency over time, is an increase in glycolytic ATP production. Thus, as cells age, the anaerobic to aerobic ATP production ratio rises, and begins to appear more like the pattern seen in cancer cells. Since most adult cells are terminally differentiated, and can no longer divide, such a ratio shift does not pose a cancer risk, as would adult stem cells, which still have functional telomeres, and could possibly become telomerase immortalized. Telomere shortening to terminal differentiation is a well established mechanism, putatively, for avoiding cancerous progression in adult cells. However, non-terminally differentiated adult cells and adult stem cells can divide, and they generally divide to replace dead or missing adult tissue, under the directive of growth factors. Under such conditions, tissues are replaced but they are not rejuvenated. Instead of young dividing new cells replacing old dead cells, old dividing cells are replacing them, in part because mitochondrial biogenesis is not occurring. Cells are being replaced, but the tissue is not being rejuvenated. It appears that as we age, the metabolic phenotype (metabotype) of the aging cell operates more and more like a cancer cell

metabotype. Somehow, it seems that that there is no great evolutionary pressure to put additional roadblocks to such a metabotype progression, especially with the, aforementioned, long list of non-metabolic quality assurance mechanisms already in the toolkit. In fact, such a progressive metabolic transition probably assists the evolutionary process. From an evolutionary perspective, it is preferred at a certain point to dispose of the old bodies, which represent yesterday’s genetic experiment, with the next generational gene mix experiment. After all, it is the genome that has a shot at perpetuity, and not the vehicles it employs to get there. In summary, it appears that adult tissues are aged metabotypes of juvenile tissues and adult stem cells are aged metabotypes of fetal stem cells, and that their metabotypes progress toward the cancer cell metabotype, irrespective of other components of the cancer cell transformational process. There appears to be a blended metabolic continuity between these basic temporal cell types, and recent science on cancer and aging are yielding insights that show metabolic crossover applications between these once disparate fields of research. It appears that we are beginning to achieve a degree of control over both aging and cancer, at least from a metabotype perspective. To a rather remarkable degree, the aging cell and cancer cell metabotype have recently been reverted to normal with dramatic increases in life extension and incidence reduction in cancer, from groups of apparently unrelated experiments that only share their fortunate results by viewing them from a shared metabolic perspective. By life extension, it is meant to mean ‘age beyond its normal well nourished maximum’ This not to be confused with achieving the natural maximum by delaying premature death, but instead, by going beyond its natural healthy maximum by a considerable extent. Also, it is far too early to talk of cancer ‘cure’, but the early data point to a significant cancer renormalization or kill. But first, there is a need for a bit of a preamble. Until recently, the only technique known to cause genuine life extension is to initiate a condition known as caloric restriction. Caloric restriction and its life extension affects hails back seven decades, but its many manifold mechanisms of action are just now coming to light, due to new technologies grown out of

the genomics revolution. Studies had shown that caloric restriction rejuvenated cells, by virtually every measure, and in virtually every tissue and organ of the body, from neuroregeneration, to delayed and reversed muscular wasting, visual impairment, skin wrinkling etc. These rejuvenation-like phenomena occurred in organisms, as lowly as yeast, upward through the multicellular organism evolutionary chain ranging from roundworms to mice. Just this past year, a 25 year caloric restriction study on rhesus monkeys extended these findings to the primate order, of which humans are a member. Preliminary results on humans are yielding parallel results. By hindsight, such findings make evolutionary sense, because feast and famine cycles go back to the dawn of time. There is a distinct survival advantage in being able to sweat out the lean times until nutrient availability of fat times allows energy availability to support cell division in single cell organisms or procreation in multicellular organisms. Hibernation or estivation may work for periodic circumstances such as winter snow or summer drought, as it does for northern bears or desert toads, but variable and unforeseen conditions have led to a much more ancient and flexible metabolic solution, as observed by caloric restriction. It seems apparent that untold millions of generations have been honed by caloric restriction to utilize it as a generic life extender as a means of avoiding extinction. In just the last couple years, the genetic mechanisms behind the caloric restriction phenomenon have become much more elucidated. First, caloric restriction turns on a gene called SIRT1 that activates a suite of, at least, 745 genes that is normally turned on in juveniles, but is turned off in adults. At the very least, SIRT1 is turning on a hugely complex gene system in adult cells that is normally only operational in juveniles, tempting the notion of ‘rejuvenation’, especially since life extension is the result of their activation. It appears obvious that this must be an ancient system for it to be composed of such a high number of orchestrated components, and in such a pan specific manifestation, with a particular bent toward extending organism life length by mimicking, or attempting to mimic the juvenile period and imposing it on adult cells. The impact seems to be most pronounced on metabolic efficiency.

For one thing, the entire metabotype of the aging cell shifts over to a juvenile metabotype when SIRT1 turns on. Surprisingly, homeostatic non-dividing adult cells undergo mitochondrial biogenesis, in which, old inefficient mitochondria become replaced by new efficient mitochondria. This is not just inefficient mitochondria dividing to produce more inefficient mitochondria, as is the case in aging adult replicating cells and cancer cells, but an actual biogenesis of young behaving mitochondria yielding cells of a juvenile metabotype. This probably, helps account for the reduction in insulin resistance, by dramatically reducing the cell’s glucose dependency, and for a host of rejuvenating effects caused by rebalancing catabolic energy production, and, probably ROS reduction, or ROS scavenging. There is support for the notion that mitochondrial biogenesis alone can have very substantial impact upon cell rejuvenation, as direct elicitors of mitochondrial biogenesis (PGC1 alpha) in mice, reduces muscular wasting and visual impairment. We can expect to see an explosion of research in this area, now that mitochondria are known to be so vital to cellular health, far more so, than was thought, in the traditional sense. However, the amount of caloric restriction needed to institute these alterations, is draconian; tantamount to humans trying to live for 110 years in hell-like semi-starvation vs. living in relative comfort for only 80 years. But, stay tuned. There are indications that caloric restriction gene systems might be, in good part, chemically inducible. There is a new kid on the block, called resveratrol. Over the last decade, or so, resveratrol has become hailed as an anti-aging super anti-oxidant. Originally touted as a component of the lycopene/tomato, omega 3/olive oil and resveratrol/ red wine Mediteranian diet, resveratrol has emerged as a heavy weight contender in its own right, and for some good reason. Shotgun gene affinity experiments, which measure up and down regulation of over 20,000 genes at a time by measuring mRNA gene transcript annealing to DNA sequence leaders, demonstrate that more than` 745 genes are regulated by

resveratrol in the same fashion as done by caloric restriction, in mouse tissues. In fact, the number and direction of regulation shows a remarkable 99.7% concordance with caloric restriction, the remaining 0.3%, which somehow bypasses SIRT1. Similar results were obtained with other tissues. The effect even occurs in fat mice, even though the jury is still out on its impact on longevity in these animals. Importantly, resveratrol initiates mitochondrial biogenesis and a laundry list of cytological and tissue regenerative affects similar, if not identical to caloric restriction. The probability of such a high gene activation concordance is so minute as to border on the incalculable. The fact that it does so via some kind of bypassing of the putative cascade initiation control genes SIRT1 and Insulin-like Growth Factor (IGF), is initiating some rather heated discussion between pharmaceutical companies with vested interests on both sides of the resveratrol gene control issue, and whether it is a true caloric restriction mimetic. Based upon the impacts seen from mitochondrial biogenesis, I would guess that a very sizeable percentage of the genes induced by SIRT1 or resveratrol, are devoted to mitochondrial biogenesis and its consequences on aging and cancer cell metabotype. There can no longer be a doubt that many, if not most cancer cells have some entrenched metabotype that is fundamentally more fixated than in normal cells. Briefly put, in-vitro studies of glucose mimicking glycolytic blocking agents such as 2-deoxy glucose and 5-thio D glucose, can kill up to 99.999% of cancer cells in a few hours while leaving normal dividing cells alive, with the cancer cells being radiation sensitive and the normal cells being radiation insensitive. These agents slam glycolytic ATP production to a halt leaving only pyruvate starved mitochondrial ATP production to remain. Being glycolysis end product dependant, much more so than most normal cells, cancer cells die, while normal cells can utilize, or switch off to alternate fuels, more readily. Unfortunately, these results don’t translate to in-vivo use because the therapeutic dose is to close to the contraindicating dose, probably because they are glucose analogues. Brain cells are highly dependant upon glucose, for instance.

In cancer cells, glycolysis often produces many times more pyruvate than the inefficient mitochondria can assimilate, so the excess pyruvate is converted to lactate for cell export to the liver, where it is converted to glucose for reexport to the tumor, in a closed loop system called the cori cycle. Other cells, such as hypoxic cells and low oxidative fast twitch muscle cells, can reversibly utilize the cori cycle, while cancer cells are much more entrenched in the cori loop. Hyperacidification by lactic acid export blockers has instituted differential cancer cell kill, in many cases, but usually fails to finally eradicate tumors that adapt to hypoxic conditions. Also, it is necessary, as with glucose feedstock blocking, to thread a narrow path between efficacy and contraindication. Bypassing cell glucose importation and lactate export systems may efficaciously demonstrate an alternative outcome. A recent strategy, applied across a broad spectrum of tumors in mice, utilizes dichloroacetate (DCA) to block a fetal puruvate kinase (FPK) enzyme in these tumors, thus renormalizing metabolic flow away from anabolism, and stopping cell growth. Apparently the switchout from adult PK to FPK is very common in such mouse tumors. Unfortunately, the results are so promising, and dichloroacetate, being a common unpatentable reagent, have led to a growing illegal market for dichloroacetate, among desperate cancer victims. Caloric restriction and antioxidants both have impact on cancer incidence and severity. Carcinogenesis is both delayed, and once initiated, growth rates are slowed. It would be very interesting to see the results of resveratrol and dichloroacetate together. One could postulate a synergistic effect, in which dichloroacetate renormalizes glycolytic flow, and resveratrol reinforces the effect via anti-oxidation and/or mitochondrial biogenesis. Then again, things may not be so simple. The FPK system might not work the same way in humans as it does in mice, because people using DCA are reporting huge cell kill rates in their tumors and the associated dead cell load problems, indicating that DCA is acting more as a blocking agent than a pathway switching agent. If true, it could all be for the better, as kill is far more preferred than renormalization, as it allows the therapy to be stopped, at some point, instead

of having to remain interminable. Besides, long term use of DCA is hepatotoxic. The DCA effect has resurrected the name of Otto Warburg, and rightly so. For the first time in 50 years, metabolism is again, moving into the forefront of cancer research. Please note that the metabotype referred to herein is only one of a number of required steps in cellular transformation to a full blown metastatic cancer. However the metabotype now assumes a required status, uniquely different from other dividing cells, such as fetal cells and adult stem cells. Cancer, it seems, is not just related to aging, but might be a mutated stepwise progression form of it. These integrated set of results give rise to an interesting evolutionary perspective. A Short Evolutionary Perspective Now that we have whole genomes, from bacteria to human beings sequenced, many obvious first questions arise. One of the most tempting is: How did something as complex as a mammal, with well over 200 different cell types, operating in a highly integrated symphony, evolve to have a few times more genes than a eukaryotic single cell life form, such as yeast. What makes this even more puzzling, is that our 25,000, or so, genes seem mostly to have arisen from a couple of duplicating of, perhaps a 5,000-7,000, or fewer, gene containing archaic genome. One notion is that duplicating whole genomes creates a regulatory opportunity for multicellularity, as it allows for differential expression of each genome in each of two cells that fail to separate after division, allowing each cell to perform unique tasks that enhance the survival of each, together. Over time, this leads to multicellularity, and simple repeating body plans, such as segmented worms, developing segments into complex body parts and plans, such as insect mandibulata or mammalian inner ear etc. Over time, gene remnants fail to transcribe, sister chromatids cross over millions of times, viral transposons shuffle DNA around, evolutionary pressures select for gene gains, losses and mutations, gene regulatory systems advance, more and more cell types and there regulatory systems evolve, and create an ever diversifying array of body plans etc, until, at present times, the

archaic genome, although readily statistically apparent, is barely still visible in the mish-mash. This is not quite like doing more with less, as it is like doing a helluva lot more with more of the same. Not to take anything away from Darwin, but nature does seem to ‘learn’ from, or pseudo-repeat, her successes. The genome duplication demonstrates how a gigantic array of regulatory elements in multicellular organisms can take an existing array of genes to new heights of diverse expression and function without actually having to create real ‘new’ genes. The new field of epigenetics is a case in point, because it pulls an apparent Lamarckian ‘acquired characteristics’, end around, the more temporally linear notion of Darwinian survival selected fitness traits resulting from serially generational genomic mutagenesis. Briefly, the epigenome responds to environmental conditions, such as famine in the sex cells of the parents, by selectively methylating genes to up and down regulate them so that these regulatory systems are passed on to the offspring, purportedly to pre-adapt the offspring to the existing environmental conditions experienced by the parent. The number of possible variations of epigenetics dwarfs the size of the genome itself. This area of research is in its infancy, but one of its discoveries is serendipitous with this narrative. One epigenomics experiment shows that feast and famine leave a differential impact on sons and daughters that significantly affects their life expectancy. More so, the epigenetic signature passes through several generations before it fades out. From a Darwinian perspective, the only way to have the appearance of a Lamarckian outcome, is for pre-adaptation to have arisen from numerous, widely spaced, earlier or many, highly selective, recent exposures to a phenomenon. The more gene systems affected in a more orchestrated fashion, the more ancient and more protracted the system must be. Open ended circular arguments apply to life, because life has ‘been there’ before, especially in the case of famine, and because, successful gene systems replicate and fan out over time. In regards to the subject focus of this document, there appears to be a much more ancient and a much larger and much more controlled famine gene manipulation system than epigenetics at work.

I broached these topics to put forth the notion that ancient systems, such as intermediary metabolism, mitochondrial function and anciently occurring phenomena, such as feast and famine, grew along with the growth of the metabolic pathways themselves. Whole suites of genes are involved with their control, but over time the system evolves to arrive at a relatively few fixed sets of ‘tried and true’ tactics that have become, more or less, successful fixed response systems. Other, more flexible life components, such as length of time as a juvenile, or length of life as an adult, may be operating in an intermediate time spectrum, as they are responding to phenomena much longer than epigenetics, but much shorter than raw metabotype, although all three systems outlined, here, from a feast and famine standpoint, can leave their mark upon metabotype expression. My point is this: even though there are hundreds of cell growth factors, the unique mixes, of which, tell each cell type when and how fast to grow and divide, there seems to be a veritable paucity of system(s) setting mitochondrial biogenesis in motion, with its apparent requisite reestablishment of the 5/95 glycolytic/Krebs ATP production ratios, and the resultant catabolic/anabolic rate shift. This rejuvenating biogenic system appears to paint with a broad brush, saying essentially ‘on’ in juvenile cells, ‘off’ in adult cells, and when in the ‘off’ position, mitochondria slowly age (as in humans), or rapidly age (as in mice), toward the inefficient cancer-like metabotype. This becomes a precancerous, or hyperplasic condition awaiting growth signal and signal related mutations, to transform it into the cancerous state. Amazingly, the juvenile system can be turned back on in adult cells, but only when the organism is in dire straights of a type recurrent from the dawn of metabolic evolution. Interestingly, most mammals fail to ovulate if they are not carrying enough fat supply, so caloric restriction carries extended life in exchange for the loss procreative capacity, thus allowing species replacement numbers to be deferred to a more nutrient plush future. Conversely, when feast times return, fat storage ensues, high ATP production efficiency is no longer a life sustaining requirement, and after a few procreative cycles, the now shorter lived adults, are disposed of. It is rather interesting that nature has also found a mechanism for failing mitochondria to act in a metabolic default toward cell

growth, the caveat of it, being linked to life saving energy supply availability, on the one hand, and the potential to assist in the death dealing carcinogenic process, on the other. There does not seem to be any real overt need to modify this ancient system, save to change the length of the juvenile and adult stages to suit the environmental conditions demanding such. These life stage length differences between mice, dogs, horses, humans etc. are independent of metabolic rate, body size, the brain to body ratio, just as is the time from birth to the elevated cancer risk incidence curve. Instead, they seem more tied to the mitochondrial decay rate as per life expectancy. It is as if the vast array of environmental constraints set the necessary life length of the organism and that cellular metabolism doesn’t have a very difficult time complying by setting juvenile and adult stage length as needed. Of mice and men; old at age three or old at age 100; several thousand percent difference in life expectancy and 99% identical metabolic pathways. This gives argument to the notion of not what, or how you do something, but when you do it. It probably works the same in mice as it does in men, with the switches being turned off and on at different times and at different strengths. The central metabolic system, and its core regulatory elements haven’t changed much since the most advanced animal was a sponge. In retrospect, glycolysis and aerobic respiration are systems entrenched in times so ancient, that it was before earth even had an oxygen atmosphere. When early oxygen producing photosynthesis first evolved in the ocean, anaerobic glycolytic bacteria, in one or more branches of life, began oxidatively extracting energy from glycolytic end products by removing hydrogen and adding oxygen in a stepwise process. Eventually this oxidative process evolved to extract the maximum obtainable energy by extending the oxidative chain all the way to the natural carbon dioxide and water, as waste product, endpoint. A symbiosis was formed when a nucleus containing archaeobacterial species housed an aerobic bacterial life form within itself, probably providing predatory protection in exchange for abundant energy.

This is a relationship similar to that which exists today when coral polyps house algae within themselves. Before the first sponge-like simple multicellular life forms evolved, a nearly billion year relationship between the host and its symbiont had been maturing. Eventually, this relationship became so inextricable that, by the time that the first eukaryotic fungal/animal precursor cell arrived, the aerobic symbiont had been mostly reduced to the status of slave to its host, giving up most of its genome in exchange for providing critical energy producing advantages to its host (except for the notable, and rather disturbing distinction of retaining a host cell killing command center, called apoptosis). We call this relic, the mitochondrion. Today, the animal cell mitochondrion contains only the genes for its respiratory chain and some of its protein synthesis and replication machinery, cell suicide mechanism, included. All other ancient pre-mitochondrial functions are now sequestered in the DNA of the cell nucleus, and their gene products are imported into the mitochondrion from the cytoplasm. This is true in even though most primitive animal life forms, and in even earlier life forms, such as single celled yeast. Thus, aerobic and anaerobic systems were intricately enmeshed, in terms of each others operation and control mechanisms for a period of time longer than the existence of the Kingdom Animalia. It is no small wonder that we see the birthings of a SIRT1 control system in organisms that predate the animal kingdom. It seems incredible how both deeply entrenched and unchanged the caloric restriction response system has remained over time. We find it throughout the entire animal kingdom, from worms, through insecta, up the vertebrate tree, from ancient to modern life forms. The system is also remarkable in its versatility, and in its short term, intermediate term and its long term applications to organismic, species and Kingdom survival. When we look at mammals, in our attempts to utilize models more akin to our human selves, we see the sweeping SIRT1 system applies to every tissue and organ tested. Although the SIRT1 cascade ‘rejuvenation’ is nowhere near to being confused with the word ‘immortalization’, it is an apt nomer, nonetheless. Among other things, the system institutes production, via mitochondrial biogenesis, of efficient catabolism, which yields survival advantages during nutrient energy

shortages, and yields energy efficient rapid growth during the growth factor directed juvenile period of rapid body expansion. It reverts adult cells to a juvenile state, inasmuch as it can within mutagenic constraints, causing a true 30% to 80% increase in life span, depending upon which model is used. After puberty, under ad libitum conditions, it becomes pretty clear that the SIRT1 system is, not only, not needed, but is not utilized, probably because it becomes disadvantageous in the statistical evolutionary survival sense. In the adult phase it, appears that cell senescence is an obligatory rule that can only be violated if survival of the next generation is at risk, due to the risk to the survival of the present generation, via starvation. Thus it does not matter if, under feast conditions, with procreation accomplished, that cancer becomes a natural outcome of declining metabolic efficiency, combined with the other required changes in cell growth control systems. In the adult system, inefficient catabolism diverts anabolic processes toward fat storage and pushes cells to become more dependant upon the cori cycle as they become more fuel dependant upon glucose by diverting excess pyruvate to lactic acid for export to the liver for gluconeogenesis. Such cells are metabotype ‘preadapted’ for cancer, as well as for organism aging or death, due to disrepair, because it really doesn’t matter how the organism is disposed of, as long as it is disposed of, usually by predation or disease, of course, due to its aged and weakened condition, Only under nutrient fuel paucity conditions does this natural decay process seem to be interrupted. The fact that SIRT gene knockout mice age rapidly and die very early, only underscores these conclusions. SIRT1 up regulation has just the opposite effect. Mitochondrial biogenesis occurs, the metabotype reverts to a juvenile metabotype , adult life span lengthens, cancer incidence declines, regain of tissue function, such as that of muscle, liver, neurological etc. takes place and total organismal energy output efficiency and general vitality ensues. The fact that resveratrol mimics this general condition, is a most fortunate discovery. The fact that the pharmaceutical giants are at war over resveratrol and its caloric restriction mimetic capacities, shows just how far human folly can escalate when so

many billions of dollars are at stake, especially if the leading candidate drug is unpatentable. Although the affinity chip data indicate that resveratrol bypasses SIRT one and IGF, the fact that it activates 99+% of its downstream cascade, in at least three tissue types, speaks volumes. The physiological and cytological studies, which support the rejuvenative effects, arise from too many sources for it to be a collusion, accident or folly. Regardless, continued research on actual SIRT1 activators, resveratrol, its analogues or SIRT1 downstream cascade activators, these recent breakthroughs should spur a huge amount of basic research in both aging and cancer, and should, especially, reinvigorate a long overdue reinvestigation of the therapeutic efficacy of cancer cell intermediary metabolic interventions. This is particularly true, in light of the preliminary results from the employment of DCA, and the pivotal position between glycolysis and the mitochondrion occupied by pyruvic acid. At the present time, the list of anti-aging supplements that need to be taken to ward off the plethora of cell systems in decline in aging organisms, is mammoth, to put it lightly. Many of these supplements (but, by no means all, such as, vitamins, anti-oxidants, phytonutrients etc.) are provided abundantly by juvenile cells, but are produced in progressively lessening quantities by aging organs, tissues and cells. However, by turning on a SIRT1 rejuvenation gene cascade, it appears that life extension occurs in the absence of such supplementation. This fact alone has incredible implications, because it proves that the vast majority of youthful cell vigor information, remains intact, and is recoverable with the right inducement(s). The implications are gargantuan, because it declares with a delightful certainty, that normal aging is not, so much so, a process of irreversible decay, as it is a form of highly controlled preplanned obsolescence. The investigation of modified supplementation, in tandem with rejuvenation gene cascade initiation and mitochondrial biogenesis control systems, could offer a fertile field for the cultivation of a broad area of applications. It is becoming progressively more clear that glycolytic and mitochondrial catabolic control systems, in and of themselves, have deep impacts upon cellular function, outside the governance of cell

growth control signaling pathways. The notion that such a system has, at its roots, control over such sweeping concepts, as aging, biogenesis, rejuvenation and cancer, is almost stupefying. After all, these systems, and their core control elements, predate multicellularity and the need for differential cell type control. Afterword I know that this a brief sketch, but I do believe that the fundamental elements and their chief interactions have been presented. I know that I cast a very broad net in an age of narrow specialists, so I beg the reader to leave the details for future investigation, while relegating present thoughts to seeing ‘the big picture’ in the context of a renaissance mind. Acknowledgement I thank Michael Wolfson for his stimulating conversations on life extension, which spurred my investigations on this topic and formed the relationship of ideas with my previous work. Without his unique perspective and irrepressible humor, I doubt that I would have undertaken this task. Gregory S. Bambeck Ph.D. Kent, Ohio, USA 44240 e. mail: February 21, 2010

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