The most reliable intervention which consistently increases the life-span of animals is calorie restriction (CR). This intervention does not only extend the ‘average life-span’ (the average number of years an animal is expected to live), but it also prolongs the ‘maximum life-span’, which is the maximum number of years a particular species can possibly reach. The maximum life-span of mice is 3 years, while that of chimpanzees is 50 years. The human average life-span is around 78 years, whereas the maximum human life-span is around 120 years. In addition, CR also prolongs the ‘health-span’ which is the number of years an organism can live without any major chronic disease.
CR is also sometimes called dietary restriction and, in simple terms is defined as under-nutrition without malnutrition. Typically, the experimental animal is kept on a diet which is 30% to 70% less compared to the amount of food taken when there are no restrictions. The quality of vitamins, minerals, protein, carbohydrate, lipids and other factors in the diet is not compromised, rather it is the amount of overall calories that is reduced. After a period of time on this diet, several biomarkers of aging return to normal levels and the animal looks and is healthy. Research performed at the National Institute of Aging shows that many of the beneficial effects of CR are seen not only in mice or rats but also in primates and even humans (1). However, many scientists want to see more research into the human effects of CR, before they accept its clinical benefits beyond doubt.
Spindler (2), working at the Department of Biochemistry, University of California, has reported the CR changes the expression of key metabolic enzymes which influence the rate of protein renewal. Normally, new proteins are constantly being formed, and damaged ones, (i.e. damaged by free radicals, glycosylation, AGEs etc.) are being eliminated all the time. This rate of formation and removal is balanced and fine tuned. With age, fewer new proteins are being created, while abnormal proteins are not being eliminated quickly enough. The result is an excessive accumulation of damaged proteins which clog-up the cell and cause further injury, contributing to the overall age-related cell dysfunction. But CR can alter this decline, by stimulating the creation of new proteins plus enhancing the effective and quick removal of any damaged ones (3). This clears up any backlog of abnormal proteins, therefore the cell is free to function again effectively. CR also modulates apoptosis (orderly cell death) by modifying chaperone levels. Chaperones are molecules which take part in the formation, repair and elimination of proteins. Specifically, CR decreases the expression of chaperone molecules in the liver and increases the rate of serum protein secretion by up to 250%. This reduces the level of damaged proteins and improves cell function (4). As I will discuss later, therapeutic agents which alter the rate of accumulation of abnormal proteins, (including those which reduce glycosylation) can be considered as having actions comparable to CR.
Due to the fact that almost all species of animals studied so far show similar responses to CR, many anti-aging scientists have supported the view that humans undergoing CR could also exhibit benefits in line similar to those seen in animals. For example, their cholesterol is reduced, blood glucose levels are normalised, glucose tolerance is improved and inflammation markers are reduced etc., (see box 1). This point of view has received a substantial boost when results from the Biosphere 2 experiment were released. Eight scientists who, for nearly two years, followed a CR regime, experienced the same physiological changes as those encountered in calorie restricted primates (5). Clearly, more human research is needed here, but the future looks promising.
Box 1: Biochemical and clinical effects of CR
CR has also hormetic effects. Hormesis is a term referring to the long term benefits of mild, repeated stress or stimulation. Mild stress such as increased external temperature, mild radiation exposure, or hypergravity, as well as nutritional stress (i.e. CR), all have been shown to improve a range of parameters associated with aging. One characteristic of hormesis is that it can be activated following a certain stimulus, but the effects of this activation are not linear (they are non-proportional). In a linear situation, if a stimulus is applied at a mild level it will cause mild stimulation effects. If it is applied at a moderate level, it will cause moderate stimulation, and if it is applied at full power it will cause maximal effects. It turns out that this does not always happen in real life. Hormetic stimulation is not linear but “U-shaped”. (see figure 1). In other words, a mild stimulus may cause strong stimulation, a medium stimulus may result into the opposite effect, (i.e. inhibition) and a further, strong increase of the same stimulus may cause the same degree of stimulation as that seen with the mild stimulus. This hormetic characteristic is important because it helps explain why sometimes an agent stimulates something, and sometimes it inhibits depending on the dose. As it will be made clear in this discussion, in the case of CR and CRM, there is both an inhibition of growth (of cancer cells), and a stimulation of growth (of healthy cells). [End of footnote]
There is one problem with CR however. Very few people are willing to undergo a life time of hunger in order to live a few extra years. Even if, according to research, CR is also effective when applied for a short period later in life, the fact remains that a few weeks or months of starvation and hunger are well beyond the capabilities of most of us in the developed world. The good news is that 70 or so years of research into CR have not gone wasted. We are now in a position to make a few educated guesses as to how exactly CR works, and try to see if we can mimic these effects by using other, less unpalatable interventions to achieve the same result.
CR works by interfering with the expression of certain genes which produce proteins, growth factors or enzymes which, in turn, influence the rate of deterioration or repair of various constituents of the body. If there was a way to influence these same genes by using a tablet or an injection, this would be a much more practical alternative compared to long periods of hunger and dietary discomfort.
Calorie restriction mimetics (CRM) are drugs or chemical compounds which mimic (reproduce) the actions of CR. In other words, the administration of a CRM results in the same physiological changes seen in CR itself. If CRM work the way they are intended to work, the big bonus in terms of human patients, would be that there is no need for lengthy fasting periods. These mimetics activate stress pathways which are also activated by calorie restriction (and possibly by other hormetic challenges). Commonly-studied mimetics are those which inhibit glycolysis or those which improve the action of insulin.
One way CRM work is by influencing specific genes which ultimately affect either cell repair or cell death. For example, one gene affected by CR is the Sir2 gene in yeast. It is activated following a short period of CR and it interacts with p53 which is a factor involved in apoptosis (cell death). When Sir2 is activated by CR, it de-acetylates (deactivates) p53 which then represses the process of excessive cell death, therefore saving cells from unnecessary death (6). See Footnote 2
The process of de-acetylating the p53 gene is called ‘gene silencing’ and it is encountered quite frequently in research aiming to identify the genes which affect aging. p53 is a gene which produces a protein of the same name, (p53) which activates apoptotic cell death. Apoptosis is a process whereby cells commit suicide in response to free radicals, glycosylation or other toxic events causing damage to the DNA. Too much apoptosis results in loss of healthy cells which causes clinical age-related symptoms. On the other hand, too little apoptosis may result in accumulation of damaged cells, (containing damaged DNA) and contributes to cancer. Therefore, a balance needs to be found between excessive and sluggish apoptosis. One way to achieve this is through regulation and re-balancing of excessive or sluggish expression of p53. Apoptosis needs to be high in organs which regenerate easily, (liver, blood, skin, epithelium) and low in organs that do not regenerate easily, (brain, muscle tissues). In the first case, the risk of cancer is increased due to rapid accumulation of damaged cells, (so a fast rate of apoptosis is necessary in order to eliminate these damaged cells and reduce the risk of cancer. These tissues can then regenerate easily with healthy cells). In the second case, the risk of cancer is low anyway, (due to slow turnover of cells), and any excessive apoptotic loss of cells will result in loss of function, (because the lost cells cannot be replaced). [End of footnote]
The yeast Sir2 gene has an equivalent in the earthworm C.elegans and, probably, in other organisms also. This has prompted scientists to look for a human equivalent. It turns out that a human gene similar to Sir2 (a homologue) is a gene called SIRT1. Anderson et al. from the Department of Pathology, Harvard Medical School, have shown that low intensity stress (hormesis) such as CR, causes SIRT1 to de-acetylate (de-activate, or ‘silence’) p53, the absence of which reduces apoptotic cell death, and hence the risk of age-related dysfunction is thus reduced (7). These researchers have also shown that another gene called PNC1 (pyrazinamide/nicotinamidase 1) encodes an enzyme which facilitates the above process, leading to life-span extension.
Langley et al. from the Wellcome Institute, University of Cambridge UK, have reported that the SIRT1 and p53 genes are present near each other, inside the nucleus of human cells, and that the SIRT1 gene regulates p53, thus being capable of modulating cellular senescence (8). Whether the p53 gene becomes activated or silenced, depends on the actual gene sensitivity and on the affinity of SIRT1 to receptors (9).
The study of how genes are affected by CR is quite laborious and time-consuming. Fortunately, new technologies have managed to provide ways which study large numbers of genes at any one moment. GeneChips (high-density DNA microarrays), make use of technology which looks at large parts of the DNA molecule in relatively short periods of time. Dhahbi et al from the Department of Biochemistry, University of California, have reported that GeneChips can study approximately 11000 genes at any one occasion, and that some of these genes are modified in diabetes (10). In this way, it has been possible to identify several genes which may play a role in age-modification through CR. Other research companies have reported that, while a CR regime lasting for two years does reverse many age-related changes, a two to four week period of CR is capable of reversing 70% of those changes. In other words, even a short CR regime lasting for up to four weeks is very effective (70%) compared to a two year CR period. Genes affected in this way are those influencing inflammation, stress, apoptosis, fibrosis, and protein turnover (11).
Calorie Restriction Mimetic number 1: Metformin
One of the most important CRM’s is the anti-diabetic drug metformin, which modulates insulin action. In order to reduce blood glucose, insulin has to be produced in sufficient amounts, but it also has to bind to insulin receptors on the cells in the body. Aging causes an increased difficulty in the smooth operation of this process, and there is a situation whereby insulin cannot effectively bind to the receptors, therefore it does not perform its duties properly. This is called ‘increased peripheral resistance’ to insulin, and it is a cardinal sign in diabetes and aging. Drugs which help mitigate this problem have existed for several years, and new ones are being studied at present.
Metformin (brand name Metforal ®), is a drug which has been in use for over 40 years against diabetes. It is considered to be a receptor sensitizer, because it enhances the sensitivity of insulin receptors on the surface of muscle and fat cells (12). In addition, it also increases the actual numbers of receptors. While other anti-diabetic drugs stimulate the pancreas to produce more insulin, metformin only increases the sensitivity to insulin and does not influence its secretion. The upside of this, is that metformin does not usually cause insulin-dependant hypoglycaemia. When the insulin receptors are as sensitive to insulin as possible, the levels of circulating glucose falls, fat metabolism becomes more balanced and the weight of the patient is reduced (13). Apart from being a receptor sensitizer, metformin also reduces glucogenesis, (glucose production by the liver) and inhibits excessive absorption of glucose by the gut, (14) thus contributing to the overall glucose-lowering effect.
Specifically, French researchers from the Laboratory of Endocrinology, Metabolism and Development in Paris, have confirmed that metformin is able to activate genes which reduce the production of glucose by the liver, thus reducing the risk of glycosylation and other age-related damage. Chemical agents such as lactate, pyruvate, alanine and galactose can be used by the liver to create new molecules of glucose. Metformin can alter the expression of genes which make this conversion possible, thus reducing glucose concentration as a whole and, especially, reducing the concentration of toxic by-products of glucose. In addition, metformin can reduce the gene expression for enzymes which increase oxidation of fatty acids. These enzymes, (such as palmitoyltransferase I) contribute to the oxidation of fats resulting in cell membrane disruption and eventual cell death. But the formation of these enzymes is blocked by metformin which ultimately saves the cell from an untimely death. At the same time, genes which encode for proteins that modulate glycolysis, (destruction of glucose) are activated by metformin.
In the French experiment, expression of genes encoding for glucokinase and liver-type pyruvate kinase, (two enzymes which are involved in glycolysis) was increased by 250% following treatment with metformin (15). It is worth remembering that CR also results in modulation of genes, (16) which affect glucose formation in the liver (high when needed, and low when not needed), influence glycolysis (i.e. glucose elimination, which is high when energy is needed by the rest of the body, and low when not needed), containment of the glycolysis by-products which may contribute to glycosylation, and reduction of tissue levels of AGEs, as well as a reduction in fatty acid oxidation, all of which correspond to the same actions of metformin genetic effects. Therefore, the case for metformin being a CRM is strengthened further.
As mentioned above, using GeneChips is a quick way to test the status of several thousand genes which may affect aging. In an experiment, scientists tested (on mice) four compounds known to affect glucose metabolism. They tested the status of 12422 genes and found that metformin was twice as effective as the other compounds in mimicking the effects of CR. It affected a total of 63 genes, particularly those involved in energy production, protein formation and degradation, cell growth, and detoxification (17).
Metformin works along several different pathways, in order to control glucose activities, modulate insulin action and reduce cell death, eventually increasing life-span. But metformin does not always operate directly via glucose and insulin modulating pathways. It has many other ‘glucose-independent’ activities. With reference to Hormesis, (see footnote 1) metformin is able to modulate the stress response, in other words, it takes part in adjusting the cellular activities following mild stress. A specific biochemical pathway is through activation of AMPK. This is a protein kinase (an enzyme) which is normally active within the cell following multiple stresses. AMPK stands for ‘Adenosine Mono Phosphate- activated protein Kinase’, and is, as the name suggests, activated by Adenosine Mono Phosphate (AMP), an energy-rich molecule (18). Normally AMPK is switched on by stresses such as hypoxia (low oxygen), glucose deprivation, ischaemia or muscle contractions (which increase the energy demands). Once activated, AMPK initiates biochemical activities which prevent and repair cell damage, by leading to a sudden bout of energy production and by switching off any energy-demanding processes which are not directly essential for the survival of the organism. For example, it blocks the long-term production of complex proteins, lipids and carbohydrates which are not needed for the immediate survival of the cell, i.e. it behaves as if the body is in ‘survival mode.’ (But when the presence of these proteins/lipids/carbohydrates becomes essential at a later stage, when the emergency is over, then other mechanisms take over to start creating them again at the right amounts and concentrations so that to keep the cells multiplying again). This is exactly what happens during CR when the body is in ‘survival mode’ and when the nutritional stress of low a calorie diet activates pathways which increase cell repair.
Metformin, and another anti-diabetic drug rosiglitazone were shown to activate AMPK (whereas insulin blocks AMPK) and, as a result, glucose metabolism and cell repair are kept in balance. This is important because a healthy AMPK status reflects an optimal heart function (19). It was also suggested that, by keeping AMPK active, metformin contributes to the beneficial effects of exercise seen in the treatment of diabetes (20). In addition, when metformin activates AMPK in the liver, the production of enzymes which help form new lipid molecules is reduced (21). In other words, metformin through the AMPK process blocks the accumulation of fat.
Summary of AMPK
CR causes a mild nutritional stress with low energy available to the cells. This stressful event activates AMPK which aims to rebalance the process of energy formation, and repairs any cell damage, (including any coincidental age-related cell damage) while switching off any processes not necessary for immediate survival. Therefore, the cell survives and the organism ultimately lives longer. Metformin, being a mimetic of CR, results in the same effect by directly activating AMPK which confers the above benefits to cell repair. The main point here is that it may not be necessary to have to undergo a period of CR to achieve cell repair, when metformin can do this itself by working on the same mechanisms as those involved in CR.
Patients with significant kidney or liver disease, or those with heart failure should avoid taking it. Common and mild side effects are nausea, vomiting or abdominal bloating. The normal anti-diabetic dosage for metformin is 500 mg twice a day, or 850 mg daily. This can be increased as necessary to a maximum of 3000 mg a day. However, the dose required for calorie restriction mimetic effects has not been calculated formally. In mice, a dose of 300 mg/kg/day has been shown to reduce body temperature (a CR mimetic effect). But this cannot be extrapolated to humans, as it will mean 21000 mg for an average male. Further research is needed to clarify this point. Healthy people who take metformin for its general anti-ageing benefits use 500 mg twice a day.
It is important to keep an eye on the blood biochemistry during metformin treatment. Tests commonly performed are fasting glucose and lipid status, liver and kidney function and haemoglobin A1c which is a glycosylated haemoglobin indicating the effectiveness of glucose control in the body. A low A1c means that the level of glucose (and therefore, indirectly, the level of glycosylation damage) in the body is well-controlled. Normal levels are those below the value of 5%. People who drink alcohol excessively should avoid metformin, or at least take it only under expert medical supervision.
Calorie Restriction Mimetic number 2: Resveratrol
Found mainly in red wine (from the skin of unripe red grapes), resveratrol is a polyphenol plant chemical with proven beneficial cardiovascular effects. What is more, resveratrol is a potent CRM. In yeast, it stimulates Sir2, increasing DNA stability and extending life-span by 70% (22). It is believed that it works the same way in humans, i.e.. by activating the human homologue SIRT1 which, as explained above results in reduced apoptosis in the liver, blood and skin, and reduced risk of age-related chronic disease. Research performed at the Hormel Institute, University of Minnesota, shows that resveratrol possesses an anticancer activity which is medicated through p53 modulation. A derivative of resveratrol can also block cells from dividing, without involving p53, thus safeguarding against unauthorised cell replication which may result in cancer (23).
A number of studies tried to explain why resveratrol sometimes induces apoptosis (and therefore eliminates damaged cells, keeping the risk of cancer low) and at other times it blocks apoptosis (and so it saves healthy cells from unnecessary death). The mechanisms governing this dual function of resveratrol are based on Hormesis and are quite complex. The effect depends on the ratio of resveratrol/related molecules, the concentration of pro and anti- apoptotic factors, the sensitivity of the target receptors, and the presence of other inhibiting or activating factors (24, 25).
Resveratrol is normally taken in 5 mg capsules once a day for prevention, and three times a day for treatment. The dose necessary to achieve CRM effects has not been calculated but, currently, there is no reason to recommend anything other than a daily dose of 5 to 10 mg. It is conceivable that, for a maximum CRM effect, resveratrol and metformin can be taken together or, perhaps even better, alternating metformin and resveratrol. There is some evidence that taking medication at irregular and ever-changing intervals has a more pronounced benefit on health (26). However, the full efficacy of this recommendation has not been evaluated clinically.
An ideal way of testing the clinical benefits of metformin and/or resveratrol used as CRM would be to measure the patient’s biomarkers by using Inner-Age ™, (Ed. See www.inner-age.com) and then try the treatment for a period of about six months. At the end of this period re-evaluate the patient’s biomarkers (by using Inner-Age ™ again) and study the difference in the scores, particularly those related to blood glucose, insulin, cardiovascular health, liver function and brain activities, all of which can be expected to show a considerable improvement. [Ed.- Details about Inner-Age are available from IAS or can be seen at www.inner-age.com] Another way would be to check whole body temperature by infra-red scanning. This is usually low in organisms undergoing CR.
Other calorie restriction mimetics
Other mimetics include agents which reduce abnormal protein accumulation. For example, agents such as aminoguanidine, carnosine and 2-deoxyglucose: These would be expected to:
A compound which inhibit glycolysis and is being used as a CRM is; 2-deoxyglucose. This has been reported to mimic some of the effects of CR, particularly increased insulin sensitivity, reduced glucose levels and other biochemical changes. Research is still under way to identify more about its possible benefits on humans (28). What is known about 2-deoxyglucose is that it can be toxic in high dosage.
Less well studied possible CRM include;
1. Modulators of NPY: The neuropeptide Y (NPY) is a small protein fragment which increases appetite, induces obesity and reduces the metabolic rate. CR stimulates the production of NPY which then encourages the organism to return to its normal eating habits and increase body weight. However, it is well known that the result of CR is weight loss, not weight gain. What happens is that CR selectively blocks some receptors, (mainly in the hippocampal region of the brain) and stimulates others, (mainly in the hypothalamic region of the brain) (29). The result, based on the concept of hormesis, (see footnote 1) is a balanced, modulating activity of CR with the known clinical benefits. The importance of this is that there are now attempts at examining artificial ways of affecting NPY and therefore mimicking the effects of CR. A manipulator of NPY release would result in exactly the same clinical effects as those seen in CR, which works by naturally modulating NPY activity.
2. Exanadin: The agent exendin, (exanatide) works by reducing plasma glucose, suppresses food intake and overall regulates glucose metabolism. It is a GLP (Glucagon-Like Peptide) antagonist, which means that it counteracts the glucose-boosting effects of the hormone glucagon. It is a promising CRM, able to increase brain function and protect the brain against toxicity, but still under investigation (30).
3. The agent PYY3-36: This is a peptide, (protein fragment) which is released from the gut following a meal. It then inhibits food intake by acting on the hypothalamus in the brain, which is considered to be the hunger centre. By reducing appetite and glucose metabolism it is believed to have at least some of the benefits seen with CR (31).
4. Leptin: Research performed over the past few years shows that leptin, a molecule which stimulates fat metabolism and reduces body weight, is an essential factor involved in CR effects (32). It is produced by adipocytes (fat cells) and it is involved in the response to fasting and in hormonal changes seen during CR. A reduction of dietary intake causes leptin levels to fall and this interferes with the secretion of testosterone, progesterone, growth hormone and thyroid hormones as a response for adaptation (33). Therefore, it is believed that leptin mediates the clinical effects of CR. As a result, agents which affect leptin production must also be classified as CRM (34). Together with insulin and ghrelin (a growth hormone stimulator) leptin balances the ratio of appetite promoters vs. appetite blockers in the hypothalamus in the brain and so regulates homeostasis and food intake. Researchers from the Radiation Biology Branch, Center for Cancer Research, National Cancer Institute in Bethesda USA, have shown that antioxidants influence leptin levels and thus reduce weight gain and reduce tumour incidence (35).
Leptin stimulates the production of AMPK and reduces the activities of enzymes which store fat. Any deficiency or resistance (unresponsiveness) to leptin, (such as that seen in aging, or in obesity), causes ectopic fat accumulation, i.e. storage of fat in tissues other than those specifically designed to store fat, (called the white adipocytes). Ectopic fat accumulation is most obvious in the intra-abdominal tissues, (which are inside the abdomen but outside the bowels). This excessive fat accumulation may cause apoptotic cell death from the pancreas and heart muscles, (where ectopic fat accumulation may also take place) worsening diabetes and heart function (36). Therefore, leptin activities share the same pathways as those of CR and metformin action.
Similarities of effects between metformin and Leptin. Both:
The increased amount of research into CR has given us promising directions into identifying effective agents which reproduce the exact benefits of CR, without the need to follow long calorie-restricted diets. The most promising and clinically relevant CR mimetics are metformin and, to a lesser degree, resveratrol, together with aminoguanidine and carnosine. Several others are in the pipeline. While research is continuing, many doctors who already recommend these compounds to their patients for other reasons, can now start considering that their treatment has an added possible bonus.
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