CALORIC RESTRICTION AND AGING

This outline and these notes provide detailed information about caloric restriction and aging.

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Definitions

• CR = dietary restriction
• CR = CR = caloric restriction
• ML = mean longevity = life expectancy = average life span
• LS = maximum life span
• higher = increases
• lower = decreases

• 1937 - Clive McCay - rats - 33% CR
• higher ML and LS
• also works with tested species of protozoans, roundworms, spiders, insects, fish, mice
• works with any "complete" diet
• undernutrition without malnutrition
• may require supplements
  
            - vitamins, minerals, bulk fiber
• late 1980s - start three studies with monkeys
• NIA - Baltimore - 160 monkeys (squirrel monkeys and rhesus monkeys)
• Univ. Maryland - Baltimore - 27 monkeys
• Univ. Wisconsin - 76 monkeys
• approx. 30% CR + supplements
  
            - different diets

• rodents - higher ML 25%+ higher LS 30%+
• results vary with;
• age of onset (young, adult, old)
• duration of CR (positive correlation)
• severity (optimum)
• strain of animal (varied)
• continuity (residual effects)
• results largely unaffected by;
• percent fat, protein, carbohyCRate
• type of fat, protein, carbohyCRate
• percent body fat
• feeding regimen

• reduces:
• rate of growth
• rate of maturation
• size at any age
• skeletal thickness
• % body fat (most times)
• total lean body mass (LBM)
• increases age of;
                   1. higher blood glucose *
                   2. lower insulin sensitivity * 
                   3. maturation ( & )
                   4. reproductive decline ()
• reduces declines in;
                  1. insulin sensitivity *
                  2. HDLs
                  3. number of muscle cells
                  4. IL-2 *
                  5. physical activity *
                  6. health (e.g., skin, cardiovascular, kidneys)
  • in rodents (so far);
                  1. protein x-links *
                   2. rate of protein synthesis
                   3. DNA repair *
                   4. melatonin *
                   5. immune function *
  
  • in monkeys
                  1. DHEA *
  
• reduces increases in;
                  1. mitochondrial damage * 
                  2. blood glucose *
                  3. blood insulin *
                  4. diabetes mellitus
                  5. blood LDLs
                  6. blood triglycerides
                  7. percent body fat (usually)
                  8. IL-6 *
                  9. kidney disease
                  10. cancers
  • in rodents (so far);
                  1. free radical production *
                  2. free radical damage * 
                  3. glycated hemoglobin * 
                  4. lipofuscin
  • in monkeys;
                  1. "low energy" muscle cells * 
                  2. blood pressure
  
  Uncertain effects on;
  GH *, TSH, prolactin, glucocorticoids *, atherosclerosis
  
  No effects on;
  BMR/LBM/HR - daytime 
  BMR/LBM/24 hrs. (monkeys)
   rate of wound healing (usually)
  
  BUT causes;
  lower body temperature
    lower BMR/LBM/HR
                      - nighttime (monkeys) *
  
   Detrimental effects
  hunger 
  lower immune function (? - monkeys) ? ? ? ? ?
   
  Effects in humans
  ? ? ? ? ?
   
  Possible mechanisms of action
       1. lower free radical formation *
       2. higher free radical destruction *
       3. higher protein turnover (replacement) *
       4. lower glycation of proteins *
       5. lower protein X-link formation *
       6. channeling energies from reproduction to survival *
       7. lower body temperature
       8. lower rate of metabolism *
       9. higher heat shock proteins *
       10. higher glucocorticoids *
       11. maintain/increase melatonin *
       12. affect other hormones *
       13. affect specific genes *
  
    CR + exercise
  CR 
     higher ML + higher LS
     lower diseases + lower aging 
  Exercise
     higher ML lower diseases 
  CR + Exercise
     higherhigher ML + higher LS
     lowerlower diseases + lower aging
  additive effects, not synergistic effects

  DETAILED NOTES

  Means for Altering ML and LS

  - LS can be altered by CR, by gene manipulations, and by "exposure to low levels of toxic agents" called "hormesis"
  - genetic means of increasing life span include (a) C. elegans {daf genes for dauer formation}, (b) Neurospora fungi and yeast, and (c) fruit flies {selective breeding or genetic manipulations increasing superoxide dismutase and catalase}
  - hormesis in C. elegans includes ionizing radiation or elevated temperatures
  - hormesis in fruit flies includes elevated temperatures
  - hormesis using ionizing radiation occurs in many animals
  - this author proposes that increasing LS depends upon mechanism(s) that increase resistance to adverse conditions (i.e., have better stress responses). This includes better resistance to injury by adverse factors and/or better repair mechanisms when injury has been sustained (better insulators and barriers and/or better negative feedback systems)
  - there may be many different interventions that can stimulate better stress responses and thereby lead to increases in LS
  - perhaps CR will be shown to have more effects in animals naturally subjected to periodic natural CR (e.g., temperate herbivores and many carnivores) than in animals that rarely experience natural CR (e.g., tropical herbivores) or in animals that go into states of low activity during natural CR (e.g., torpor, hibernation, lower body temperature)

  History of DR

  - first CR experiments in 1937 at Cornell by Clive McCay with rats
  - 33% DR
  - CR works with diets where mostly the carbohydrates, especially the simple carbohydrates, are reduced
  - CR = 30% decrease in calories but with vitamin and mineral supplements
  - CR works just as well even if there are reasonable variations in the proportions of fat, carbohydrate and protein, including the types of protein
  - CR works if there are vitamin and mineral supplements since the lower food intake with CR does not provide adequate vitamins and minerals unless VERY carefully controlled by using high nutrient density foods carefully selected to provide proper balance as well as proper amounts of macronutirents and micronutrients. therefore, simply eating much less will not work since there is high risk of malnutrition
  - supplementation with vitamins or minerals or antioxidants does not extend LS
  - CR works when started at any age except in the very young, when growth is very important and rapid
  - CR has greater beneficial effects when started in late youth or early adulthood rather than in later adulthood
  - CR works in a dose-dependent manner, so there does not seem to be a threshold needed to get beneficial effects. however, when CR is extreme, detrimental effects of undernutrition and malnutrition outweigh the benefits from the CR, so ML and LS are shortened
  - CR in rodents can increase ML by 25% or more and can increase LS by 30% or more, depending upon the CR regimen and the type of animals used (e.g., genetics alters the efficacy of DR)
  - beneficial effects of CR in rodents include delayed onset of age-related declines in blood glucose regulation (insulin sensitivity); loss of reproductive functioning in females; declines in DNA repair; immune decline; learning ability, muscle mass; rate of total body protein synthesis
  - in rodents, CR slows the age-related changes in protein cross-linking, free-radical production; damage by free-radicals
  - the three CR studies with monkeys are at NIA in Baltimore with Lane, Ingram, Roth, et al., at University of Wisconsin (UW) with Kemnitz, et al., and at UM,B in Baltimore with Bodkin, et al.
  - CR work at UW (76 monkeys - 11-14 years old at start - 10% dietary fat - 30% of individual AL intake) and at UM,B (27 monkeys - 11-12 years old at start - 13% dietary fat - CR to maintain constant weight in CR animals) use only older monkeys
  - NIA study in Baltimore
  - started in 1987
  - 30% DR
  - NIA used 30% CR on RMs (rhesus monkeys)
  - CR work at NIA uses monkeys of a variety of ages (160 monkeys - 5% dietary fat - 30% CR reduction w/t controls)
  - rhesus monkeys have LS of 40 years (Macaca mullata)
  - squirrel monkeys have LS of 20 years (Saimiri sciuris)

  Effects of CR on ML and on LS

  - CR increases ML and LS in insects, works, protozoans, and other animals
  - with CR, rats live longer, have greater ML and greater LS, retain health and activity and vigor better
  - CR in rats increases ML and LS as long as the CR is not very severe (e.g., <60% AL) and as long as the CR does not begin too soon after birth
  -DR increases ML and LS despite when during youth, maturation, or adulthood it begins
  - CR begun early in life has a residual effect on increasing ML and LS even if the CR is ended during adulthood
  - CR has its effect any time the total calorie intake is reduced adequately regardless of which main organic macronutrients are reduced (i.e., protein, fat carbohydrate) as long as there is adequate supply of calories and essential nutrients (e.g., AAs, FFAs, vitamins, minerals)
  - CR increases ML and LS by shifting the Gompertz equation plots to the right overall. With proper CR diet, there is a reduction in mortality rates at all ages when CR is applied. Thus, even in cases where CR affects ML but not LS, more individuals in the study population reach very old age, not just pass "middle age".
  - only CR increases both ML and LS
  - CR resulted in healthy monkeys and a lower mortality rate and a higher ML when compared with AL monkeys
  - some of the essential, universal or at least wide spread effects of CR that reduce aging rates as shown by increased ML and increased LS and decreased disease have not been identified yet
  - even if CR proves not to increase LS in primates as it does in rodents and many other species, it will probably increase ML by reducing factors the increase risk for or actually cause many age-related diseases

  Overall effects of DR

  - CR works in roundworms and fish, spiders, guppies
  - CR works in rotifers
  - CR works when started at any age except in the very young, when growth is very important and rapid
  - CR has greater beneficial effects when started in late youth or early adulthood rather than in later adulthood
  - CR works in a dose-dependent manner, so there does not seem to be a threshold needed to get beneficial effects. however, when CR is extreme, detrimental effects of undernutrition and malnutrition outweigh the benefits from the CR, so ML and LS are shortened
  - CR works regardless of the feeding regimen (e.g., skipping days, different times of feeding during the day, lowered amount of food each day, "step regimen" with period of CR for days or weeks following by AL followed by CR for another period, etc.
  - CR works even if there is not a decrease in percent body fat compared with AL
  - the degree of effectiveness of CR depends upon the strain of animal used within each species (e.g., long-lived rat strain respond differently to different CR regimens and have different degrees of effectiveness on different CR regimens {timing, degree of CR, stage of life applied)
  - beneficial effects of CR in rodents include delayed onset of age-related declines in blood glucose regulation (insulin sensitivity); loss of reproductive functioning in females; declines in DNA repair; immune decline; learning ability; muscle mass; rate of total body protein synthesis
  - in rodents, CR slows the age-related changes in protein cross-linking, free-radical production; damage by free-radicals
  - in rodents, CR postpones the onset of many age-related diseases (e.g., many types of cancer, kidney disease, diabetes mellitus, cataracts)
  - in rodents, CR slows the cross-linking of proteins; reduces the age-related decrease in the rate of protein synthesis; slows the age-related decrease in muscle mass (perhaps by sustaining more activity); helps maintain parameters of age-related decreases in "mental" activities
  - CR resulted in lower triglycerides and higher HDLs. This effect is produced in part by CR causing less age-related decreases in regulation of glucose and in part by CR causing smaller body size
  - in some studies of CR in rodents, there is a decrease in % body fat, which did not occur in these monkeys
  - CR slows the rate of maturation, leads to lower body weight, delays reproductive maturation, can result in lower percent body fat
  - CR has similar effects on monkeys and on rodents including slowing age-related increases in body weight, body fat, lean body mass, sexual maturation, skeletal growth, blood glucose, blood insulin, blood triglycerides, LDL levels, IL-6, IGF-1; and age-related decreases in insulin sensitivity, DHEA levels (in monkeys - rodents have no DHEA), HDL levels, and voluntary physical activity (actually becomes higher in some cases)
  - CR did not affect amount of activity of the animals
  - CR monkeys have lower BP
  - short term CR in monkeys does not affect blood pressure or heart rate
  - CR in monkeys results in improved blood lipid profiles (lower triglycerides, higher HDLs)
  - beneficial changes in blood chemistry from CR in monkeys account for only a portion of improved biomarkers of aging, so other unidentified biomarkers must also be affected by DR. These other might include adrenal steroid levels, immune responses, and thermoregulation, glyoxidation of proteins
  - CR resulted in greater retention the number and the types of skeletal muscle fibers in the vastus lateralis muscle of rats.
  - the effects of CR on pituitary hormones, adrenal hormones, pancreatic hormones, TSH, prolactin, GH, and IGFS are reviewed. Basically, these are not studied well, and the impression is that if there are any changes, they are unclear or not very relevant to the effects of CR on ML or on LS.

  Effects on monkeys

  - monkeys gain weight as they age throughout life
  - CR allowed gain in weight throughout life, but reduced the degree of gain in body weight.
  - some CR work with monkeys does show a decrease in % body fat, but there are differences among types of CR with monkeys. (e.g., CR to maintain a certain body weight such as young adult body weight even with increasing age, which is usually associated with increasing weight; CR with monkeys of different ages; age at which CR begins; duration of DR; type of diet; amount of body fat in animals when the CR began)
  - CR causes decreases in gain of body weight, in gain of LBM, in gain in body fat, and in rate of sexual maturation
  - CR monkeys are smaller, take longer to reach maturity, have lower blood glucose levels, have lower insulin levels, have greater voluntary daytime activity
  - CR in rhesus monkeys delays skeletal maturation.
  - the CR regimen used seemed to have no adverse effects on the health of the monkeys as determined by many parameters of blood

  Effects of CR plus exercise

  - exercise increases ML but does not affect LS. CR increases ML and LS. Exercise seems to increase ML by reducing risk factors or physiological age changes that increase the likelihood of certain age-related disease, such as circulatory system diseases. The mechanisms by which exercise increases ML are not related to the mechanisms by which CR increases ML and LS. The additive effects of CR and exercise on ML are due to the additive effects of the different mechanisms of the two conditions. The mechanisms by which CR increases ML and LS are not known but they involve alterations of basic aging processes. The mechanisms by which exercise increases ML are not clearly known, but they are by increasing the age of onset, decreasing the incidence, and/or decreasing the severity of age-related diseases.

  DR in humans

  - for humans, people on Okinawa, who historically work hard physically and consume lower calorie balanced diets have many more very old people and much lower incidences of certain age-related diseases (e.g., cancers)
  - in short term experiments with humans in Biosphere 2, Roy Walford showed that short term CR in humans resulted in lower BP and lower blood glucose and lower blood pressure
  - potential risks with CR in humans are decreased stress response and other defense mechanisms
  - recommendations for humans include that CR starts at about age 20, start gradually (even more gradually if started at higher ages), reaching ideal maximum of 65% "normal" intake to sustain weight at "individual normal weight" during 20s = 10%-25% body "set point" using well-balanced diet with little sugar, adequate protein and fat, and adjusting calories by adjusting complex carbohydrates
  - if people cannot follow CR, if the mechanism by which CR works can be found, maybe alternative methods of producing the same effects can be found

  Possible mechanisms of CR action

  - hypotheses about how CR works include different metabolism of glucose; less free radical damage; decrease glycation of molecules; altered gene expression; altered stress hormones; altered melatonin levels; altered oxidative pathways in mitochondria; more efficient energy transduction from nutrients; shifting energies to homeostasis rather than to growth and reproduction; altered body temperature; altered rate of metabolism ( rate of living ); improving adaptive responses to environmental stressors and adverse condition (e.g., more heat shock protein, slightly elevated glucocorticoids)
  - CR may work by limiting free radical damage in mitochondria by reducing free radical production through more efficient oxidative reactions. results would include less damage to mitochondrial DNA and mitochondrial proteins, thus allowing them to work better, and further reducing the formation of free radicals

  DR working by affecting free radicals and oxidative damage

  - fruit flies with mutations that increase antioxidant enzymes have longer LS and have less free radical damage
  - LS in fruit flies is inversely related to the rates of free radical production by mitochondria (superoxide free radical {O2.} and H2O2)
  - conclusion is the free radicals "cause" aging
  - some experiments showed no effect of antioxidant supplementation on LS. but by mutation studies, fruit flies with mutations increasing the formation of antioxidant enzymes (e.g., superoxide dismutase and catalase) had greater LS by 14% -34%
  - since giving rodents antioxidants increases ML but not LS, CR may not work by altering free radical damage
  - antioxidant defense mechanisms include those that reduce free radical damage such as free-radical scavenger enzymes, antioxidants, proteins the chelate (bind) metal ions (thereby reducing metal-promoted free radical reactions such as from Fe). Other mechanisms repair free radical damage, such as DNA repair and enzymes that remove damaged protein or lipid molecules
  - note that free radicals promote glycation and that glycation promotes free radicals and that both reduce free radical removal and free radical repair by damaging the molecules in the free radical removal and repair reactions. Meanwhile all the damaged and cross-linked molecules are disturbing all other metabolic processes and damaging other biological cellular and intercellular components, yielding biological aging.
  - this author that since CR decreases the formation of free radicals of all types, that CR upgrades free radical destroying mechanisms and enzymes, that CR animals show less free radical and oxidative damage in all areas of cells (e.g., mitochondrial membranes) studied, and that by-products of free radical and glycation and Maillard products, etc., are lower in CR animals, that the main if not the only way by which CR increases ML and LS is by reducing free radical damage. This also leads to the conclusion the aging is caused by free radicals.

  Effects on glucose and insulin in monkeys

  - CR results in lower blood glucose levels in rodents
  - with regard to insulin and glucose regulations in rodents, CR animals have lower levels of insulin and better glucose tolerance test results. This is probably because AL animals develop insulin insensitivity with aging and that CR prevents the age-related increase in insulin insensitivity (i.e., age-related decrease in insulin sensitivity). The CR animals also had lower fasting blood glucose levels, even though they may be eating the same amount of glucose.
  - in rodents, CR results in decreased levels of liver enzymes for glycolysis and for lipid metabolism (perhaps because the animals are CR and need to use glucose in different ways).
  - in monkeys as in humans, during the development of diabetes mellitus, the organism goes through a stage of excessive secretion of insulin in response to a glucose load (e.g., OGTT). As the pathogenesis of diabetes continues, there seems to be exhaustion of the beta cells resulting in a dramatic decrease in insulin production from a glucose challenge, which marks the onset of "full blown" diabetes mellitus with very poor OGTT results and hypoinsulinemia necessitating insulin supplementation (i.e., transition from low glucose tolerance to NIDDM to IDDM). CR stops this pathogenesis.
  - Syndrome X = onset of obesity, decreased insulin sensitivity, decreased glucose tolerance, high blood pressure, and abnormal blood lipids which increases with aging in monkeys
  - these monkeys began on CR in approximately 1986, so they were on CR for 9 years in 1995
  - having lower blood glucose levels and better insulin levels powers risk of several diseases in humans including atherosclerosis, coronary heart disease, microvascular pathologies (e.g., kidneys, retina), thicker basement membranes (e.g., capillaries), cellular immune responses, hypertension
  - CR greatly reduces or entirely prevents the development of all aspects of Syndrome X in monkeys.
  - in monkeys with low insulin sensitivity or with IDDM, insulin has less effect on stimulating skeletal muscle glycogen synthetase, resulting in less removal of glucose from the blood. This is part of the lowered insulin sensitivity in these animals. This same effect is seen in humans at high risk for diabetes but who have not developed diabetes.
  - CR results in different metabolism of glucose in glycogen synthesis in monkey skeletal muscle
  - I do not understand the significance of the changes in enzyme levels and in the responsiveness of the enzymes to insulin, and in the changes in levels of glucose-6-phosphate, except to say that this work shows that CR alters glucose metabolism in skeletal muscle by altering the levels and regulatory responses of several enzymes related to blood glucose regulation and glucose storage and breakdown.
  - CR resulted in older monkeys having % body fat similar to young adult monkeys
  - CR increases insulin sensitivity in all CR monkey studies, even CR monkeys with the same % body fat as AL monkeys, showing that changes in % body fat is not necessary for CR to increase insulin sensitivity
  - with age, there is an increase in the maximum blood glucose levels reached during an IVGTT (intravenous glucose tolerance test). CR resulted in animals reaching a lower maximum blood glucose during an IVGTT
  - with CR, there were age-related increases in basal blood levels of insulin, insulin levels reached during IVGTT, and total amount of insulin secreted during a IVGTT
  - the CR monkeys metabolize glucose at the same rates as did the AL animals during a IVGTT as reflected in the rate of decline of blood glucose during a IVGTT
  - with CR, the basal levels, maximum levels, and total amounts of insulin were lower in CR animals than in AL animals during a IVGTT
  - at first for squirrel monkeys and for rhesus monkeys, blood glucose and blood insulin levels were the same for CR and for AL animals. After 3-4 years of CR, the CR animals had lower basal blood glucose and lower blood insulin levels. Essentially the same results occur in the UW monkeys.
  - CR in this study prevented age-related development of obesity, hyperglycemia, hyperinsulinemia, and low glucose tolerance
  - CR monkeys eat 60% of calories consumed by AL monkeys (i.e., 40% less calories in CR than in AL monkeys)
  - monkeys develop spontaneous obesity and spontaneous NIDDM with aging when fed AL
  - with AL over nine years, AL monkeys develop increased % body fat, obesity, declining glucose tolerance, declining insulin sensitivity of glycogen synthetase in skeletal muscle and in adipose tissue, hyperinsulinemia, increased and then decreased B-cell sensitivity to glucose load with normal fasting PGL followed by increasing fasting PGL, and then reduced insulin response to glucose and NIDDM
  - in addition, the CR monkeys have had longer mean longevity and few pathologies. The AL monkeys have had more pathologies including skin rashes, skin ulcers, edema, kidney failure, and cardiac pathologies. The CR monkeys had the same metabolic rate on a per total kgm basis as did the AL monkeys, but being lighter in weight, the CR monkeys consumed 40% fewer calories per day than did the AL monkeys. However, the metabolic rate per kgm of lean body mass for CR and AL monkeys was not determined, and may have been less in CR monkeys.
  - CR might delay the onset of diabetes mellitus by decreasing the chances of obesity, but even in CR animals with the same % body fat as AL animals, CR still has beneficial effects on blood glucose, insulin levels, and insulin responsiveness (sensitivity)

  DR working by altering glycation and glucose cross-links

  - glycation reactions can lead to aging by forming cross-links among molecules (e.g., collagen, other structural and enzymatic protein). In the reactions involved, free radicals speed up glycation, and byproducts of glycation reaction produce free radicals. (note importance of Amadori products -> Maillard reactions -> Maillard products = cross-linked molecules including damaged free radical scavengers and enzymes that remove free radical-damaged molecules or repair DNA. This reduces free radical destruction and free radical repair, which can lead to increased glycation, etc., etc. Some of these detrimental processes are speeded up or aided by Fe and by CU ions. Some of the reactions forming Amadori products and Maillard products produce free radical and highly reactive compounds, leading to more molecular damage and reduced free radical defense and repair, leading to more free radical effects, -> more glycation -> -> etc., etc.
  - note that free radicals promote glycation and that glycation promotes free radicals and that both reduce free radical removal and free radical repair by damaging the molecules in the free radical removal and repair reactions. Meanwhile all the damaged and cross-linked molecules are disturbing all other metabolic processes and damaging other biological cellular and intercellular components, yielding biological aging
  - there is an age-related increase in tendon-breaking time as measured by the amount of time it takes for a tendon to break when under a constant tension and being chemically affected by enzymes or by urea. An increase in breaking time means that the collagen is more resistant to breakdown showing that there are more chemical bonds (i.e., cross-links) holding the collagen molecules and fibers together. Therefore, an increase in tendon breaking time is a measure of collagen cross-link formation with a direct proportion between break time and amount of cross-links.
  - some of the age-related increases in cross-links in collagen are probably from sugars by non-enzymatic glycosylation (i.e., glycation) followed by oxidation forming pentosidine if the amino acids linked are lysine and arginine.
  - the formation of protein cross-links by glucose is called glycation, forming an Amadori product or glycated protein. This cross-link may be oxidized to form pentosidine by the Maillard reaction, so the total reaction of forming and oxidizing a glucose cross-link in a protein is called glycoxidation. The resulting cross-link gives the protein a brownish color
  - with aging, collagen changes from being easily solubilized by urea or enzymes to being more slowly solubilized by urea or enzymes because of age-related increase in cross-links
  - with aging, collagen becomes less elastic because of increases in cross-links
  - some of the age-related increases in cross-links in collagen are probably from sugars by non-enzymatic glycosylation (i.e., glycation) followed by oxidation forming pentosidine if the amino acids linked are lysine and arginine.
  - the formation of protein cross-links by glucose is called glycation, forming an Amadori product or glycated protein. This cross-link may be oxidized to form pentosidine by the Maillard reaction, so the total reaction of forming and oxidizing a glucose cross-link in a protein is called glycoxidation. The resulting cross-link gives the protein a brownish color.
  - in humans there is an age-related increase in pentosidine indicating an age-related increase in glycoxidation of proteins. This is shown in skin, cartilage and the dura mater
  - animals in this study included rats, shrews, beagle dogs, cows, pigs, squirrel monkeys, rhesus monkeys, and humans because they have very different LS increasing in this order (rats = shrews = 3-4 years), (cows = dogs = 20 years), (squirrel monkeys = 21 years), (pigs = 27 years), (rhesus = 40 years), (humans = 120 years)
  - in all species, pentosidine increases with age in a curvilinear fashion with increasing rates with increasing age
  - in general, the rate of pentosidine formation is inversely proportional to the LS of the species
  - though there is an age-related increase in pentosidine and the rate of formation is inversely related to LS, the amount of pentosidine accumulated does not have an effect on LS, though its mechanism or formation must be related to whatever mechanisms determines LS
  - it seems that CR may have its aging-attenuating effects through some effects on glucose metabolism and glucose regulation
  - with CR, there is better control of glucose blood levels, which may limit damage from glucose by glycation and other mechanisms seen in diabetics.
  - this author concludes that since CR decreases the formation of free radicals of all types, that CR upgrades free radical destroying mechanisms and enzymes, that CR animals show less free radical and oxidative damage in all areas of cells (e.g., mitochondrial membranes) studies, and that by-products of free radical and glycation and Maillard products, etc., are lower in CR animals, that the main if not the only way by which CR increases ML and LS is by reducing free radical damage. This also leads to the conclusion the aging is caused by free radicals
  - CR did not result in a difference in the amount of glycated hemoglobin
  - CR in rodents results in lower levels of glycated hemoglobin
  - if by genetic engineering, genes from organisms having enzymes that remove glycation cross-links could be put into cells of animals. Without such enzymes, LS might be increased by reducing glycation and glyoxidation

  DR working by affecting mitochondria

  - there is an age-related increase in free radical; formation by mitochondria in many animals, including fruit flies, houseflies, pigs, and cows
  - there is an age-related decrease in ATP production in tissues such as brain, heart, and muscle. This may be due to free radical damage to mitochondria.
  - the age-related increase in mtDNA deletions in skeletal muscle in monkeys is heterogeneous among muscle fibers. Some fibers accumulate a disproportionately large amount of mtDNA deletions
  - conclusion is that although the total number of mtDNA deletions in a skeletal muscle is not great, since their occurrence is much higher in some fibers than in others, the mtDNA deletions may become significant in producing significant age-related deleterious changes in specific fibers, resulting in age-related deleterious effects in the muscle as a whole
  - there is an age-related increase in mtDNA deletions in skeletal muscle
  - in rodents, CR resulted in fewer mtDNA (mitochondrial DNA) deletions in adductor longus and in soleus muscles but had no effect in two other muscles
  - in rodents, CR resulted in (a) less age-related loss of skeletal muscle fibers, (b) less age-related deleterious changes in mitochondrial enzymes (loss of cytochrome c oxidase, increase in succinate dehydrogenase), and (c) fewer mtDNA deletions
  - 50% CR had greater effects than did 35% CR
  - CR in mice skeletal muscle
  - there is an age-related decrease in the activity of mitochondrial electron transport complexes (Complexes I, II, III, IV) by 33-64%
  - CR resulted in virtually stopping the age-related decreases in activity of the four mitochondrial complexes
  - in monkeys, there is an age-related increase in mtDNA deletions in skeletal muscle
  - the age-related increase in mtDNA deletions in skeletal muscle in monkeys is heterogeneous among muscle fibers. Some fibers accumulate a disproportionately large amount of mtDNA deletions
  - conclusion is that although the total number of mtDNA deletions in a skeletal muscle is not great, since their occurrence is much higher in some fibers than in others, the mtDNA deletions may become significant in producing significant age-related deleterious changes in specific fibers, resulting in age-related deleterious effects in the muscle as a whole

  DR working through glucocorticoids

  - CR may work in several ways including providing adaptive mechanisms and strategies to allow animals to better cope with the natural fluctuations in food availability. By directing energies more toward survival rather than to reproduction during times of natural dietary restriction, animals have increased survivability to endure the food shortage and to spend energy looking for or moving to food sources. In a general way, these alterations increase the effectiveness of insulators and barriers to harm while increasing negative feedback mechanisms for repair of sustained damage. In mammals, this may be having CR increase glucocorticoid levels somewhat and thereby initiating the beneficial consequences of stress responses. Thus far evidence is circumstantial, such as increasing fruit fly ML by giving them glucocorticoids, inhibition of certain tumors by slight elevations in glucocorticoids, and smaller age-related decreases in pulmonary function in individuals with above-average levels of glucocorticoids.
  - in animals with glucocorticoids, increasing glucocorticoids may not only increase defense and repair aspects of the stress response but also limit damage from excessive inflammatory response and excessive immune responses and autoimmune responses (e.g., those involved in Alzheimer's disease, arthritis G.I. tract diseases)
  - though there may be several ways by which CR has its effects on ML and LS, one of them is probably through neuroendocrine mechanisms. This article suggests that since CR causes a mild increase in glucocorticoids, especially at certain times of day as related to circadian rhythms and to times of feeding, and that slightly elevated levels of glucocorticoids has been shown to be beneficial and increase ML and possibly LS, and the slightly elevated levels of glucocorticoids are beneficial by modulating inflammatory and immune responses to allow them to occur but not in excessive degrees, and that some age changes seems to be related to detrimental effects from inflammation and from immune responses. Furthermore, slight stress responses, which are accompanied by slight increases in glucocorticoids, seem to increase ML and possible LS by helping animals over come detrimental environmental conditions in the wild, which has an evolutionary advantage and therefore would make sense in terms of survival advantage. Note that while CR elevated glucocorticoid levels, it reduces levels of GH and insulin and sometimes T4 or T3. Note also that any consideration of the effects of CR through altering levels of hormones must also take into consideration changes in sensitivity to hormones, changes in the active/inactive forms of hormones, (and changes in ratios of hormone that have interactive effects).
  - in summary, this author proposes the hypothesis that CR will work in many animals by upgrading glucocorticoid levels and/or stimulating heat shock protein synthesis (and possibly by other mechanisms) in animals naturally subjected to periods of unpredictable DR. IN animals not subjected to natural unpredictable CR, if CR extends their ML or LS, it will do so by other means. There may be still other animals that do not experience natural unpredicatbale CR in which CR has no effect on ML or LS. This is because of the different outcomes from different types of natural selection to the need to withstand natural unpredictable periods of DR.
  - this author refutes the glucocorticoid theory of aging, which states that elevated levels of glucocorticoids, as found in severe stress, cause age changes though a variety of detrimental effects on various cells, such as cell damage and cell death in brain neurons, especially in the hippocampus. This article states that in CR animals, there is no major change in glucocorticoids, so CR cannot be working by keeping glucocorticoids low (Note, though, that the glucocorticoid theory of aging states that LOWER chronic elevated glucocorticoids helps increase ML and LS by stimulating defense mechanisms, even though HIGH levels of glucocorticoids have detrimental effects. So in evaluating the effect of glucocorticoids on increasing or decreasing ML and LS, keep in mind whether you are dealing with slightly elevated or very elevated levels of glucocorticoids).
  - this author concludes that CR does not act by altering levels or responses to glucocorticoids, even though the author presents evidence from reports on both sides of the argument. The author seems to put more faith in the reports suggesting that CR does not work by modulating blood levels of glucocorticoids, or the responses of cells to glucocorticoids, or to varying diurnal rhythms of glucocorticoids, or in the stress response in terms of elevations in glucocorticoid levels, or in the reduction of glucocorticoid levels after the stressor is removed. The overall conclusion of this author is the parameters related to glucocorticoids are not significantly different or are not different at all when comparing CR animals with AL animals. (Note that others take the opposite opinion using the same data, which is scanty.)

  DR working by affecting protein metabolism and rate of turnover

  - there is an age-related decline in the rates of protein synthesis and protein degradation and, therefore, and age-related decrease in the rate of protein turnover. This is true in most individual tissue types and organs in the body as well as for the total protein turnover in the whole body. AN exception may be the lung, where there may be no decline in the rate of protein turnover. In addition to using protein turnover to recycle amino acids, turnover permits adjustments in enzyme and structural proteins and receptors and modulators as well as for destruction of damaged proteins (e.g., oxidative damage) and replacement with normal proteins.
  - there is an age-related increase in the concentration of abnormal proteins due to post-translational modification errors (the main metabolic cause of abnormal proteins) and in oxidatively damaged proteins. With CR, there is less age-related increase in abnormal proteins form both sources (i.e., post-transnational modifications and oxidative damage). There is an intracellular protein complex that specifically degrades oxidatively damaged proteins. This complex is called a "proteasome".
  - CR produces an increase in the rate of protein synthesis and an increase in the rate of protein degradation, and therefore causes an increase in the rate of protein turnover. The age-related decline in the rate of protein turnover is not stopped or slowed by CR, but since young CR animals have 30%-40% greater protein turnover and the age-related decline in the rate of protein turnover is essentially the same in CR and in AL animals, at any age, CR animals always have substantially higher rates of protein turnover than do AL animals. The benefit of higher rates of protein turnover include faster responses when alterations in proteins are needed (e.g., receptors or regulators) and faster elimination of damaged proteins (e.g., oxidative damage). This may be one mechanism by which CR increases ML and LS. Other types of "problematic" proteins can develop as a result of metabolic errors (e.g., gene mutations, errors in transcription, mRNA processing, translation, or post-transnational processing of proteins as well as damage caused by environmental factors (e.g., bacteria, radiation, heat, toxins).
  - CR may increase ML and LS in many animals by increasing the synthesis of protects substances such as heat shock proteins, which increase protection against a variety of adverse factors in saddition to elevated temperature

  Effects on temperature in rodents

  - CR in rodents results in lowered body temperatures in mice and, to a lesser extent, in rats

  Effects on monkey temperature

  - CR lowers body temperature in rhesus monkeys as it does in rodents
  - CR causes less decrease in body temperature in rhesus monkeys than it does in mice
  - the short term decreases in body temperature and in 24 hour metabolic rate in rhesus monkeys is probably due to reductions in synthetic metabolic activities as the animals lose weight and due to energy-conserving metabolic strategies and lowering of body temperature, since activity levels as measured by heart function and by activity levels do not change with DR.
  - CR which is long term in rhesus monkeys results in a lowering of body temperature which is not temporary but lasts for many years. It takes several (2 years) years on CR before the lowering of body temperature when measured rectally, perhaps because rectal temperature measurements are not accurate due to the procedure and time of day of measuring. When body temperature measured by skin temperature is measured, even short term CR causes a lowering of body temperature in rhesus monkeys

  DR working by effects on BMR

  - in rodent studies, some studies show that CR leads to slower metabolic rate per kgm, while other studies show only temporary change with CR in metabolic rate per kgm. IN OTHER LABS (e.g., Lane, M.A., et al., NIA, Baltimore, MD), CR monkeys have greater decline in body temperature than AL monkeys, suggesting that in primates as in rodents, CR may lead to a slower metabolic rate with a higher efficiency in use of calories consumed. This slower metabolic rate may be related to the increase in mean longevity and decreased pathology in CR animals compared to AL animals. The slower metabolic rate may indicate a shift in use of calories from survival of the individuals plus reproduction to only survival of the individuals. ALSO, this lab is trying to identify better biomarkers of aging (e.g., blood chemistry parameters) using CR and AL monkeys in longitudinal studies.
  - CR does not have a long-term effect on metabolic rate per unit of fat free mass or lean body mass. However, there may be short term reductions in metabolic rate when CR is first instituted.
  - CR does not work by altering the metabolic rate per unit of lean body mass (essentially muscle mass). CR may cause a short-term increase or decrease in metabolic rate, but overall metabolic rate per unit of fat free mass is the same in CR and in AL mammals. For example, for some days or weeks after CR is started, there may be a decrease in metabolic rate, but it resumes AL rates thereafter. In CR animals, they may metabolize glucose faster after eating than do AL animals, but this may be because CR animals have a higher insulin sensitivity and maintain a lower blood glucose level than do AL animals. Note that this may not apply to poikilotherms (e.g., flies). In poikilotherms, a decrease in metabolic rate, such as by lowering environmental temperature (and therefore body temperature) or in homeotherm animals by going into states of torpor, ML and LS may be increased, but only because there is less "living" during the time given.
  - CR does not lead to increases in ML and LS by altering metabolic rates per unit of fat free body weight per unit time (e.g., Kcal/kg/hr)
  - CR resulted in a decrease in metabolic rate per unit of lean body mass (per LBM) AT NIGHT but not during the day and not in the total 24 hour metabolic rate per LBM
  - CR may have its effects through mechanisms that decrease metabolic rate when the animals are resting or asleep.

  Effects on BMR in monkeys

  - the lowering of body temperature by CR in rhesus monkeys is accompanied by a decrease in 24 hour metabolic rate, (but other work showed that the lowered metabolic rate is restored to AL levels after a few years on CR, so CR causes only a temporary decrease in 24 hour metabolic rate.
  - CR does not have a long-term effect on metabolic rate per unit of fat free mass or lean body mass. However, there may be short term reductions in metabolic rate when CR is first instituted.
  - CR causes a temporary decrease in 24 hour metabolic rate for some months or years until the animals adjust to the DR. Then 24 hour metabolic rate rises to that of AL animals, showing that the beneficial effects from CR are not due to a decrease in overall metabolic rate or "rate of living"

  DR working by affecting genes

  - CR may help by limiting free radical damage to mitochondria by limiting free radical production and/or by sustaining or promoting formation of natural antioxidants (e.g., antioxidant enzymes)
  - CR alters the activity of specific genes. The effect of CR on the activity of one gene may occur at a different age than does the effect of CR on another gene. In many cases, once CR causes an increase in the activity of a gene, that gene retains higher activity throughout life as compared with ad. lib. controls. Among the genes activated by CR are those for synthesis of antioxidant enzymes (i.e., catalase, glutathione peroxidase, superoxide dismutase. CR also increases genes activity for IL-2, which can increase immune responses, and in heat shock proteins, which assist in resisting a number of detrimental environmental conditions in addition to elevated temperature. At the same time, CR decreases the activity of other genes, including some that synthesis proteins known to adversely affect the cells in an age-related way. Furthermore, CR does not affect the activity of still other genes. In altering the activity of genes, CR alters the rate of synthesis of the mRNA as well as the amount of the protein synthesized by that gene. In other words, CR alters transcription and translation rates. CR seems to alter the expression of genes and the synthesis of their proteins by affecting the rate of transcription of the gene's mRNA. This may occur by affecting the DNA-associated proteins or by affecting transcription factors (i.e., proteins that regulate gene expression). There is some evidence showing that CR can act by both mechanisms.

  Effects of CR on immune system

  - in rats and in humans there is an age-related decline in immune system functioning. Among the changes are a decrease in the response of T cells to mitogens, an increase in old memory cells, a decrease in the production of IL-2, and increase in the production of IL-6, an increase in indicators of autoimmune responses. Problems from the increase in old memory cells is that these cells are not as responsive to signals and that these cells MAY be adversely affected in an age-related manner so they function incorrectly, such as by producing less IL-2, more IL-6, and more autoimmune activities.
  - in rodents, CR helps sustain immune functions by reducing age-related decreases in immune function and age-related increases in autoimmune reactions
  - CR in rats and in mice slows the age-related decreases in mitogen-stimulated proliferation of lymphocytes, T-cell cytolysis, antibody responses, production and responses to IL-2
  - CR decreases the age-related decline in immune system functioning, including reducing age-related declines in mitogen responsiveness by T cells, IL-2 production and increases in IL-6 production. The beneficial effects of CR on limiting age-related changes in immune system may also help explain how CR limits age-related incidence of certain cancers.
  - CR also increases genes activity for IL-2, which can increase immune responses
  - CR had no effect on peripheral blood cell lymphocyte counts
  - in this work, the monkeys had 2-4 years of DR. This work studied the effects of CR on monkeys with regard to mitogen stimulation of lymphocyte proliferation, activity of NK cells against leukemia, expression of CD cell surface antigens on T-cells, antibody production against influenza vaccine, and peripheral blood counts of lymphocytes. Results were that CR not only did not slow or stop any age-related changes in certain parameters, but CR actually REDUCED mitogen stimulation of lymphocyte proliferation, activity of NK cells against leukemia, and antibody production against influenza vaccine. Expression of CD cell surface antigens on T-cells, and peripheral blood counts of lymphocytes were not affected by DR. Reasons for the differences in effects in monkeys in this study, suggesting detrimental effects of CR on immune function, and results in rodents, showing beneficial results of CR on immune functioning, may be that the CR used here has been relatively short compared with the life span of the monkeys and that no very old monkeys were included in the study. In the rodent studies, the CR is much longer relative to the animals' life span, and the beneficial results of CR were observed in very old animals

  - IN OTHER LABS, (e.g., Kemnitz, J.W., et al., Wisconsin) the CR monkeys had lower blood pressure, but also had similar (not better) immune function for some parameters (stimulation of mitosis in immune cells) or lower immune function (e.g., lower NK activity and antibody responses) when compared to AL monkeys.

  - CR resulted in lower immune system activity as indicated by lower response to mitogen-stimulated cell reproduction
  - CR may help sustain immune function by limiting oxidative damage to membrane or other components of T cells such as the lipid components of the cell membranes

  Effects on monkey bones

  - CR does not affect blood calcium levels. In rats there is no age-related changes in blood calcium levels in AL animals or in CR animals. Therefore, while there are age-related changes in bone matrix and in mechanisms regulating bone matrix, there are not age-related changes in the ability of homeostatic mechanisms to maintain normal blood calcium levels. This is important because calcium levels play important roles in many enzymatic, signaling, and regulatory mechanisms.
  - Age-related bone changes in rats are very different from those in humans for several reasons. One is that rats often develop an age-related nephropathy that increases bone resorption. A second is the rats do not have a menopause as found in humans. A third is that in humans there is an age-related decrease in calcitonin while in rats there is an age-related increase in calcitonin.
  - CR can reduce age-related losses of bone matrix in rats, probably because certain types of CR (e.g., with low amounts of kidney-damaging proteins in rats) limit age-related nephropathy. Other than this, CR seems to have no significant effect on bone matrix in aging rats. However, CR with the proper types of protein that limits age-related renal disease also limits the age-related increase in parathormone, which also contributes to less age-related loss on bone matrix. Thus, in rats, CR may help reduce age-related loss of bone matrix by helping maintain normal kidney function.
  - in rodents and in monkeys, CR also inhibits skeletal development during youth
  - CR slowed growth in young monkeys, resulting in shorter monkeys with less total body bone mineral content but with no change in bone mineral density (?strength)
  - CR slowed the development of the skeleton but though it reached smaller overall dimensions, the bone quality was as good as in AL animals
  - since CR animals are lighter in weight than are AL animals, having a smaller skeleton should not be an adverse effect of DR
  - CR seemed to have no effect on blood levels of phosphorous, calcium, and parathormone, showing that CR did not have effects on calcium or phosphorous regulation
  - there is an age-related decrease in osteocalcin
  - CR did not affect the age-related decrease in osteocalcin, showing there was no effect of CR on reducing the age-related decline in bone formation
  - CR slowed the rate of bone formation and growth, but in a balance manner, so though the animals were smaller, the quality of the bones was the same as in AL animals. This is logically consistent with the lack of difference in osteocalcin levels.
  - there is an age-related decrease in AP (total blood alkaline phosphatase from all sources)
  - CR delayed the onset of age-related increase in total alkaline phosphatase (AP) (an index of bone formation) and in IL-6 levels (IL-6 seems to inhibit bone formation and to promote bone resorption)
  - CR reduced the age-related decline in AP

  Effects of CR on animal activity

  - in monkeys, there is an age-related decrease in voluntary physical activity
  - CR in rats results in increased voluntary activity
  - CR resulted in less age-related decreases in voluntary physical activity in monkeys
  - CR monkeys show eagerness to eat and spend much time searching for food. CR monkeys eat "voraciously" when fed

  Effects of CR on lipofuscin

  - the age-related increase in lipofuscin in brain cells of mice is not linear. The lipofuscin accumulates at a faster rate as age increases.
  - in rodents, CR that was severe (52% CR) resulted in less age-related increases in lipofuscin in brain cells of mice but less severe CR (25%) did not have this effect

  Effects of CR on DHEA

  - there is an age-related decrease in DHEA in humans and in monkeys. The age-related decrease in DHEA in monkeys is twice as fast as in humans
  - DHEA lowers LDLs in rhesus monkeys on a diet with modest fat content (12% calories from fat) but if the monkeys eat a high fat diet (30% calories from fat).
  - CR might have some of its effects by reducing the age-related decline in DHEA, which seems to have a protective effect on the cardiovascular system, on cancer formation, and on development of obesity
  - CR reduces or stops the age-related decreases in DHEA in monkeys

  Effects of CR on wounds

  - there is an age-related decrease in the rate of wound closure in rats and in monkeys
  - CR does not affect the rate of wound repair in mice
  - though CR does not affect the rate of wound repair in mice, CR mice that are fed AL after receiving a wound have wound healing like younger mice
  - though CR does not slop the age-related slowing of skin wound repair, CR animals retain the ability to have youthful rates of wound repair if they are fed more during the wound repair process
  - the retention of ability to have youthful rates of wound repair when CR mice are fed more after wounding is due in part to retention of youthful proliferation and migration by skin fibroblasts and secretion of insulin-like binding protein 3
  - CR does not affect the rate of wound CLOSURE and nail growth in monkeys or in wound closure in rats. The rates of wound closure and of nail growth decreases with age in AL and in CR animals
  - CR does not affect the age-related decrease in wound closure in rats or in monkeys

  Effects of CR on IL-6

  - elevated levels of IL-6 may be contributory to lymphoma, Alzheimer's disease, and osteoporosis
  - in studies with rodents, CR limits the age-related increase in IL-6, possible having beneficial effect in limiting potential adverse effects of IL-6
  - though there is not an age-related change in IL-6, CR animals had lower levels of IL-6
  - most studies with mammals show an age-related increase in IL-6, but none of the animals in this study showed age-related changes in IL-6. However, these animals may simply not be old enough to show the usual age-related increase in IL-6

  DR effects on cancer

  - a hypothesis is that CR may have beneficial effects on cancers by slowing cell reproduction
  - in rodents, CR not only delays the onset of many age-related diseases, but it also slows the progress of many of them. Certain forms of cancer are exceptions, where either the age of onset or the progress of the disease is not affected by DR. however, even in these exceptions, either the age of onset or the rate of progression is affected beneficially by DR

  Effects of CR on atherosclerosis

  - CR is now being studied in rodents as it pertains to atherosclerosis by Cefalu et al.

  Return to Notes on Aging

     © Copyright 2000 - Augustine G. DiGiovanna - All rights reserved.
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  AGING: BIOLOGICAL PERSPECTIVES by Augustine G. DiGiovanna, The McGraw-Hill
  Companies, New York, 1994 or 2000; (2) If prior written permission is obtained from Augustine G.
  DiGiovanna, Ph.D., Salisbury University  - agdigiovanna@Salisbury.edu