First experiments:

Osborne and McKay

Osborne, Mendel, Ferry

• First scientific experiment suggesting that caloric restriction could extend lifespan

• During experiments on nutrition, selected rats were fed a calorie restricted diet ("stunted") over various period of time.

• Normally-fed rats mortality rate (91 rats total):• 17 (19%) < 1yr.• 48 (53%) < 2 yrs.• 26 (29%) > 2 yrs.• Oldest lived to 34 months.

Four calorie restricted females

• More than 2/3 of stock rats die within 2 yrs.• All 4 stunted females lived longer than 2 years.• All four were breeding at a time when rats are

typically in menopause.• They produced from 3 to 6 litters; all as vigorous

as offspring from younger mothers.• Osborne suggested that stunting growth in

young rats via nutrition extends lifespan.• Sample size (4 rats) was insufficient to give a

statistically significant demonstration.

• Osborne knew he couldn’t make any firm conclusions from an experiment with only four rats. But he thought the observation was interesting and published it in the journal Science (which Thomas Edison had founded a few years earlier). In that Science article Osborne said, “it appears as if the preliminary stunting period lengthened the total span of their life.”

• That same year, J. Northrop showed that he could extend the lifespan of fruit flies when he restricted their food intake during the larval stage.

McCay, Crowell, and Maynard

• First scientific experiment that demonstrated caloric restriction extended the lifespan of rats

• “to test the assumption that an individual within a species that grows to maturity slowly will have a greater lifespan than one that grows rapidly.”

• designed a diet “to satisfy the nutritional requirements of the body in every respect, except that body would have insufficient calories to permit growth when the food intake was restricted. … the food ingested every each day provided every recognized constituent to insure the health of the animal.”

• “At the time of weaning 106 rats were divided into three groups … The members of one group were allowed to grow normally, ..members of the second group were forced to grow slowly by limiting their daily allowance of food from the time of weaning. The third group of rats was allowed to grow normally for two weeks after weaning. They were then restricted in their food allowance.

McCay described the results:

• “… the slow climb to maturity of the lucky rats or victims (as your philosophy dictates) can be seen.”

• Thirteen of the original 106 rats (18%) were still alive after 1,200 days, and all thirteen were in the caloric restricted groups.

• None of the free-feeding rates survived 1,200 days.

Males and females differed

• Median lifespan for free-feeding male rats was 522 days, versus 919 days for the male rats on CR beginning two weeks after weaning, a gain of 76%.

• Median lifespan for free-feeding female rats was 820 days, versus 894 days for the male rats on CR beginning two weeks after weaning, a gain of 9%.

Results replicated in other species

• In the decades since McCay’s experiments, many researchers have shown that CR extends lifespan in a diverse range of relatively short-lived species, including nematode worms, spiders, fish, mice and rats.

• CR typically increases lifespan by 10% to 50%, with greater restriction (up to a reduction of calories to half of normal) giving greater lifespan extension.

• These researchers have also shown that CR delays the onset or reduces the incidence of many diseases, including cancer, diabetes, and heart disease

Calorie Restriction in Rhesus Monkeys

Mattison, Lane, Roth, Ingram

• CR extends lifespan, reduces incidence of age related disease in some animal models.

• Research has so far concentrated on rodents and other short-lived animals.

• Interest in the effects on long-lived primates.• Typical average life span of rhesus macaques estimated

at 25 years.• In 1987 NIA began study of 30% CR in male and female

rhesus macaques of 1-17 years of age.• Range of ages allowed assessment of CR on various

age groups.• Number of animals was eventually 60 of each sex.

Effects of CR

• decreased body weight and fat mass• improved glucoregulatory function• decreased blood pressure and blood lipids• lowered body temperature• males:• delayed skeletal and sexual maturation• attenuated decline in DHEA and melatonin• females:• no effect on bone mass, reproductive hormones,

menstrual cycle

CR and skeletal health

• Decline in serum alkaline phosphatase (AP) a product of bone-forming cells

• AP parallels bone growth and is at lowest levels when growth is complete.

• AP levels in humans increase during adolescence then rapidly decline to adult levels.

• Same results found in rodents and may indicate slowing of aging.

Reproduction

• Juveniles: Reproductive maturation delayed in prepubescent monkeys on CR early in life.

• Adult males: Testosterone level changes delayed by at least one year.

• Adult females: Little difference in CR vs non-CR monkeys.

Blood sugar, insulin

• CR monkeys able to regulate glucose better than controls.

• Lower fasting glucose and insulin levels after 3 years.

• During intravenous glucose tolerance tests, max glucose level in CR less than in Control.

• The acute insulin response was also lower.• Possible that CR increases insulin sensitivity

and may postpone type II diabetes.

• CR may alter basic mechanism of fuel use.

• Short term CR reduced fasting and peak insulin level prior to changes in adiposity.

• In fact, CR induced changes in lean (<22% fat) monkeys.

• Suggests CR affects insulin levels independent of body weight and fat levels.

Cardiovascular disease

• Leading cause of death in US. but can be mitigated by diet and exercise.

• CR has protective and beneficial effects in this study. • • Monkeys fed a diet low in total fat, saturated fat and

cholesterol.• Both CR and Control actually have lower plasma

cholesterol than normal animals.• Benefits:• lower triglyceride levels in young and adults• increased levels of high density lipoprotein (HDL)• (reduced cardiovascular disease in humans)

Body temperature

• CR over 6 years reduced colonic body temperature about 0.5 Celsius.

• This agrees with rodent studies.• Is consistent across age range of 7-13 years.• Additional test using implanted thermometers

using radio telemetry for 24 hr monitoring.• Temperature decreased as food intake reduced.• At 30% restriction, temp was significantly

(p<0.003) lower than controls group.• Circadian patterns were maintained.

Activity

• Monitored locomotor activity and basic behavior patterns in males after 6 yrs of CR.

• Used ultra-sonic motion detectors and video.• Found daily activities and behaviors typical for

captive primates.• CR males displayed more pacing, gross

movement and less passivity than control.• CR female juveniles (6-8yrs) less active than

control.

Biomarkers

• A biomarker for aging is a measurement that predicts survival.

• Could help to reduce cost and time involved in evaluation of anti-aging interventions.

• There is debate over what constitutes a marker and how it should be evaluated.

• That aside, the following are some candidates.

DHEA

• Precursor of testosterone and estrogen. • Elevated DHEA associated with protection function in

aging diseases, diabetes, heart disease and cancer.• Peak levels in humans occur at about 20 years of age in

men and women.• In 792 normal rhesus monkeys, DHEA levels dropped

90% from infancy to 3 years followed by an average decline of 4.2% per yr

• In this study: DHEA declined 3% in CR males between 3-6 yrs versus 30% decline for controls.

• CR may postpone age-related hormonal decline.

Melatonin

• A horomone secreted by the pineal gland• Secreted in a pulsatile pattern peaking in early

morning • Decreases with age as pineal gland shrinks.• Reported to improve sleep, increase longevity,

strengthen immune system, lower blood pressure and act as an anti-oxidant.

• Cross sectional data from 52 male and female control rhesus monkeys confirmed age related decline in melatonin levels.

• CR monkeys had significantly higher levels.

CR effect on lifespan and disease

• Preliminary mortality data are not yet statistically significant, but indicate that mortality to date is lower in CR animals (15%) than in controls (24%).

• The CR group has lower incidence of chronic diseases including cancer, cardiovascular disease, diabetes, endometriosis, fibrosis, amyloidosis, ulcers, cataracts and kidney failure.

Biomarkers of caloric restriction may predict longevity in humans

Roth, G. S., et al

• Caloric restriction (CR) slows aging and maintains health and function in a diverse array of species ranging from worms and flies to rodents.

• Studies on CR in primates (rhesus monkeys) were initiated in the 1980’s, but have not yet reached their endpoints.

• The rhesus monkey data show hallmarks of the effects of CR in lower animals, in particular attenuation of age changes in plasma triglycerides, melatonin, oxidative damage and glucose tolerance.

• Preliminary mortality data are not yet statistically significant, but indicate that mortality to date is lower in CR animals (15%) than in controls (24%).

• Previous research showed that two highly-reproducible biomarkers of CR in rodents, reduced body temperature and reduced plasma insulin, also occur in CR rhesus monkeys.

• Serum dehydroepiandrosterone sulfate (DHEAS, a steroid hormone related to testosterone) levels decline in aging monkeys and humans. CR also slows the rate of decline of DHEAS in CR monkeys.

• These three markers, body temperature, plasma insulin, and DHEAS may be important in health maintenance and may serve as biomarkers of potential longevity.

Methods

• The authors studied data from the Baltimore Longitudinal Study of Aging in Male Humans (BLSA).

• They obtained records of subjects’ body temperature, plasma insulin, and DHEAS, and divided the subjects into those in the upper half or those in the lower half of measurements of each biomarker. They compared the survival of men in each group.

• To the authors’ knowledge, BLSA men are not CR.

Results

• Significant longer survival in men with lower body temperature, lower plasma insulin, and higher DHEAS (Figure 1) (p < 0.05).

• The environmental or genetic factors that cause CR-like effects on body temperature, plasma insulin, and DHEAS in these men appear to be related to longevity.

• Suggest that the same mechanisms that control aging in animals are likely at work in humans, and that modifying these mechanisms may extend lifespan in humans.

Demography of Dietary Restriction and Death in

DrosophilaMair, Goymer, Pletcher, Partridge

Hypothesis

• Definition: Mortality rate: the probability of dying in any given interval, given the animal is alive at the start of the interval

• Hypothesis: CR begun at any stage of life reduces the mortality rate to that of animals on lifetime CR

Methods

• Four groups of flies:– CR for life– Fully fed for life– Fully fed, then switched to CR– CR, then switched to fully fed

Results

• Two days after starting CR for the first time, and at various ages, fully fed flies are no more likely to die than flies of the same age who have experienced long-term CR.

• Switching from CR to fully fed resulted in rapid increase of mortality levels. CR animals who switch to fully-fed have the same mortality rate as long-term fully fed flies.

Conclusion

• It doesn’t matter when CR is started. Switching to CR late in life reduces mortality to the same rate as long-term CR.

Anson RM, Guo Z, et al

• Intermittent fasting dissociates beneficial effects of dietary restriction on glucose metabolism and neuronal resistance to injury from calorie intake.

Methods & Results

• Animals either fully-fed or fed only on alternate days (fast / free-feeding)

• Fasting animals had metabolic effects very similar to those seen in CR animals, specifically lower serum glucose, lower insulin, higher neuronal resistance to stress.

Gene Expression Profiling of Aging Using DNA Microarrays

Weindruch, Kayo, Lee, Prolla

DNA microarrays

• A new tool that lets us measure biological age on a tissue specific basis.

• Allow evaluation of interventions at the molecular level.

• Allow study of 10,000 genes within a single experimental set-up.

Biomarkers of aging

• There is a need for tissue specific biomarkers, because long-term survival studies difficult in long lived organisms.

• 2 approaches:– 1. DNA microarrays that measure changes in

gene expression– 2. Proteomics that measure protein

abundance and state.

• CR induces hundreds (thousands) of biological changes.

• It is difficult to separate cause and effect.

• Arrays provide a simultaneous analysis of gene expression patterns.

• Large scale analysis of transcriptional responses.

Methods

• Skeletal muscle used because it is primarily composed of long-lived, high oxygen-consuming post-mitotic cells - similar to aging targets such as the heart and brain.

• Also, a comparison of gastrocnemius muscle form 5 to 30 month old mice showed aging is associated with alterations at the mRNA level which may reflect changes in gene expression, mRNA stability or both.

Methods

• mice 30 months old housed individually• fed non purified diet and acidified water AL for 1week• then split into 2 groups and fed same isocaloric,

semipurified diets• Control mice fed 84kcal/week about 20% less than

standard diet• this necessary to avoid obesity and maintain motor

activity• CR mice fed 62kcal/week (26% less)• food enriched with protein, vitamins and minerals to

match Control diet for nutrition

Results

• 6347 genes surveyed• 58 (0.9%) displayed greater than 2fold increase

in gene expression• 55 (0.9%) displayed geater then 2fold decrease

in expression• Therefore gene expression patterns appear fairly

stable during adult mammal lifespan• Contrasts with hypothesis that aging is due to

large and widespread alterations.

Stress Response

• Of 58 genes expressed 9 (16%) were assigned to stress responses

• Mitochondrial sarcomeric creatine kinase expressed 3.8fold, the largest differential recorded between young and old animals.

• Sarcomeric creatine kinase may be a compensatory response to increased free radicals (Reactive oxygen species or ROS) in aged animals.

• Overall, this supports idea that increased ROS at the transcription level results in macromolecular damage during aging.

Other gene groups affected

• Motor neuron genes

• Metabolism genes

Reversing age related changes

• 6 mice from 2 to 30 months of age fed 24% less than control animals.

• Age related changes were attenuated by CR– 29% of largest age associated alterations (>2fold)

completely prevented– 34% partially suppressed– 84% of genes involved in stress response,

biosynthesis, protein metabolism and energy metabolism were completely or partially suppressed.

Additional findings

• Evidence that aging is characterized by:– activation of an adaptive stress response

mechanism.– increased levels of ROS in skeletal muscle

and brain– activation of neuronal and myogenic

responses to injury in skeletal muscle– activation of transcriptional responses found

in human neurodegenerative disorders