Daily Fasting Improves Health and Survival in Male Mice Independent of Diet Composition and Calories.

The importance of dietary composition and feeding patterns in aging remains largely unexplored, but was implicated recently in two prominent nonhuman primate studies. Here, we directly compare in mice the two diets used in the primate studies focusing on three paradigms: ad libitum (AL), 30% calorie restriction (CR), and single-meal feeding (MF), which accounts for differences in energy density and caloric intake consumed by the AL mice. MF and CR regimes enhanced longevity regardless of diet composition, which alone had no significant impact within feeding regimens. Like CR animals, MF mice ate quickly, imposing periods of extended daily fasting on themselves that produced significant improvements in morbidity and mortality compared with AL. These health and survival benefits conferred by periods of extended daily fasting, independent of dietary composition, have major implications for human health and clinical applicability.

Caloric Restriction Study Design Limitations in Rodent and Nonhuman Primate Studies.

For a century, we have known that caloric restriction influences aging in many species. However, only recently it was firmly established that the effect is not entirely dependent on the calories provided. Instead, rodent and nonhuman primate models have shown that the rate of aging depends on other variables, including the macronutrient composition of the diet, the amount of time spent in the restricted state, age of onset, the gender and genetic background, and the particular feeding protocol for the control group. The field is further complicated when attempts are made to compare studies across different laboratories, which seemingly contradict each other. Here, we argue that some of the contradictory findings are most likely due to methodological differences. This review focuses on the four methodological differences identified in a recent comparative report from the National Institute on Aging and University of Wisconsin nonhuman primate studies, namely feeding regimen, diet composition, age of onset, and genetics. These factors, that may be influencing the effects of a calorie restriction intervention, are highlighted in the rodent model to draw parallels and elucidate findings reported in a higher species, nonhuman primates.

Effects of Sex, Strain, and Energy Intake on Hallmarks of Aging in Mice.

Calorie restriction (CR) is the most robust non-genetic intervention to delay aging. However, there are a number of emerging experimental variables that alter CR responses. We investigated the role of sex, strain, and level of CR on health and survival in mice. CR did not always correlate with lifespan extension, although it consistently improved health across strains and sexes. Transcriptional and metabolomics changes driven by CR in liver indicated anaplerotic filling of the Krebs cycle together with fatty acid fueling of mitochondria. CR prevented age-associated decline in the liver proteostasis network while increasing mitochondrial number, preserving mitochondrial ultrastructure and function with age. Abrogation of mitochondrial function negated life-prolonging effects of CR in yeast and worms. Our data illustrate the complexity of CR in the context of aging, with a clear separation of outcomes related to health and survival, highlighting complexities of translation of CR into human interventions.

Gene-specific and mitochondrial repair of oxidative DNA damage.

The Southern blot gene-specific DNA damage and repair assay is a robust and flexible method for quantifying many kinds of induced damage and repair with high reproducibility. Specific nicking and loss of a restricted DNA fragment at the site of induced damage is visualized by Southern blot and quantified against a control; since the blot is gene specific, only the damage of interest is measured. Here we show how the assay may be adapted to assess mitochondrial DNA (mtDNA) damage. In the mitochondrion, 8-oxoguanine is a significant oxidative lesion; in the laboratory, photoactivated methylene blue may be used to introduce this lesion into cells. Other lesions may also be studied by using different DNA damaging agents. We find that damage induction by methylene blue is consistently far greater in the mitochondrion than the nucleus. Thus advantageously, mitochondrial 8-oxoguanine repair may be studied without mtDNA isolation or preparation, which are processes known to induce DNA damage and skew measurements. This chapter gives detailed instructions for using methylene blue and the gene-specific repair assay to accurately measure mitochondrial oxidative damage and repair rates.

Caloric restriction modulates insulin receptor signaling in liver and skeletal muscle of rat.

OBJECTIVE: We investigated how the insulin/insulin-like growth factor-1 signaling pathway is involved in the robust antiaging effects produced by caloric restriction. METHODS: We subjected male rats to feeding ad libitum or calorie restriction, i.e., 60% of the ad libitum amount, for 2 and 25 mo and then assessed the effects of calorie restriction on insulin receptor (IR) signaling in liver and skeletal muscle. RESULTS: The results indicated that aging was accompanied by a significant decrease in IR tyrosine phosphorylation after insulin stimulation in live and skeletal muscle, which was associated with a significant increase in the activity of protein tyrosine phosphatase-1B. However, these age-related alterations were attenuated by long-term calorie restriction. Expression profile of mRNA showed an increased expression of mRNAs for IR and insulin-like growth factor-1 receptor in both tissues of calorie-restricted rats, but increased expression of IR mRNA was dissociated with the IR gene product in rats maintained on long-term calorie-restricted diet. CONCLUSION: IR signaling may play an important role in aging and its retardation by calorie restriction, and normal function of IR in liver and skeletal muscle is required for healthy aging and extending lifespan in mammals.

The diet restriction paradigm: a brief review of the effects of every-other-day feeding.

It has been known since the early 1900s that restriction of dietary intake relative to the ad libitum (AL) level increases stress resistance, cancer resistance, and longevity in many species. Studies investigating these phenomena have used three paradigms for dietary restriction. In the first, the AL intake of a control group is measured, and an experimental group is fed less than that amount in a specified proportion, e.g., 40%. In the second, food is provided AL to both the control and experimental groups: however, the experimental group is subjected to periods of fasting. Recent studies using this paradigm provide food every other day (EOD). Both of these paradigms have been in use since the early 1900s. A third paradigm that combines them was developed in the early 1970s: one or more days of fasting separate the provision of a limited amount of food. It was assumed for many years that the physiological responses to these paradigms were due exclusively to a net decrease in energy intake. Recently, however, it was found that some species and strains of laboratory animals, when fed AL every other day, are capable of gorging so that their net weekly intake is not greatly decreased. Despite having only a small deficit in energy intake relative to control levels, however, these animals experience enhanced longevity and stress resistance is enhanced in comparison to AL controls as much in animals enduring daily restriction of diet. These observations warrant renewed interest in this paradigm and suggest that comparisons of the paradigms and their effects can be used to determine which factors are critical to the beneficial effects of caloric restriction.

Development of calorie restriction mimetics as a prolongevity strategy.

By applying calorie restriction (CR) at 30-50% below ad libitum levels, studies in numerous species have reported increased life span, reduced incidence and delayed onset of age-related diseases, improved stress resistance, and decelerated functional decline. Whether this nutritional intervention is relevant to human aging remains to be determined; however, evidence emerging from CR studies in nonhuman primates suggests that response to CR in primates parallels that observed in rodents. To evaluate CR effects in humans, clinical trials have been initiated. Even if evidence could substantiate CR as an effective antiaging strategy for humans, application of this intervention would be problematic due to the degree and length of restriction required. To meet this challenge for potential application of CR, new research to create "caloric restriction mimetics" has emerged. This strategy focuses on identifying compounds that mimic CR effects by targeting metabolic and stress response pathways affected by CR, but without actually restricting caloric intake. Microarray studies show that gene expression profiles of key enzymes in glucose (energy) handling pathways are modified by CR. Drugs that inhibit glycolysis (2-deoxyglucose) or enhance insulin action (metformin) are being assessed as CR mimetics. Promising results have emerged from initial studies regarding physiological responses indicative of CR (reduced body temperature and plasma insulin) as well as protection against neurotoxicity, enhanced dopamine action, and upregulated brain-derived neurotrophic factor. Further life span analyses in addition to expanded toxicity studies must be completed to assess the potential of any CR mimetic, but this strategy now appears to offer a very promising and expanding research field.

Absolute versus relative caloric intake: clues to the mechanism of calorie/aging-rate interactions.

It has been suggested that the influence of caloric intake on aging rate is not due to the absolute number of calories ingested. Instead, aging rate is altered only when there is a disparity between the actual caloric intake and that which would be ingested if the food supply were unlimited. This review will discuss a few of the studies supporting this viewpoint.

Mitochondrial influence on aging rate in Caenorhabditis elegans.

Virtually every model of mitochondrial involvement in aging shares the underlying proposition that mitochondrial dysfunction will accelerate the rate of aging. Caenorhabditis elegans is a post-mitotic organism with limited capacity for replacement and repair, and there is a great deal of evidence that interventions which decrease the induction of damage extend lifespan in this model. However, decreased availability of ubiquinone in adulthood has also been found to promote longevity and stress resistance, and evidence tentatively supports decreased mitochondrial function under these conditions. In addition, gene silencing experiments and mutations that target mitochondrial electron transport have also been found to increase lifespan and stress resistance in C. elegans, as has treatment with the mitochondrial inhibitor antimycin A. The involvement of damage by reactive oxygen species has been suggested, and yet many of these manipulations would be expected to increase the production of reactive oxygen species. The extension of lifespan by these interventions seems paradoxical and the mechanism, when it is elucidated, promises to have far-reaching significance.

An in vitro model of caloric restriction.

The mechanisms underlying the ability of caloric restriction (CR) to extend life span and enhance stress responsiveness remain elusive. Progress in this area has been slow due to the complexities of using animals for CR studies and assessing life span as the measure of CR effectiveness. It is therefore of great interest to develop in vitro models of CR. Here we use sera obtained from either Fisher 344 rats or Rhesus monkeys that were fed ad libitum (AL) or CR diets to culture various cell types. We show that treatment of cultured cells with CR sera caused reduced cell proliferation, enhanced tolerance to oxidants and heat, and heightened expression of stress-response genes. These phenotypic features mirror the effects of CR in animals. Supplementation of CR serum with insulin and insulin-like growth factor (IGF)-1 partially restored the proliferative and stress-response phenotype that was seen in cells cultured with AL serum, indicating that reduced levels of insulin and IGF-1 likely contribute to the CR-related effects. This in vitro cell culture model recapitulates key in vivo proliferative and stress-response phenotypic features of CR, and further suggests that endocrine mechanisms contribute to the enhanced stress responsiveness observed in CR animals.

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

Dietary restriction has been shown to have several health benefits including increased insulin sensitivity, stress resistance, reduced morbidity, and increased life span. The mechanism remains unknown, but the need for a long-term reduction in caloric intake to achieve these benefits has been assumed. We report that when C57BL6 mice are maintained on an intermittent fasting (alternate-day fasting) dietary-restriction regimen their overall food intake is not decreased and their body weight is maintained. Nevertheless, intermittent fasting resulted in beneficial effects that met or exceeded those of caloric restriction including reduced serum glucose and insulin levels and increased resistance of neurons in the brain to excitotoxic stress. Intermittent fasting therefore has beneficial effects on glucose regulation and neuronal resistance to injury in these mice that are independent of caloric intake.