R. Michael Anson

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 C57BL/6 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.

Development of Calorie Restriction Mimetics as a Prolongevity Strategy

Abstract: 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.

DNA damage, mutation and fine structure DNA repair in aging

The primary focus of this review is on correlations found between DNA damage, repair, and aging. New techniques for the measurement of DNA damage and repair at the level of individual genes, in individual DNA strands and in individual nucleotides will allow us to gain information regarding the nature of these correlations. Fine structure studies of DNA damage and repair in specific regions, including active genes, telomeres, and mitochondria have begun. Considerable intragenomic DNA repair heterogeneity has been found, and there have been indications of relationships between aging and repair in specific regions. More studies are necessary, however, particularly studies of the repair of endogenous damage. It is emphasized that the information obtained must be viewed from a perspective that takes into account the total responses of the cell to damaging events and the inter-relationships that exist between DNA repair and transcription.

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.

Mitochondrial endogenous oxidative damage has been overestimated

The oxidatively induced DNA lesion 8‐oxo‐dG in mitochondrial DNA (mtDNA) is commonly used as a marker for oxidative damage to mitochondria, which in turn is thought to be a fundamental cause of aging. For years, mitochondrial levels of 8‐oxo‐dG were believed to be ~10‐fold higher in mtDNA than in nuclear DNA even in normal, young animals. However, studies in our own and other laboratories have shown that this lesion is efficiently repaired. Also, mutational consequences specific to 8‐oxo‐dG (G to T transversions) are rarely reported. In the present study, we showed that the levels of damage measured using high‐pressure liquid chromatography/electrochemical detection and an enzymatic/Southern blot assay were comparable. The latter assay does not require isolation of mitochondria, and so this assay was then used to determine the level of in vivo damage present in rat liver mtDNA both with and without organelle isolation. Levels of 8‐oxo‐dG are approximately threefold higher when measured in mtDNA purified from isolated mitochondria than when measured without prior mitochondrial isolation. Furthermore, most genomes were free of endogenous enzyme‐sensitive sites (i.e., they did not contain 8‐oxo‐dG), and only after mitochondrial isolation were levels higher in mtDNA than in a nuclear sequence. Anson, R. M., Hudson, E., Bohr, V. A. Mitochondrial endogenous oxidative damage has been overestimated. FASEB J. 14, 355–360 (2000)

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 nuclear and mitochondrial repair of formamidopyrimidine DNA glycosylase-sensitive sites in Chinese hamster ovary cells

This study examines the capacity of a mammalian cell to repair, at the gene level, DNA base lesions generated by photoactivation of acridine orange. Chinese hamster ovary fibroblasts were exposed to acridine orange and visible light, and gene-specific DNA repair was measured in the dihydrofolate reductase (DHFR) gene and in the mitochondrial genome. DNA lesions were recognized by Escherichia coli formamidepyrimidine-DNA glycosylase (FPG) which removes predominantly 8-oxodG and the corresponding formamidopyrimidine ring opened bases, and subsequently cleaves the DNA at the resulting apurinic site. FPG-recognized DNA lesions increased linearly with increasing photo-activation of AO, while cell survival was not affected by light alone and was negligibly affected by preincubation with AO in the dark. The frequency of induction of FPG-sensitive DNA damage by photoactivation of AO was similar in the transcribed and non-transcribed nuclear DNA as well as in the mitochondrial DNA. FPG-sensitive sites in the DHFR gene were repaired quickly, with 84% of adducts repaired within 4 h. The lesion frequency, kinetics and percent of repair of non-transcribed genomic DNA did not differ significantly from repair in the active DHFR gene up to 1 h postexposure. At late time points, transcribed DNA was repaired faster than the non-transcribed DNA. Mitochondrial DNA was efficiently repaired, at a rate similar to that in the active nuclear DNA.

Oxidative DNA damage processing and changes with aging.

Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, and we are able to examine the repair of different types of lesions and to measure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. Contrary to widely held notions, mitochondria have efficient DNA repair of oxidative DNA damage and we are exploring the mechanisms. In a human disorder, Cockayne syndrome (CS), characterized by premature aging, there appear to be deficiencies in the repair of oxidative DNA damage in the nuclear DNA, and this may be the major underlying cause of the disease.

Measurement of oxidatively induced base lesions in liver from Wistar rats of different ages

Mitochondrial and nuclear DNA were isolated from the livers of young (6-7 month) and old (23-24 month) Wistar rats and the levels of 10 different oxidatively induced lesions were analyzed by gas chromatography/mass spectrometry. This is the first study to measure several different oxidatively induced base lesions in both mitochondrial and nuclear DNA as a function of age. No significant age effects were observed for any lesion. Furthermore, contrary to expectations, we did not observe elevated levels of oxidatively induced base lesions in mitochondrial DNA. This contrasts with 50-fold differences reported for several lesions between mitochondrial and nuclear DNA from porcine liver (Zastawny et al., Free Radic. Biol. Med. 24:722-725, 1998). The fact that different lesion levels are observed even when similar techniques are employed emphasizes that the role of oxidative mitochondrial DNA damage and its repair in aging must continue to be the subject of intense investigation. Questions concerning endogenous levels of damage should be revisited as existing methods are improved and new methods become available.

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.