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There is a pressing need to develop new strategies for enhancing health in the elderly and preventing the rise in age-related diseases. Calorie restriction without malnutrition (CR) stands among the different antiaging interventions. Lifelong CR leads to increased expression and activity of plasma membrane CYB5R3, and male mice overexpressing CYB5R3 exhibit some beneficial adaptations that are also seen with CR. However, the mechanisms involved in both interventions could be independent since key aspects of energy metabolism and tissue lipid profile do not coincide, and many of the changes induced by CR in mitochondrial abundance and dynamics in the liver and skeletal muscle could be counteracted by CYB5R3 overexpression. In this study, we sought to elucidate the impact of CR on key markers of metabolic status, mitochondrial function, and pro-oxidant/antioxidant balance in transgenic (TG) female mice overexpressing CYB5R3 compared to their WT littermates. In females fed ad libitum, CYB5R3 overexpression decreased fat mass, led to a preferred utilization of fatty acids as an energy source, upregulated key antioxidant enzymes, and boosted respiration both in skeletal muscle and liver mitochondria, supporting that CYB5R3 overexpression is phenotypic closer to CR in females than in males. Whereas some markers of mitochondrial biogenesis and dynamics were found decreased in TG females on CR, as also found for the levels of Estrogen Receptor α, mitochondrial abundance and activity were maintained both in skeletal muscle and in liver. Our results reveal overlapping metabolic adaptations resulting from the overexpression of CYB5R3 and CR in females, but a specific crosstalk occurs when both interventions are combined, differing from the adaptations observed in TG males., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Caloric restriction and intermittent fasting prolong the lifespan and healthspan of model organisms and improve human health. The natural polyamine spermidine has been similarly linked to autophagy enhancement, geroprotection and reduced incidence of cardiovascular and neurodegenerative diseases across species borders. Here, we asked whether the cellular and physiological consequences of caloric restriction and fasting depend on polyamine metabolism. We report that spermidine levels increased upon distinct regimens of fasting or caloric restriction in yeast, flies, mice and human volunteers. Genetic or pharmacological blockade of endogenous spermidine synthesis reduced fasting-induced autophagy in yeast, nematodes and human cells. Furthermore, perturbing the polyamine pathway in vivo abrogated the lifespan- and healthspan-extending effects, as well as the cardioprotective and anti-arthritic consequences of fasting. Mechanistically, spermidine mediated these effects via autophagy induction and hypusination of the translation regulator eIF5A. In summary, the polyamine-hypusination axis emerges as a phylogenetically conserved metabolic control hub for fasting-mediated autophagy enhancement and longevity., (© 2024. The Author(s).)

Chaperone-mediated autophagy (CMA) is part of the mammalian cellular proteostasis network that ensures protein quality control, maintenance of proteome homeostasis, and proteome changes required for the adaptation to stress. Loss of proteostasis is one of the hallmarks of aging. CMA decreases with age in multiple rodent tissues and human cell types. A decrease in lysosomal levels of the lysosome-associated membrane protein type 2A (LAMP2A), the CMA receptor, has been identified as a main reason for declined CMA in aging. Here, we report constitutive activation of CMA with calorie restriction (CR), an intervention that extends healthspan, in old rodent livers and in an in vitro model of CR with cultured fibroblasts. We found that CR-mediated upregulation of CMA is due to improved stability of LAMP2A at the lysosome membrane. We also explore the translational value of our observations using calorie-restriction mimetics (CRMs), pharmacologically active substances that reproduce the biochemical and functional effects of CR. We show that acute treatment of old mice with CRMs also robustly activates CMA in several tissues and that this activation is required for the higher resistance to lipid dietary challenges conferred by treatment with CRMs. We conclude that part of the beneficial effects associated with CR/CRMs could be a consequence of the constitutive activation of CMA mediated by these interventions., Competing Interests: Competing interests statement:A.M.C. is a co-founder and scientific advisor for the autophagy program at Life Biosciences. This activity is not related to the work reported here and has not been supported by this company, nor has the data been presented/discussed with them. A.M.C. has co-authored positioning papers in the field of aging and of autophagy with Drs. Kennedy and Hansen, respectively. Those manuscripts do not represent collaborative research and are only focused on opinions on the current status of both fields. The rest of the authors declare no competing interests in relation with this work.

The lifespan extension induced by 40% caloric restriction (CR) in rodents is accompanied by postponement of disease, preservation of function, and increased stress resistance. Whether CR elicits the same physiological and molecular responses in humans remains mostly unexplored. In the CALERIE study, 12% CR for 2 years in healthy humans induced minor losses of muscle mass (leg lean mass) without changes of muscle strength, but mechanisms for muscle quality preservation remained unclear. We performed high-depth RNA-Seq (387-618 million paired reads) on human vastus lateralis muscle biopsies collected from the CALERIE participants at baseline, 12- and 24-month follow-up from the 90 CALERIE participants randomized to CR and "ad libitum" control. Using linear mixed effect model, we identified protein-coding genes and splicing variants whose expression was significantly changed in the CR group compared to controls, including genes related to proteostasis, circadian rhythm regulation, DNA repair, mitochondrial biogenesis, mRNA processing/splicing, FOXO3 metabolism, apoptosis, and inflammation. Changes in some of these biological pathways mediated part of the positive effect of CR on muscle quality. Differentially expressed splicing variants were associated with change in pathways shown to be affected by CR in model organisms. Two years of sustained CR in humans positively affected skeletal muscle quality, and impacted gene expression and splicing profiles of biological pathways affected by CR in model organisms, suggesting that attainable levels of CR in a lifestyle intervention can benefit muscle health in humans., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Emerging new evidence highlights the importance of prolonged daily fasting periods for the health and survival benefits of calorie restriction (CR) and time-restricted feeding (TRF) in male mice; however, little is known about the impact of these feeding regimens in females. We placed 14-month-old female mice on five different dietary regimens, either CR or TRF with different feeding windows, and determined the effects of these regimens on physiological responses, progression of neoplasms and inflammatory diseases, serum metabolite levels, and lifespan. Compared with TRF feeding, CR elicited a robust systemic response, as it relates to energetics and healthspan metrics, a unique serum metabolomics signature in overnight fasted animals, and was associated with an increase in lifespan. These results indicate that daytime (rest-phase) feeding with prolonged fasting periods initiated late in life confer greater benefits when combined with imposed lower energy intake., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Cancer incidence increases with age and is a leading cause of death. Caloric restriction (CR) confers benefits on health and survival and delays cancer. However, due to CR's stringency, dietary alternatives offering the same cancer protection have become increasingly attractive. Short cycles of a plant-based diet designed to mimic fasting (FMD) are protective against tumorigenesis without the chronic restriction of calories. Yet, it is unclear whether the fasting time, level of dietary restriction, or nutrient composition is the primary driver behind cancer protection. Using a breast cancer model in mice, we compare the potency of daily CR to that of periodic caloric cycling on FMD or an isocaloric standard laboratory chow against primary tumor growth and metastatic burden. Here, we report that daily CR provides greater protection against tumor growth and metastasis to the lung, which may be in part due to the unique immune signature observed with daily CR., (© 2021. The Author(s).)

The impact of chronic caloric restriction (CR) on health and survival is complex with poorly understood underlying molecular mechanisms. A recent study in mice addressing the diets used in nonhuman primate CR studies found that while diet composition did not impact longevity, fasting time and total calorie intake were determinant for increased survival. Here, integrated analysis of physiological and multi-omics data from ad libitum, meal-fed, or CR animals was used to gain insight into pathways associated with improved health and survival. We identified a potential involvement of the glycine-serine-threonine metabolic axis in longevity and related molecular mechanisms. Direct comparison of the different feeding strategies unveiled a pattern of shared pathways of improved health that included short-chain fatty acids and essential PUFA metabolism. These findings were recapitulated in the serum metabolome from nonhuman primates. We propose that the pathways identified might be targeted for their potential role in healthy aging., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Calorie restriction without malnutrition (CR) is considered as the most effective nongenetic nor pharmacological intervention that promotes healthy aging phenotypes and can extend lifespan in most model organisms. Lifelong CR leads to an increase of cytochrome b 5 reductase-3 (CYB5R3) expression and activity. Overexpression of CYB5R3 confers some of the salutary effects of CR, although the mechanisms involved might be independent because key aspects of energy metabolism and lipid profiles of tissues go in opposite ways. It is thus important to study if some of the metabolic adaptations induced by CR are affected by CYB5R3 overexpression. CYB5R3 overexpression greatly preserved body and liver weight in mice under CR conditions. In liver, CR did not modify mitochondrial abundance, but lead to increased expression of mitofusin Mfn2 and TFAM, a transcription factor involved in mitochondrial biogenesis. These changes were prevented by CYB5R3 overexpression but resulted in a decreased expression of a different mitochondrial biogenesis-related transcription factor, Nrf1. In skeletal muscle, CR strongly increased mitochondrial mass, mitofusin Mfn1, and Nrf1. However, CYB5R3 mice on CR did not show increase in muscle mitochondrial mass, regardless of a clear increase in expression of TFAM and mitochondrial complexes in this tissue. Our results support that CYB5R3 overexpression significantly modifies the metabolic adaptations of mice to CR.

Caloric restriction (CR) is the leading non-pharmaceutical dietary intervention to improve health- and lifespan in most model organisms. A wide array of cellular pathways is induced in response to CR and CR-mimetics, including the transcriptional activator Nuclear factor erythroid-2-related factor 2 (Nrf2), which is essential in the upregulation of multiple stress-responsive and mitochondrial enzymes. Nrf2 is necessary in tumor protection but is not essential for the lifespan extending properties of CR in outbred mice. Here, we sought to study Nrf2-knockout (KO) mice and littermate controls in male C57BL6/J, an inbred mouse strain. Deletion of Nrf2 resulted in shortened lifespan compared to littermate controls only under ad libitum conditions. CR-mediated lifespan extension and physical performance improvements did not require Nrf2. Metabolic and protein homeostasis and activation of tissue-specific cytoprotective proteins were dependent on Nrf2 expression. These results highlight an important contribution of Nrf2 for normal lifespan and stress response., Competing Interests: Declaration of competing interest None to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Although survival has been the focus of aging research for many years, the field is rapidly evolving towards incorporating healthspan and health indices in studies that explore aging-related outcomes. Frailty is one such measure that is tightly correlated with human aging. Several frailty measures have been developed that focus on phenotypes of aging, including physical, cognitive and metabolic health that define healthspan. The extent at which cumulative deficits associated with frailty predict functional characteristics of healthy aging and longevity is currently unknown. A growing consensus for the use of animal models has emerged to evaluate a composite measure of frailty that provides a translational basis to understanding human frailty. In this review, we will focus on the impact of several anti-aging interventions, some of which have been characterized as caloric restriction (CR) mimetics such as metformin, rapamycin, and resveratrol as well as more novel approaches that are emerging in the field - nicotinamide adenine dinucleotide precursors, small molecule activators of sirtuins, and senolytics - on a number of frailty measurements associated with aging-related outcomes in mice and discuss the translatability of such measures to human frailty., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Loss of skeletal muscle mass and function is a hallmark of aging. This phenomenon has been related to a dysregulation of mitochondrial function and proteostasis. Calorie restriction (CR) has been demonstrated to delay aging and preserve function until late in life, particularly in muscle. Recently, we reported the type of dietary fat plays an important role in determining life span extension with 40% CR in male mice. In these conditions, lard fed mice showed an increased longevity compared to mice fed soybean or fish oils. In this article, we analyze the effect of 40% CR on muscle mitochondrial mass, autophagy, and mitochondrial dynamics markers in mice fed these diets. In CR fed animals, lard preserved muscle fibers structure, mitochondrial ultrastructure, and fission/fusion dynamics and autophagy, not only compared to control animals, but also compared with CR mice fed soybean and fish oils as dietary fat. We focus our discussion on dietary fatty acid saturation degree as an essential predictor of life span extension in CR mice., (© The Author(s) 2018. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Caloric restriction (CR) is the most potent nonpharmacological intervention known to both protect against carcinogenesis and delay aging in laboratory animals. There is a growing number of anticarcinogens and CR mimetics that activate NAD(P)H:quinone oxidoreductase 1 (NQO1). We have previously shown that NQO1, an antioxidant enzyme that acts as an energy sensor through modulation of intracellular redox and metabolic state, is upregulated by CR. Here, we used NQO1-knockout (KO) mice to investigate the role of NQO1 in both the aging process and tumor susceptibility, specifically in the context of CR. We found that NQO1 is not essential for the beneficial effects of CR on glucose homeostasis, physical performance, metabolic flexibility, life-span extension, and (unlike our previously observation with Nrf2) chemical-induced tumorigenesis.

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., (Published by Elsevier Inc.)

Background Aging exponentially increases the incidence of morbidity and mortality of quintessential cardiovascular disease mainly due to arterial proinflammatory shifts at the molecular, cellular, and tissue levels within the arterial wall. Calorie restriction ( CR ) in rats improves arterial function and extends both health span and life span. How CR affects the proinflammatory landscape of molecular, cellular, and tissue phenotypic shifts within the arterial wall in rats, however, remains to be elucidated. Methods and Results Aortae were harvested from young (6-month-old) and old (24-month-old) Fischer 344 rats, fed ad libitum and a second group maintained on a 40% CR beginning at 1 month of age. Histopathologic and morphometric analysis of the arterial wall demonstrated that CR markedly reduced age-associated intimal medial thickening, collagen deposition, and elastin fractionation/degradation within the arterial walls. Immunostaining/blotting showed that CR effectively prevented an age-associated increase in the density of platelet-derived growth factor, matrix metalloproteinase type II activity, and transforming growth factor beta 1 and its downstream signaling molecules, phospho-mothers against decapentaplegic homolog-2/3 (p- SMAD -2/3) in the arterial wall. In early passage cultured vascular smooth muscle cells isolated from AL and CR rat aortae, CR alleviated the age-associated vascular smooth muscle cell phenotypic shifts, profibrogenic signaling, and migration/proliferation in response to platelet-derived growth factor. Conclusions CR reduces matrix and cellular proinflammation associated with aging that occurs within the aortic wall and that are attributable to platelet-derived growth factor signaling. Thus, CR reduces the platelet-derived growth factor-associated signaling cascade, contributing to the postponement of biological aging and preservation of a more youthful aortic wall phenotype.

Calorie restriction (CR) is one of the most robust means to improve health and survival in model organisms. CR imposes a metabolic program that leads to increased stress resistance and delayed onset of chronic diseases, including cancer. In rodents, CR induces the upregulation of two NADH-dehydrogenases, namely NAD(P)H:quinone oxidoreductase 1 (Nqo1) and cytochrome b 5 reductase 3 (Cyb5r3), which provide electrons for energy metabolism. It has been proposed that this upregulation may be responsible for some of the beneficial effects of CR, and defects in their activity are linked to aging and several age-associated diseases. However, it is unclear whether changes in metabolic homeostasis solely through upregulation of these NADH-dehydrogenases have a positive impact on health and survival. We generated a mouse that overexpresses both metabolic enzymes leading to phenotypes that resemble aspects of CR including a modest increase in lifespan, greater physical performance, a decrease in chronic inflammation, and, importantly, protection against carcinogenesis, one of the main hallmarks of CR. Furthermore, these animals showed an enhancement of metabolic flexibility and a significant upregulation of the NAD + /sirtuin pathway. The results highlight the importance of these NAD + producers for the promotion of health and extended lifespan., (© 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

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., (Published by Oxford University Press on behalf of The Gerontological Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

The calorie restriction paradigm has provided one of the most widely used and most useful tools for investigating mechanisms of aging and longevity. By far, rodent models have been employed most often in these endeavors. Over decades of investigation, claims have been made that the paradigm produces the most robust demonstration that aging is malleable. In the current review of the rodent literature, we present arguments that question the robustness of the paradigm to increase lifespan and healthspan. Specifically, there are several questions to consider as follows: (1) At what age does CR no longer produce benefits? (2) Does CR attenuate cognitive decline? (3) Are there negative effects of CR, including effects on bone health, wound healing, and response to infection? (4) How important is schedule of feeding? (5) How long does CR need to be imposed to be effective? (6) How do genotype and gender influence CR? (7) What role does dietary composition play? Consideration of these questions produce many caveats that should guide future investigations to move the field forward., (Published by Elsevier B.V.)

Caloric restriction (CR) without malnutrition extends lifespan and delays the onset of age-related disorders in most species but its impact in nonhuman primates has been controversial. In the late 1980s two parallel studies were initiated to determine the effect of CR in rhesus monkeys. The University of Wisconsin study reported a significant positive impact of CR on survival, but the National Institute on Aging study detected no significant survival effect. Here we present a direct comparison of longitudinal data from both studies including survival, bodyweight, food intake, fasting glucose levels and age-related morbidity. We describe differences in study design that could contribute to differences in outcomes, and we report species specificity in the impact of CR in terms of optimal onset and diet. Taken together these data confirm that health benefits of CR are conserved in monkeys and suggest that CR mechanisms are likely translatable to human health., Competing Interests: R.W. is a member of the board of LifeGen Technologies, a company focused on nutritional genomics. G.S.R. is Chief Executive Officer of GeroScience, Inc. and Vice President of Prolongevity Technologies. D.K.I. serves as Chief Scientific Officer for GeroScience, Inc., and Prolongevity Technologies, Inc. D.B.A. serves on the board of IKEA and has received consulting fees from multiple government, not for profit, and for profit organizations with interests in obesity and nutrition. None of these activities benefit directly from this research and no competing financial interests are declared. The remaining authors declare no competing financial interests.

Calorie restriction (CR) has been repeatedly shown to prevent cancer, diabetes, hypertension, and other age-related diseases in a wide range of animals, including non-human primates and humans. In rodents, CR also increases lifespan and is a powerful tool for studying the aging process. Recently, it has been reported in mice that dietary fat plays an important role in determining lifespan extension with 40% CR. In these conditions, animals fed lard as dietary fat showed an increased longevity compared with mice fed soybean or fish oils. In this paper, we study the effect of these dietary fats on structural and physiological parameters of kidney from mice maintained on 40% CR for 6 and 18 months. Analyses were performed using quantitative electron microcopy techniques and protein expression in Western blots. CR mitigated most of the analyzed age-related parameters in kidney, such as glomerular basement membrane thickness, mitochondrial mass in convoluted proximal tubules and autophagic markers in renal homogenates. The lard group showed improved preservation of several renal structures with aging when compared to the other CR diet groups. These results indicate that dietary fat modulates renal structure and function in CR mice and plays an essential role in the determination of health span in rodents., (© 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

This article investigates the effect on the mouse frailty index (FI), of factors known to influence lifespan and healthspan in mice: strain (short-lived DBA/2J mice vs long-lived C57BL/6J mice), calorie restriction (CR), and resveratrol treatment. The mouse FI, based on deficit accumulation, was recently validated in C57BL/6J mice by Whitehead JC, Hildebrand BA, Sun M, et al. (A clinical frailty index in aging mice: comparisons with frailty index data in humans. J Gerontol A Biol Sci Med Sci. 2014;69:621-632) and shares many characteristics of the human FI. FI scores were measured in male and female aged (18 months) ad-libitum fed and CR DBA/2J and C57BL/6J mice, as well as male aged (24 months) C57BL/6J mice ad-libitum fed with or without resveratrol (100 mg/kg/day) in the diet for 6 months. Mean scores of two raters were used, and the raters had excellent inter-rater reliability (ICC = 0.88, 95% CI [0.80, 0.92]). Furthermore, the interventions of CR and resveratrol were associated with a significant reduction in FI scores in C57BL/6J mice, compared to age-matched controls. The short-lived DBA/2J mice also had slightly higher FI scores than the C57BL/6J mice, for the male calorie-restricted groups (DBA/2J FI = 0.16±0.03, C57BL/6J FI = 0.11±0.03, p = .01). This study uses the mouse FI developed by Whitehead JC, Hildebrand BA, Sun M, et al. (A clinical frailty index in aging mice: comparisons with frailty index data in humans. J Gerontol A Biol Sci Med Sci. 2014;69:621-632) in a different mouse colony and shows that this tool can be applied to quantify the effect of dietary and pharmaceutical interventions on frailty., (© The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Membrane Theory of Aging proposes that lifespan is inversely related to the level of unsaturation in membrane phospholipids. Calorie restriction (CR) without malnutrition extends lifespan in many model organisms, which may be related to alterations in membrane phospholipids fatty acids. During the last few years our research focused on studying how altering the predominant fat source affects the outcome of CR in mice. We have established four dietary groups: one control group fed 95 % of a pre-determined ad libitum intake (in order to prevent obesity), and three CR groups fed 40 % less than ad libitum intake. Lipid source for the control and one of the CR groups was soybean oil (high in n-6 PUFA) whereas the two remaining CR groups were fed diets containing fish oil (high in n-3 PUFA), or lard (high in saturated and monounsaturated fatty acids). Dietary intervention periods ranged from 1 to 18 months. We performed a longitudinal lifespan study and a cross-sectional study set up to evaluate several mitochondrial parameters which included fatty acid composition, H(+) leak, activities of electron transport chain enzymes, ROS generation, lipid peroxidation, mitochondrial ultrastructure, and mitochondrial apoptotic signaling in liver and skeletal muscle. These approaches applied to different cohorts of mice have independently indicated that lard as a fat source often maximizes the effects of 40 % CR on mice. These effects could be due to significant increases of monounsaturated fatty acids levels, in accordance with the Membrane Theory of Aging.

Both caloric restriction (CR) and low-protein, high-carbohydrate (LPHC) ad-libitum-fed diets increase lifespan and improve metabolic parameters such as insulin, glucose, and blood lipids. Severe CR, however, is unsustainable for most people; therefore, it is important to determine whether manipulating macronutrient ratios in ad-libitum-fed conditions can generate similar health outcomes. We present the results of a short-term (8 week) dietary manipulation on metabolic outcomes in mice. We compared three diets varying in protein to carbohydrate ratio under both CR and ad libitum conditions. Ad libitum LPHC diets delivered similar benefits to CR in terms of levels of insulin, glucose, lipids, and HOMA, despite increased energy intake. CR on LPHC diets did not provide additional benefits relative to ad libitum LPHC. We show that LPHC diets under ad-libitum-fed conditions generate the metabolic benefits of CR without a 40% reduction in total caloric intake., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Astrocytes are key cells in brain aging, helping neurons to undertake healthy aging or otherwise letting them enter into a spiral of neurodegeneration. We aimed to characterize astrocytes cultured from senescence-accelerated prone 8 (SAMP8) mice, a mouse model of brain pathological aging, along with the effects of caloric restriction, the most effective rejuvenating treatment known so far. Analysis of the transcriptomic profiles of SAMP8 astrocytes cultured in control conditions and treated with caloric restriction serum was performed using mRNA microarrays. A decrease in mitochondrial and ribosome mRNA, which was restored by caloric restriction, confirmed the age-related profile of SAMP8 astrocytes and the benefits of caloric restriction. An amelioration of antioxidant and neurodegeneration-related pathways confirmed the brain benefits of caloric restriction. Studies of oxidative stress and mitochondrial function demonstrated a reduction of oxidative damage and partial improvement of mitochondria after caloric restriction. In summary, caloric restriction showed a significant tendency to normalize pathologically aged astrocytes through the activation of pathways that are protective against the age-related deterioration of brain physiology., (© 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Caloric restriction (CR) without malnutrition is one of the most consistent strategies for increasing mean and maximal lifespan and delaying the onset of age-associated diseases. Stress resistance is a common trait of many long-lived mutants and life-extending interventions, including CR. Indeed, better protection against heat shock and other genotoxic insults have helped explain the pro-survival properties of CR. In this study, both in vitro and in vivo responses to heat shock were investigated using two different models of CR. Murine B16F10 melanoma cells treated with serum from CR-fed rats showed lower proliferation, increased tolerance to heat shock and enhanced HSP-70 expression, compared to serum from ad libitum-fed animals. Similar effects were observed in B16F10 cells implanted subcutaneously in male C57BL/6 mice subjected to CR. Microarray analysis identified a number of genes and pathways whose expression profile were similar in both models. These results suggest that the use of an in vitro model could be a good alternative to study the mechanisms by which CR exerts its anti-tumorigenic effects.

Imbalance between proliferation and cell death accounts for several age-linked diseases. Aging, calorie restriction (CR), and fat source are all factors that may influence apoptotic signaling in liver, an organ that plays a central metabolic role in the organism. Here, we have studied the combined effect of these factors on a number of apoptosis regulators and effectors. For this purpose, animals were fed diets containing different fat sources (lard, soybean oil, or fish oil) under CR for 6 or 18 months. An age-linked increase in the mitochondrial apoptotic pathway was detected with CR, including a decrease in Bcl-2/Bax ratio, an enhanced release of cytochrome c to the cytosol and higher caspase-9 activity. However, these changes were not fully transmitted to the effectors apoptosis-inducing factor and caspase-3. CR (which abated aging-related inflammatory responses) and dietary fat altered the activities of caspases-8, -9, and -3. Apoptotic index (DNA fragmentation) and mean nuclear area were increased in aged animals with the exception of calorie-restricted mice fed a lard-based fat source. These results suggest possible protective changes in hepatic homeostasis with aging in the calorie-restricted lard group., (© The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The cumulative effects of cellular senescence and cell loss over time in various tissues and organs are considered major contributing factors to the ageing process. In various organisms, caloric restriction (CR) slows ageing and increases lifespan, at least in part, by activating nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases of the sirtuin family. Here, we use an in vitro model of CR to study the effects of this dietary regime on replicative senescence, cellular lifespan and modulation of the SIRT1 signaling pathway in normal human diploid fibroblasts. We found that serum from calorie-restricted animals was able to delay senescence and significantly increase replicative lifespan in these cells, when compared to serum from ad libitum fed animals. These effects correlated with CR-mediated increases in SIRT1 and decreases in p53 expression levels. In addition, we show that manipulation of SIRT1 levels by either over-expression or siRNA-mediated knockdown resulted in delayed and accelerated cellular senescence, respectively. Our results demonstrate that CR can delay senescence and increase replicative lifespan of normal human diploid fibroblasts in vitro and suggest that SIRT1 plays an important role in these processes.

In this paper we analyzed changes in hepatocyte mitochondrial mass and ultrastructure as well as in mitochondrial markers of fission/fusion and biogenesis in mice subjected to 40% calorie restriction (CR) for 18 months versus ad libitum-fed controls. Animals subjected to CR were separated into three groups with different dietary fats: soybean oil (also in controls), fish oil and lard. Therefore, the effect of the dietary fat under CR was studied as well. Our results show that CR induced changes in hepatocyte and mitochondrial size, in the volume fraction occupied by mitochondria, and in the number of mitochondria per hepatocyte. Also, mean number of mitochondrial cristae and lengths were significantly higher in all CR groups compared with controls. Finally, CR had no remarkable effects on the expression levels of fission and fusion protein markers. However, considerable differences in many of these parameters were found when comparing the CR groups, supporting the idea that dietary fat plays a relevant role in the modulation of CR effects in aged mice., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Calorie restriction (CR) is a lifestyle intervention employed to reduce body weight and improve metabolic functions primarily via reduction of ingested carbohydrates and fats. Taste perception is highly related to functional metabolic status and body adiposity. We have previously shown that sweet taste perception diminishes with age; however, relatively little is known about the effects of various lengths of CR upon taste cell morphology and function. We investigated the effects of CR on taste bud morphology and expression of sweet taste-related modulators in 5-, 17-, and 30-month-old rats. In ad libitum (AL) and CR rats, we consistently found the following parameters altered significantly with advancing age: reduction of taste bud size and taste cell numbers per taste bud and reduced expression of sonic hedgehog, type 1 taste receptor 3 (T1r3), α-gustducin, and glucagon-like peptide-1 (GLP-1). In the oldest rats, CR affected a significant reduction of tongue T1r3, GLP-1, and α-gustducin expression compared with age-matched AL rats. Leptin receptor immunopositive cells were elevated in 17- and 30-month-old CR rats compared with age-matched AL rats. These alterations of sweet taste-related modulators, specifically during advanced aging, suggest that sweet taste perception may be altered in response to different lengths of CR.

The SIRT1 deacetylase is one of the best-studied putative mediators of some of the anti-aging effects of calorie restriction (CR), but its role in CR-dependent lifespan extension has not been demonstrated. We previously found that mice lacking both copies of SIRT1 displayed a shorter median lifespan than wild-type mice on an ad libitum diet. Here, we report that median lifespan extension in CR heterozygote SIRT1(+/-) mice was identical (51%) to that observed in wild-type mice, but SIRT1(+/-) mice displayed a higher frequency of certain pathologies. Although larger studies in additional genetic backgrounds are needed, these results provide strong initial evidence for the requirement of SIRT1 for the lifespan extension effects of CR, but suggest that its high expression is not required for CR-induced lifespan extension., (© 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

The levels of microRNAs (miRNAs) are altered under different conditions such as cancer, senescence, and aging. Here, we have identified differentially expressed miRNAs in skeletal muscle from young and old rhesus monkeys using RNA sequencing. In old muscle, several miRNAs were upregulated, including miR-451, miR-144, miR-18a and miR-15a, while a few miRNAs were downregulated, including miR-181a and miR-181b. A number of novel miRNAs were also identified, particularly in old muscle. We also examined the impact of caloric restriction (CR) on miRNA abundance by reverse transcription (RT) followed by real-time, quantitative (q)PCR analysis and found that CR rescued the levels of miR-181b and chr1:205580546, and also dampened the age-induced increase in miR-451 and miR-144 levels. Our results reveal that there are changes in expression of known and novel miRNAs with skeletal muscle aging and that CR may reverse some of these changes to a younger phenotype.

We analyzed ultrastructural changes and markers of fission/fusion in hepatocyte mitochondria from mice submitted to 40% calorie restriction (CR) for 6 months versus ad-libitum-fed controls. To study the effects of dietary fat under CR, animals were separated into three CR groups with soybean oil (also in controls), fish oil, and lard. CR induced differential changes in hepatocyte and mitochondrial size, in the volume fraction occupied by mitochondria, and in the number of mitochondria per hepatocyte. The number of cristae per mitochondrion was significantly higher in all CR groups compared with controls. Proteins related to mitochondrial fission (Fis1 and Drp1) increased with CR, but no changes were detected in proteins involved in mitochondrial fusion (Mfn1, Mfn2, and OPA1). Although many of these changes could be attributed to CR regardless of dietary fat, changing membrane lipid composition by different fat sources did modulate the effects of CR on hepatocyte mitochondria.

Caloric restriction (CR) and down-regulation of the insulin/IGF pathway are the most robust interventions known to increase longevity in lower organisms. However, little is known about the molecular adaptations induced by CR in humans. Here, we report that long-term CR in humans inhibits the IGF-1/insulin pathway in skeletal muscle, a key metabolic tissue. We also demonstrate that CR induces dramatic changes of the skeletal muscle transcriptional profile that resemble those of younger individuals. Finally, in both rats and humans, CR evoked similar responses in the transcriptional profiles of skeletal muscle. This common signature consisted of three key pathways typically associated with longevity: IGF-1/insulin signaling, mitochondrial biogenesis, and inflammation. Furthermore, our data identify promising pathways for therapeutic targets to combat age-related diseases and promote health in humans., (© 2013 John Wiley & Sons Ltd and the Anatomical Society.)

Significance: Calorie restriction (CR) is a known intervention that delays most aging processes. Most of the beneficial effects of CR are mediated by improved maintenance of mitochondrial performance in aged individuals. The control of mitochondrial biogenesis, apoptosis, and protein turnover is required for healthy aging. CR is able to induce molecular mechanisms that preserve oxidative capacity and decrease oxidative damage., Recent Advances and Critical Issues: Published data indicate that peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) is activated in old animals under CR conditions compared to ad libitum counterparts, enhancing mitochondrial biogenesis. Molecular regulation of PGC-1α has recently attracted significant research interest. We discuss the master regulators of energy metabolism such as AMP-activated protein kinase and sirtuin 1 among others that have been demonstrated to activate mitochondrial biogenesis through increased PGC-1α activity at transcriptional and post-translational levels. Additionally, we describe the latest findings that explain how CR promotes mitochondrial efficiency and decreases mitochondrial-derived oxidative damage., Future Directions: Understanding the beneficial mitochondrial changes conferred by CR will aid design of therapies for age-related diseases and help slow the aging process. Given the difficulty for humans to adhere to CR, we also explore new molecules that have been proposed during the last years to mimic the CR phenotype and their potential as future therapeutics.

Resveratrol, a polyphenolic compound, has been shown to extend lifespan in different organisms. Emerging evidence suggests that the prolongevity effect of resveratrol depends on dietary composition. However, the mechanisms underlying the interaction of resveratrol and dietary nutrients in modulating lifespan remain elusive. Here, we investigated the effect of resveratrol on lifespan of Drosophila melanogaster fed diets differing in the concentrations of sugar, yeast extract, and palmitic acid representing carbohydrate, protein, and fat, respectively. Resveratrol at up to 200 μM in diets did not affect lifespan of wild-type female flies fed a standard, restricted or high sugar-low protein diet, but extended lifespan of females fed a low sugar-high protein diet. Resveratrol at 400 μM extended lifespan of females fed a high-fat diet. Lifespan extension by resveratrol was associated with downregulation of genes in aging-related pathways, including antioxidant peroxiredoxins, insulin-like peptides involved in insulin-like signaling and several downstream genes in Jun-kinase signaling involved in oxidative stress response. Furthermore, resveratrol increased lifespan of superoxide dismutase 1 (sod1) knockdown mutant females fed a standard or high-fat diet. No lifespan extension by resveratrol was observed in wild-type and sod1 knockdown males under the culture conditions in this study. Our results suggest that the gender-specific prolongevity effect of resveratrol is influenced by dietary composition and resveratrol promotes the survival of flies by modulating genetic pathways that can reduce cellular damage. This study reveals the context-dependent effect of resveratrol on lifespan and suggests the importance of dietary nutrients in implementation of effective aging interventions using dietary supplements.

Calorie restriction is the most powerful method currently known to delay aging-associated disease and extend lifespan. Use of this technique in combination with genetic models has led to identification of key metabolic regulators of lifespan. Limiting energy availability by restricting caloric intake leads to redistribution of energy expenditure and storage. The signaling required for these metabolic changes is mediated in part by the sirtuins at both the posttranslational and transcriptional levels, and consequently, sirtuins are recognized as instigating factors in the regulation of lifespan. This family of class III protein deacetylases is responsible for directing energy regulation based on NAD(+) availability. However, there are many effectors of NAD(+) availability, and hence sirtuin action, that should be considered when performing experiments using calorie restriction. The methods outlined in this chapter are intended to provide a guide to help the aging community to use and interpret experimental calorie restriction properly. The importance of healthspan and the use of repeated measures to assess metabolic health during lifespan experiments are strongly emphasized.

Glucocorticoids (GC)--corticosterone (CORT) in rodents and cortisol in primates--are stress-induced hormones secreted by adrenal glands that interact with the hypothalamic pituitary axis. High levels of cortisol in humans are observed in neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), as well as in diabetes, post-traumatic stress syndrome, and major depression. Experimental models of diabetes in rats and mice have demonstrated that reduction of CORT reduces learning and memory deficits and attenuates loss of neuronal viability and plasticity. In contrast to the negative associations of elevated GC levels, CORT is moderately elevated in dietary restriction (DR) paradigms which are associated with many healthy anti-aging effects including neuroprotection. We demonstrate here in rats that ablating CORT by adrenalectomy (ADX) with replenishment to relatively low levels (30% below that of controls) prior to the onset of a DR regimen (ADX-DR) followed by central administration of the neurotoxin, kainic acid (KA), significantly attenuates learning deficits in a 14-unit T-maze task. The performance of the ADX-DR KA group did not differ from a control group (CON) that did not receive KA and was fed ad libitum (AL). By contrast, the sham-operated DR (SHAM-DR KA) group, SHAM-AL KA group, and ADX-AL KA group demonstrated poorer learning behavior in this task compared to the CON group. Stereological analysis revealed equivalent DR-induced neuroprotection in the SH-DR KA and ADX-DR KA groups, as measured by cell loss in the CA2/CA3 region of the hippocampus, while substantial cell loss was observed in SH-AL and ADX-AL rats. A separate set of experiments was conducted with similar dietary and surgical treatment conditions but without KA administration to examine markers of neurotrophic activity, brain-derived neurotrophic factor (BDNF), transcriptions factors (pCREB), and chaperone proteins (HSP-70). Under these conditions, we noted elevations in both BDNF and pCREB in ADX DR rats compared to the other groups; whereas, HSP-70, was equivalently elevated in ADX-DR and SH-DR groups and was higher than observed in both SH-AL and ADX-AL groups. These results support findings that DR protects hippocampal neurons against KA-induced cellular insult. However, this neuroprotective effect was further enhanced in rats with a lower-than control level of CORT resulting from ADX and maintained by exogenous CORT supplementation. Our results then suggest that DR-induced physiological elevation of GC may have negative functional consequences to DR-induced beneficial effects. These negative effects, however, can be compensated by other DR-produced cellular and molecular protective mechanisms., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Calorie restriction (CR), a reduction of 10–40% in intake of a nutritious diet, is often reported as the most robust non-genetic mechanism to extend lifespan and healthspan. CR is frequently used as a tool to understand mechanisms behind ageing and age-associated diseases. In addition to and independently of increasing lifespan, CR has been reported to delay or prevent the occurrence of many chronic diseases in a variety of animals. Beneficial effects of CR on outcomes such as immune function, motor coordination and resistance to sarcopenia in rhesus monkeys have recently been reported. We report here that a CR regimen implemented in young and older age rhesus monkeys at the National Institute on Aging (NIA) has not improved survival outcomes. Our findings contrast with an ongoing study at the Wisconsin National Primate Research Center (WNPRC), which reported improved survival associated with 30% CR initiated in adult rhesus monkeys (7–14 years) and a preliminary report with a small number of CR monkeys. Over the years, both NIA and WNPRC have extensively documented beneficial health effects of CR in these two apparently parallel studies. The implications of the WNPRC findings were important as they extended CR findings beyond the laboratory rodent and to a long-lived primate. Our study suggests a separation between health effects, morbidity and mortality, and similar to what has been shown in rodents, study design, husbandry and diet composition may strongly affect the life-prolonging effect of CR in a long-lived nonhuman primate.

During aging there is an increasing imbalance of energy intake and expenditure resulting in obesity, frailty, and metabolic disorders. For decades, research has shown that caloric restriction (CR) and exercise can postpone detrimental aspects of aging. These two interventions invoke a similar physiological signature involving pathways associated with stress responses and mitochondrial homeostasis. Nonetheless, CR is able to delay aging processes that result in an increase of both mean and maximum lifespan, whereas exercise primarily increases healthspan. Due to the strict dietary regime necessary to achieve the beneficial effects of CR, most studies to date have focused on rodents and non-human primates. As a consequence, there is vast interest in the development of compounds such as resveratrol, metformin and rapamycin that would activate the same metabolic- and stress-response pathways induced by these interventions without actually restricting caloric intake. Therefore the scope of this review is to (i) describe the benefits of CR and exercise in healthy individuals, (ii) discuss the role of these interventions in the diseased state, and (iii) examine some of the promising pharmacological alternatives such as CR- and exercise-mimetics., (Published by Elsevier B.V.)

Multiple health benefits of calorie restriction (CR) and alternate day fasting (ADF) regimens are widely recognized. Experimental data concerning the effects of calorie restriction on cardiac health are more controversial, ranging from evidence that ADF protects heart from ischemic damage but results in developing of diastolic dysfunction, to reports that CR ameliorates the age-associated diastolic dysfunction. Here we investigated the effects of chronic CR on morphology and function of the cardiovascular system of aged rats and cardioprotective effect of CR against ischemic damage in the experimental rat model of MI. Cardiovascular fitness of 24-month old Fisher 344 rats maintained through life on ad libitum (AL) or CR diets was extensively evaluated via echocardiography, dobutamine stress test, pressure-volume loop analyses, pulse wave velocity measurements, and histology. Groups of 2-month old AL and 29-month old CR rats were studied for comparison. Myocardial infarction (MI) was induced by a permanent ligation of the anterior descending coronary artery in 5-month old rats maintained for 3 months on CR or AL. MI size was evaluated histologically 24 hrs following coronary ligation. Cardiac remodeling was followed-up via echocardiography. Age-associated changes in 24-month old rats consisted of 33% increase of fibrosis in the myocardium and more than 2 fold increase of the collagen in the tunica media of the aorta. There was a significant decrease in the density and total number of cardiomyocytes, while their size was increased. These morphological changes were manifested in a decline of systolic and diastolic cardiac function, increase of left ventricular and aortic stiffness, and arterio-ventricular uncoupling. Tachycardic response to dobutamine challenge was absent in the old rats. Compared to AL rats, 24-month old CR rats had reduced levels of cardiac and aortic fibrosis, increased density of cardiomyocytes that were smaller in size, attenuated diastolic dysfunction, normal systolic function and arterio-ventricular coupling. Tachycardic response to dobutamine was also intact in CR 24-month old rats and aortic stiffness was reduced. Adjustment for body weight differences through ratiometric or allometric scaling did not affect the overall pattern of differences between AL and CR rats. Attenuation of morphological and functional age-associated changes in 24-month old CR rats either was not observed at all or was smaller in 29-month old CR rats. Size of MI induced by a permanent coronary ligation as well as post-MI cardiac remodeling and function were similar in CR and AL rats. CR does not increase tolerance of myocardium to ischemic damage, but attenuates the age-associated changes in the heart and major vessels. The attenuation of age-associated changes by CR cannot be explained by the effect of lower body weight but are attributable to more intimate cellular mechanisms of CR itself. Attenuation of age-associated changes by CR waned with advancing age, and is consistent with the idea that CR postponed senescence., (Published by Elsevier Ltd.)

Calorie restriction (CR) is known to have profound effects on tumor incidence. A typical consequence of CR is hunger, and we hypothesized that the neuroendocrine response to CR might in part mediate CR's antitumor effects. We tested CR under appetite suppression using two models: neuropeptide Y (NPY) knockout mice and monosodium glutamate-injected mice. While CR was protective in control mice challenged with a two-stage skin carcinogenesis model, papilloma development was neither delayed nor reduced by CR in the monosodium glutamate-treated and NPY knockout mice. Adiponectin levels were also not increased by CR in the appetite-suppressed mice. We propose that some of CR's beneficial effects cannot be separated from those imposed on appetite, and that NPY neurons in the arcuate nucleus of the hypothalamus are involved in the translation of reduced intake to downstream physiological and functional benefits., (No claim to original US government works. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

The societal impact of obesity, diabetes, and other metabolic disorders continues to rise despite increasing evidence of their negative long-term consequences on health span, longevity, and aging. Unfortunately, dietary management and exercise frequently fail as remedies, underscoring the need for the development of alternative interventions to successfully treat metabolic disorders and enhance life span and health span. Using calorie restriction (CR)-which is well known to improve both health and longevity in controlled studies-as their benchmark, gerontologists are coming closer to identifying dietary and pharmacological therapies that may be applicable to aging humans. This review covers some of the more promising interventions targeted to affect pathways implicated in the aging process as well as variations on classical CR that may be better suited to human adaptation.

Apyrimidinic/apurinic endonuclease (APE) is a key protein involved in the base-excision DNA repair (BER) pathway of oxidative DNA lesions. Using a novel oligonucleotide substrate, we demonstrate that APE activity in the frontal/parietal cortex (F/PCTX), cerebellum, brainstem, midbrain and hypothalamus declined with age in rats on an ad libitum (AL) diet. In contrast, APE activity for these brain regions was approximately 1.5-3 times higher in young, caloric restricted (CR) rats. Despite continuous CR treatment in all animals since six weeks of age, APE activity in the CR group started to decline by middle-age and continued into old age. However, CR maintained APE activity at a level that was significantly higher than that in AL rats across age and in the brain regions examined. Because Western analysis of APE, DNA polymerase beta and DNA ligase III levels in the F/PCTX of both CR and AL rats remained unchanged with age, this suggests that the increased APE activity in CR rats is the result of differential post-translational modification of APE., (Copyright 2009 Elsevier Inc. All rights reserved.)

Endothelial dysfunction, oxidative stress and inflammation are associated with vascular aging and promote the development of cardiovascular disease. Caloric restriction (CR) mitigates conditions associated with aging, but its effects on vascular dysfunction during aging remain poorly defined. To determine whether CR exerts vasoprotective effects in aging, aortas of ad libitum (AL) fed young and aged and CR-aged F344 rats were compared. Aging in AL-rats was associated with impaired acetylcholine-induced relaxation, vascular oxidative stress and increased NF-kappaB activity. Lifelong CR significantly improved endothelial function, attenuated vascular ROS production, inhibited NF-kappaB activity and down-regulated inflammatory genes. To elucidate the role of circulating factors in mediation of the vasoprotective effects of CR, we determined whether sera obtained from CR animals can confer anti-oxidant and anti-inflammatory effects in cultured coronary arterial endothelial cells (CAECs), mimicking the effects of CR. In CAECs cultured in the presence of AL serum TNFalpha elicited oxidative stress, NF-kappaB activation and inflammatory gene expression. By contrast, treatment of CAECs with CR serum attenuated TNFalpha-induced ROS generation and prevented NF-kappaB activation and induction of inflammatory genes. siRNA knockdown of SIRT1 mitigated the anti-oxidant and anti-inflammatory effects of CR serum. CR exerts anti-oxidant and anti-inflammatory vascular effects, which are likely mediated by circulating factors, in part, via a SIRT1-dependent pathway.

Laboratory studies consistently demonstrate extended lifespan in animals on calorie restriction (CR), where total caloric intake is reduced by 10-40% but adequate nutrition is otherwise maintained. CR has been further shown to delay the onset and severity of chronic diseases associated with aging such as cancer, and to extend the functional health span of important faculties like cognition. Less understood are the underlying mechanisms through which CR might act to induce such alterations. One theory postulates that CR's beneficial effects are intimately tied to the neuroendocrine response to low energy availability, of which the arcuate nucleus in the hypothalamus plays a pivotal role. Neuropeptide Y (NPY), a neurotransmitter in the front line of the arcuate response to low energy availability, is the primary hunger signal affected by CR and therefore may be a critical mechanism for lifespan extension.

Calorie restriction (CR) produces several health benefits and increases lifespan in many species. Studies suggest that alternate-day fasting (ADF) and exercise can also provide these benefits. Whether CR results in lifespan extension in humans is not known and a direct investigation is not feasible. However, phenotypes observed in CR animals when compared to ad libitum fed (AL) animals, including increased stress resistance and changes in protein expression, can be simulated in cells cultured with media supplemented with blood serum from CR and AL animals. Two pilot studies were undertaken to examine the effects of ADF and CR on indicators of health and longevity in humans. In this study, we used sera collected from those studies to culture human hepatoma cells and assessed the effects on growth, stress resistance and gene expression. Cells cultured in serum collected at the end of the dieting period were compared to cells cultured in serum collected at baseline (before the dieting period). Cells cultured in serum from ADF participants, showed a 20% increase in Sirt1 protein which correlated with reduced triglyceride levels. ADF serum also induced a 9% decrease in proliferation and a 25% increase in heat resistance. Cells cultured in serum from CR participants induced an increase in Sirt1 protein levels by 17% and a 30% increase in PGC-1alpha mRNA levels. This first in vitro study utilizing human serum to examine effects on markers of health and longevity in cultured cells resulted in increased stress resistance and an up-regulation of genes proposed to be indicators of increased longevity. The use of this in vitro technique may be helpful for predicting the potential of CR, ADF and other dietary manipulations to affect markers of longevity in humans.

A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging, including reduced albuminuria, decreased inflammation, and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at 12 months of age. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started midlife.