Mechanisms of Dietary Restriction in Aging and Disease (Interdisciplinary Topics in Gerontology)

Mechanisms of Dietary Restriction in Aging and Disease

Interdisciplinary Topics in Gerontology Vol. 35

Series Editors

Patrick R. Hof, New York, N.Y. Charles V. Mobbs, New York, N.Y.

Editorial Board

Constantin Bouras, Geneva Caleb E. Finch, Los Angeles, Calif. Kevin Flurkey, Bar Harbor, Me. Terry Fulmer, New York, N.Y. Jack Guralnik, Bethesda, Md. Jeffrey H. Kordower, Chicago, Ill. Bruce S. McEwen, New York, N.Y. Diane Meier, New York, N.Y. Jean-Pierre Michel, Geneva John H. Morrison, New York, N.Y. Mark Moss, Boston, Mass. Nancy Nichols, Melbourne S. Jay Olshansky, Chicago, Ill. James L. Roberts, San Antonio, Tex. Albert Siu, New York, N.Y. John Q. Trojanowski, Philadelphia, Pa.

Mechanisms of Dietary Restriction in Aging and Disease Volume Editors

Charles V. Mobbs, New York, N.Y. Kelvin Yen, New York, N.Y. Patrick R. Hof, New York, N.Y.

23 figures and 5 tables, 2007

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney

Charles V. Mobbs, PhD

Patrick R. Hof, MD

Department of Neuroscience Mount Sinai School of Medicine New York, N.Y., USA

Department of Neuroscience Mount Sinai School of Medicine New York, N.Y., USA

Kelvin Yen, BA Department of Neuroscience Mount Sinai School of Medicine New York, N.Y., USA Library of Congress Cataloging-in-Publication Data Mechanism of dietary restriction in aging and disease / volume editors, Charles V. Mobbs, Kelvin Yen, Patrick R. Hof. p. ; cm – (Interdisciplinary topics in gerontology, ISSN 0074-1132 ;v. 35) Includes bibliographical references and index. ISBN-13: 978-3-8055-8170-7 (hardcover : alk. paper) ISBN-10: 3-8055-8170-X (hardcover : alk. paper) 1. Nutrition disorders in old age. 2. Older people–Nutrition. I. Mobbs, Charles V. II. Yen, Kelvin. III. Hof, Patrick R. IV. Series. [DNLM: 1. Caloric Restriction. 2. Longevity–physiology. 3. Aging–physiology. W1 IN679 v.35 2007 / WT.116 M486 2007] RC620.6.M43 2007 618.97–dc22 2006022533

Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and MEDLINE/Index Medicus. Disclaimer. The statements, options and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2007 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 0074–1132 ISBN–10: 3–8055–8170–X ISBN–13: 978–3–8055–8170–7

Contents

VII Preface

1 The Role of Hormesis in Life Extension by Dietary Restriction Masoro, E.J. (San Antonio, Tex.) 18 Metabolic Reprogramming in Dietary Restriction Anderson, R.M.; Weindruch, R. (Madison, Wisc.) 39 Secrets of the lac Operon Glucose Hysteresis as a Mechanism in Dietary Restriction, Aging and Disease Mobbs, C.V.; Mastaitis, J.W.; Zhang, M.; Isoda, F.; Cheng, H.; Yen, K. (New York, N.Y.) 69 Effects of Dietary Restriction on the Expression of InsulinSignaling-Related Genes in Long-Lived Mutant Mice Bartke, A.; Masternak, M.M.; Al-Regaiey, K.A.; Bonkowski, M.S. (Springfield, Ill.) 83 Anti-Inflammatory Mechanisms of Dietary Restriction in Slowing Aging Processes Morgan, T.E.; Wong, A.M.; Finch, C.E. (Los Angeles, Calif.) 98 Dietary Restriction in the Nematode Caenorhabditis elegans Houthoofd, K. (Ghent/London); Gems, D. (London); Johnson, T.E. (Boulder, Colo.); Vanfleteren, J.R. (Ghent)

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115 Diet Restriction in Drosophila melanogaster Design and Analysis Tatar, M. (Providence, R.I.) 137 Dietary Restriction in Aging Nonhuman Primates Mattison, J.A.; Roth, G.S.; Lane, M.A.; Ingram, D.K. (Baltimore, Md.) 159 Caloric Intake and Alzheimer’s Disease Experimental Approaches and Therapeutic Implications Pasinetti, G.M.; Zhao, Z.; Qin, W.; Ho, L.; Shrishailam, Y.; MacGrogan, D.; Ressmann, W.; Humala, N.; Liu, X.; Romero, C.; Stetka, B.; Chen, L.; Ksiezak-Reding, H.; Wang, J. (New York, N.Y./Bronx, N.Y.) 176 Can Short-Term Dietary Restriction and Fasting Have a Long-Term Anticarcinogenic Effect? Klebanov, S. (New York, N.Y.)

193 Author Index 194 Subject Index

Contents

VI

Preface

This volume arose from a program announcement made by the National Institutes on Aging (NIA) requesting applications to study the basic mechanisms by which dietary restriction decreases disease burden and increases life span (RFA: AG–01–002, ‘Molecular and neural mechanisms underlying the effects of caloric restriction on health and longevity’). Most of the applicants who were funded had long been fascinated with the significance of this phenomenon, potentially the most far-reaching in biomedical science. A particularly valuable aspect of this program announcement was the requirement that funded applicants, along with a few other selected invitees, were to meet near the NIH on an annual basis to present and discuss their most recent results, published and unpublished. As these meetings progressed, it became clear that the field had experienced profound progress since the classic volume presenting a comprehensive view of the field, The Retardation of Aging and Disease by Dietary Restriction, by Richard Weindruch and Roy Walford, published in 1988, now sadly out of print. While it would be impossible for a multi-author volume to capture the coherence, tone and focus that made that classic so valuable to so many of us early in our careers, nevertheless it seemed as if some kind of review along those lines would be of some value. Therefore we contacted the participants in the NIA workshops, as well as many of the other leading authorities in the field, and many were gracious enough to accept our invitations to contribute reviews. We are particularly grateful to Dr. Masoro and Dr. Weindruch, who did so much to create this field, and Dr. Finch, who has

VII

contributed so much to so many areas of gerontology, for agreeing to contribute papers representing their current thoughts on the subject. Indeed, we very much appreciate the time and effort that all our contributors made, and hope that our readers benefit as much from reading the articles as we did editing them. Charles V. Mobbs, Kelvin Yen, Patrick R. Hof New York, N.Y.

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Mobbs CV, Yen K, Hof PR (eds): Mechanisms of Dietary Restriction in Aging and Disease. Interdiscipl Top Gerontol. Basel, Karger, 2007, vol 35, pp 1–17

The Role of Hormesis in Life Extension by Dietary Restriction Edward J. Masoro Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, Tex., USA

Abstract The level of food restriction that results in life extension and retarded aging in rodents also enhances their ability to cope with intense stressors. Moreover, this level of dietary restriction (DR) leads to a modest increase in the daily peak concentration of plasma free corticosterone, which strongly points to DR as a low-intensity stressor. These findings suggest that hormesis plays a role in the life-extending and anti-aging actions of DR. The evidence for and against this possibility is considered, and it is concluded that hormesis does have an important role. Copyright © 2007 S. Karger AG, Basel

It is some 70 years since McCay et al. [1] showed that long-term restriction of food intake markedly extends the life of rats. This finding has been confirmed many times in studies involving a variety of strains of rats and mice as well as a spectrum of other species, including hamsters, dogs, invertebrate animals and yeast [2]. The dietary factor responsible for the life extension of rats is a reduction of caloric intake rather than a specific nutrient [2]. The relevant studies have not been done to determine if this is also true of mice. One study indicates that in the case of Drosophila melanogaster, a decreased intake of protein or fat or both may underlie the life-prolonging action of food restriction [3]. The term dietary restriction (DR) will be used in this article when referring to life extension due to food restriction, since in some species, dietary factors other than reduction in caloric intake could be responsible for life extension. The mechanisms underlying the anti-aging and life-extending actions of DR remain to be defined, although many hypotheses have been proposed over the past 70 years. These include, but are not limited to, the effects of the following: retardation of growth; reduction of body fat content; reduction of

50 Cancer incidence per 1,000

45 40 35 30 25 20 15 10 5 0 0

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20 30 40 50 Concentration of carcinogen

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Fig. 1. Effects of a hypothetical carcinogen on cancer incidence. Low concentrations of the hypothetical carcinogen decrease and high doses increase cancer incidence, a phenomenon termed hormesis. The horizontal dashed line refers to no effect on cancer incidence.

metabolic rate; decreased body temperature; increased physical activity; enhancement of apoptosis; increased protein turnover; attenuation of oxidative stress; attenuation of glycation and glycoxidation, and attenuation of insulinIGF-1 signaling [2]. In 1998, Masoro [4] and Turturro et al. [5] independently proposed the hormesis hypothesis. This hypothesis, which may embrace many of the specific hypotheses just mentioned, will be discussed in detail in this article.

Concept of Hormesis

Hormesis refers to the phenomenon in which the response of an organism to a chemical or physical agent is qualitatively different when the agent is of high intensity than when it is of low intensity. An example would be a carcinogen that promotes the occurrence of cancer when administered at medium or high levels but protects the organism from cancer when used at low levels (fig. 1). Indeed, a variety of toxic chemical agents involving a spectrum of endpoints (growth, metabolic effects, reproduction, disease processes and longevity) have been shown to have hormetic actions at low concentrations in a wide range of taxonomic groups [6]. Ionizing radiation has also been claimed to exhibit hormesis, e.g. high doses of X-rays and gamma rays have been found to decrease and low doses to increase the life spans of mice, rats, houseflies, flour

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beetles, codling moths and house crickets [7, 8]. However, a recent National Research Council report claims that such is not the case for the human response to ionizing radiation [9]. Within the realm of toxicology, hormesis has come to be viewed as a beneficial phenomenon, i.e. there are many examples of low doses of toxic compounds resulting in protective or other favorable actions. Indeed, hormesis is now generally used to refer to the low-dose beneficial effects of otherwise harmful agents. Rattan [10] has proposed the following definition of hormesis as it relates to aging: hormesis in aging is characterized by the beneficial effects resulting from the cellular responses to mild repeated stress. That definition serves as the basis for this discourse.

Dietary Restriction – A Low-Intensity Stressor

In considering the possibility that hormesis plays an important role in DR’s anti-aging and life-extending actions, the first question that must be addressed is whether DR is a low-intensity stressor. It has long been known that stressors markedly elevate the plasma level of glucocorticoids in mammalian species, and the elevation of the plasma level of this class of steroid hormones is now generally considered to be a signature response to a stressor [11]. Sabatino et al. [12] compared the daily circadian rhythms of plasma glucocorticoid levels of ad libitum fed male rats with those of male rats on a DR regimen; they found that the daily afternoon peak plasma concentration of free corticosterone of the DR rats is moderately but significantly greater throughout life than that of ad libitum fed rats. Armario et al. [13] reported that the concentration of plasma total corticosterone is elevated in young male rats on a DR regimen for 34 days. Stewart et al. [14] found that DR elevated the plasma total corticosterone concentration in 5-month-old rats but not in 24-month-old rats. In the study of Sabatino et al. [12], plasma total corticosterone was also found to be elevated only in young rats, while plasma free corticosterone exhibits a modest daily elevation throughout life. The reason for the apparent discrepancy is the decrease in the level of plasma corticosteroid-binding globulin with increasing age in the rats on a DR regimen, resulting in an elevation of free corticosterone in the absence of an increase in total corticosterone. That the plasma free corticosterone remains elevated is important because it is not the total concentration but rather the free corticosterone concentration that is believed responsible for the physiological actions of this hormone [15]. Indeed, Han et al. [16] reported that as the rat ages, there are changes in the mechanism underlying the increase in the level of plasma free corticosterone induced by DR. The fact that two or more mechanisms are employed to sustain the life-long DR-induced daily

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elevation of the peak level of plasma free corticosterone is indicative of its importance. In summary, the evidence is convincing that long-term DR in rats causes the animal to experience daily periods of mild stress throughout life. Although not as thoroughly studied, it appears that such is also the case for mice [17]. Thus, the answer is yes, long-term DR is a sustained low-intensity stressor.

Dietary Restriction and Coping with Acute Intense Stressors

Rattan [18] proposed that the concept of hormesis as an aging retardant is based on the principle that repeated exposure to mild stress stimulates maintenance and repair processes. In their consideration of aging, from the viewpoint of far from equilibrium thermodynamics, Toussaint et al. [19] have come to a similar conclusion; they state that conditions of moderate stress that enhance the efficiency of the stress response are known as hormesis. Assuming that DR has a hormetic action, it should enhance the ability of the organism to cope with intense stressors. There is much evidence that DR, indeed, has this action. In both young and old rats, DR attenuates the acute loss of body weight due to surgical stress [4]. It also reduces the inflammatory response in young mice following the injection of an inflammatory substance into their footpad [17], and it increases the ability of rats to survive a sudden marked increase in environmental temperature [20]. DR protects rodents from the damaging action of a spectrum of toxic chemicals [21]. Testing the effects of four potentially toxic drug candidates on Sprague-Dawley rats, Keenan et al. [22] found that DR increased the maximum tolerated dose of each. Berg et al. [23] reported that following the administration of ganciclovir sodium, B6C3F1 mice on a DR regimen had a markedly reduced mortality, compared to those fed ad libitum. Thus, DR meets the classic criteria of hormesis. A marked reduction in food intake is clearly harmful to the point of being lethal, while a long-term moderate reduction in food intake enables the organisms to more successfully cope with damaging environments and toxic agents.

Dietary Restriction, Hormesis and Aging Processes

So DR does have a hormetic action. How is that relevant to aging? Aging, defined in terms of senescence, is characterized by a progressive deterioration of function throughout most of the adult life of an organism. Most biological gerontologists believe that this functional deterioration is caused by the accumulation of molecular damage from both endogenous (e.g. the metabolic

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generation of reactive oxygen molecules) and exogenous (e.g. damage due to toxic chemicals) sources, which is not successfully prevented by the organism’s protective and repair processes. Kirkwood’s Disposable Soma of Aging Theory [24] poses that less energy is used for somatic maintenance than is needed for indefinite survival. Indeed, it is likely that the rate of aging is determined by the extent of imbalance in favor of damaging processes over repair and protective processes. Thus DR, acting as a moderate stressor, protects the organisms from acute, intense stressors, which suggests that it may also protect against the longterm damage that underlies aging. Moreover, it is likely that DR does so, in part, by augmenting protective and repair processes, i.e. Rattan’s view [18] of the role of hormesis in the retardation of aging. Indeed, there is evidence that moderate stressors, other than DR, extend life. In 1958, Maynard Smith [25] reported that a transient heat stressor increased the life span of female fruit flies; Khazaeli et al. [26] confirmed that finding and, in addition, showed that it occurs in males as well. Shama et al. [27] observed that a transient mild heat stressor extends the replicative life span of Saccharomyces cerevisiae. Lithgow et al. [28] and Yashin et al. [29] reported a similar effect of transient, mild heat stressors on the longevity of Caenorhabditis elegans; this, they propose, is because of an increased adaptation of the organism resulting from the mild heat stress stimulus. Indeed, Cypser and Johnson [30] found that several different low-intensity stressors extend the life of C. elegans. Moreover, upon a review of the literature, Johnson et al. [31] concluded that it is not uncommon for longevity to be increased in organisms exposed to moderately stressful conditions. The study of Kapahi et al. [32] also links longevity to the ability to cope with a damaging environment; in their study on culture of fibroblasts derived from eight mammalian species, resistance to damaging agents correlated positively with the life span of the species. There is concern that unlike the modest effects of other low-intensity stressors, DR markedly extends life. It has been suggested that this quantitative difference tends to negate a role for hormesis in the life-extending action of DR. In my opinion, this view is illusory. For example, although DR from 6 weeks of age on increased the median length of life of male rats by 51%, those on this dietary regime from 6 weeks to 6 months of age exhibited only a 15% increase [33]. In the report of Le Bourg et al. [34], exposing young D. melanogaster males for 14–24 days to a moderate hypergravity stressor frequently resulted in a 10–15% increase in mean length of life. Clearly, the quantitative difference between these two stressors is not great when each is applied for only a fraction of the life span. There is genetic evidence of a link between longevity and the ability to cope with intense stressors. Organisms with genetic manipulations that result in

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increased longevity usually, though not always, have an increased ability to cope with intense stressors [2, 35]. For example, single-gene mutations experimentally generated in C. elegans [36, 37], yeast [38] and D. melanogaster [39] have yielded organisms with this characteristic. Thus, DR extends life and enhances the ability to cope with intense stressors. Other moderate stressors extend life and increase the ability to cope with intense stressors. These findings suggest that hormesis plays a role in the lifeextending action of DR. What is missing is an understanding of the molecular, cellular and organismic processes involved in the hormesis-based life-extending actions of DR. There are, however, promising leads. The study of Anderson et al. [40] has provided a partial description of the DR-induced hormetic pathway responsible for the increase in the replicative life span of a PSY strain of S. cerevisiae. They found that both a functional PNC1 gene and Sir2 gene are required for DR to induce replicative life extension in this yeast strain. PNC1 encodes the PNC1 protein, a nicotinamidase, and DR was found to increase the amount of this enzyme. Sir2 encodes the SIR2 protein, which has deacetylase activity; a product of the deacetylase reaction is nicotinamide, an inhibitor of the SIR2 deacetylase activity. Thus, by increasing the level of the nicotinamidase protein, DR maintains a low level of nicotinamide in the cells of this yeast strain, thereby increasing SIR2 deacetylase activity. It is this increased deacetylase activity that plays a key role in the DR-induced replicative life extension. Moreover, Anderson et al. [40] found that the same pathway is involved in replicative life extension induced by two other low-intensity stressors, heat stress and osmotic stress. Thus, Anderson et al. have begun to delineate the hormetic pathway in this yeast strain that connects the stressor to an increase in SIR2 deacetylase activity. The pathway linking the increased deacetylase activity to an increase in replicative life span remains to be defined. However, the finding that DR can extend the replicative life span of yeast strains lacking the SIR2 protein has cast doubt on the general relevancy of the work of Anderson et al. Recently, Lamming et al. [41] reported that one yeast strain lacking SIR2 contains HST2, a sirtuin protein homologue of SIR2, and that this homologue is also a deacetylase and functions in the actions of DR in a fashion similar to SIR2. The question that arises is whether sirtuin proteins play a similar role in the action of DR in other species. Indeed, there is such evidence [42]. Rogina and Helfand [43] reported that SIR2 is directly involved in the DR-induced life extension of Drosophila. Also, Tissenbaum and Guarente [44] reported that increasing the dosage of the Sir2.1 gene, which codes for a sirtuin protein, extends the life of C. elegans and that this effect requires an active DAF-16 gene. Nevertheless, the role of sirtuin proteins in DR-induced life span extension in C. elegans is in doubt since Lakowski and Hekimi [45] found that mutation of the eat-2 gene extends the life of this species, presumably by

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decreasing food intake, and that the life extension does not require an active DAF-16 gene. Furthermore, several very recent findings have to be reconciled with the view that sirtuin proteins play a key role in the pathway linking low-intensity stressors to life extension. In contrast to its role in the replicative life span, Fabrizio et al. [46] reported that Sir2 decreases the chronological life of yeast rather than increasing it. Kaeberlein et al. [47] have found that mutations of Tor1 or Sch9, as well as other genes in their nutrient-sensing pathways, extend the replicative life of yeast independent of Sir2. However, it is not clear whether HST2 or some other sirtuin homologue of SIR2 is required. Tor and Sch9 mutations have also been found to extend the life of D. melanogaster [48] and C. elegans [49]. Stress response genes, which protect against cellular damage [50], are also likely to be part of the DR-induced hormetic pathway. For example, DR enhances the expression of heat shock proteins in rats exposed to damaging agents [19, 51–55]. Moreover, several studies, which do not involve DR, strongly implicate stress response gene expression in life extension. Tatar et al. [56] studied a transgenic strain of D. melanogaster with an extended life span and found that this strain exhibits an enhanced expression of stress response genes. Garigan et al. [57] presented evidence that heat shock proteins slow the rate of aging of C. elegans. Indeed, several studies show that stress response proteins and the transcription factor promoting their expression have an antiaging and life-extending action in this species of worm. Hsu et al. [58] reported a regulatory relationship between heat shock transcription factor and the aging of C. elegans, and Yokoyama et al. [59] found that the constitutive overexpression of hsp70F extended the life of this organism. Morley and Morimoto [60] expanded our understanding by showing that HSF-1, a transcriptional factor that regulates stress-inducible gene expression, enhances the ability of C. elegans to cope with intense stressors and, in addition, extends the life of these worms. Significantly, Walker and Lithgow [61] found that insulin-like signaling plays a regulatory role in the life-extending action of stress response genes in C. elegans. Thus, there is strong evidence to support the view that an enhancement of the expression of stress response genes is a component of the hormetic pathway by which DR retards aging processes and extends life. An enhancement of repair processes is also likely to be a component of DR-induced hormesis. Indeed, DR has been found to retard the age-associated decrease in the ability of rats to repair the transcribed strands of DNA [62, 63]. Also, DR increases the rate of whole-body protein turnover in adult and old rats [64, 65]. These actions should slow the accumulation with increasing age of damaged DNA and protein molecules; indeed, that has been found to be the case for both rats and mice [66–72].

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In addition to its effects at a cellular level, DR also has potential hormetic actions at the organismic level. As mentioned above, DR causes a lifetime elevation of the daily peak concentration of plasma free corticosterone in rats [12], the major glucocorticoid in this species. The hypothalamic-hypophysealadrenal cortical glucocorticoid system plays a key role in enabling mammals to cope with damage [11]. Thus, the hormetic pathway for the DR-induced antiaging activity may well involve the daily elevation in the level of plasma free glucocorticoid, which, in turn, retards aging by its effects on target cells. Indeed, Leakey et al. [73] proposed that increased levels of glucocorticoids contribute to the anti-aging actions of DR. The work of Pashko and Schwartz [74] on carcinogenesis provides experimental support for this proposal. They found that DR’s ability to protect mice against chemically induced tumors is lost if the animals have been adrenalectomized. Schwartz and Pashko [75] hypothesized that elevated levels of adrenal steroids underlie the ability of DR to inhibit both carcinogen-induced tumors and those that occur spontaneously with advancing age. Birt et al. [76] have also found that the ability of DR to inhibit skin carcinogenesis in mice requires elevated levels of glucocorticoids.

Analyses of Findings Opposing the Hormesis Hypothesis

Up to this point, the focus has been primarily on findings that support the hormesis hypothesis. In this section, an assessment is made of findings that question its validity. Neafsey [77] compared the lifetime age-specific mortality pattern in the hormesis-based increase in longevity induced by DR with that induced by low levels of methylene chloride or gamma radiation. Because the pattern in the DR studies that she assessed differed from that of the other two stressors, Neafsey concluded that hormesis does not underlie the life-extending actions of DR. However, it has recently become clear that the lifetime age-specific mortality pattern in response to DR exhibits interspecies as well as intraspecies variation [69, 78–80]. Moreover, the classic interpretation of agespecific mortality analyses has recently been questioned [81] and needs to be reevaluated [82]. Thus, the basis of Neafsey’s challenge [77] is currently open to question. Although DR enhances the ability of organisms to cope with several intense stressors, it does not do so for all of them. Indeed, animals on a DR regimen cope less effectively with some intense stressors. Of course, such occurrences do not lessen the fact that DR has beneficial actions in response to many stressors. Even in the case of those stressors where organisms fare less well, other effects of DR may mask the beneficial hormetic action.

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The healing of skin wounds appears to be a case in point. It is known that DR adversely affects the healing of such wounds in mice and rats [83, 84]. Reed et al. [85] investigated the effect of age on the healing of such wounds in ad libitum fed mice and found, as have others, that this ability deteriorates with increasing age. However, if mice that had been on DR until advanced ages were fed ad libitum starting 4 weeks prior to wounding, their wounds healed as rapidly as those of young mice. Wound healing requires the expenditure energy for the biosynthesis of collagen and other matrix molecules and for augmented cell proliferation; it appears that the reduced energy intake by the mouse on DR masks its beneficial effect on wound healing. The effect of DR on the response of rats to cold stress may be another example. Campbell and Richardson [86] reported that rats on DR are less able to cope with cold stress. It is well known that an increase in metabolic heat production is the major way that small mammals like rats cope with cold stress. It is likely that this is another case where the reduced energy intake and storage mask a beneficial hormetic action of DR. A study, such as that done by Reed et al. [85] on wound healing, has yet to be done in regard to cold stress. The findings are mixed regarding the effect of DR on coping with the challenge of infectious agents. In 1975, Gerbase-Delima et al. [87] reported that in young C57BL/6 mice, DR decreases the in vitro proliferative response of their splenic lymphocytes to mitogens; in contrast, it enhances the in vitro proliferative responses of splenic lymphocytes from middle-aged and old mice. It was concluded that DR initiated in young mice slows the maturation of the immune system, but enhances its function once maturation is achieved. Subsequent studies using a variety of mouse and rat strains have yielded similar results as well as other indicators of immune function enhancement. See Pahlavani [88] for a review of these many findings. However, other studies have yielded conflicting results. Roecker et al. [89] found that adult rhesus monkeys on DR for 2–4 years exhibit a reduction in the mitogen-induced proliferative response of peripheral blood mononuclear cells. Weindruch et al. [90] studied the immune function of rhesus monkeys that had been on DR for 7 years starting at 1 year of age or 3–5 years of age; they found that DR decreased the peripheral blood mononuclear cell proliferative response in the younger but not the older group, and that both groups exhibited lymphopenia. Sun et al. [91] tested 6-month-old C57BL/6 mice for their response to polymicrobial sepsis induced by cecal ligation and puncture; they found that mice on DR for 5 months died earlier than those fed ad libitum. This may have been due to DR’s effect on the maturation of the immune system since DR was started at 1 month of age in these mice. The findings on the effect of DR on the response of animal models to influenza are also conflicting. Effros et al. [92] reported that long-term DR enhances the immune response of mice to influenza vaccination, as evidenced by increased

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antigen-specific lymphoproliferation, antigen presentation, antibody production and T-cell function. In contrast, Roecker et al. [89] found that adult rhesus monkeys on DR for 2–4 years exhibit a reduced plasma antibody response to influenza vaccine. Recently, Gardner [93] has reported that long-term DR decreased the survival of mice after primary influenza infection. Further research is clearly needed to understand the basis of these conflicting findings. The impressive studies of Sapolsky [94], showing that glucocorticoids can adversely affect the aging of the nervous system, have led to some doubt that the increased daily elevation in the level of plasma free glucocorticoid in DR rodents plays a role in the life extension. However, this concern is countered by the fact that adrenalectomy results in a loss in the ability of DR to retard carcinogenesis in mice [74]. Indeed, it seems likely that a level of glucocorticoid, either too low or too high, adversely affects longevity, and that the level in DR rodents is nearly optimal. Further detailed studies are needed to fully define the relationship between long-term glucocorticoid levels and longevity.

Conclusions

Although the currently available database strongly supports the concept that hormesis plays an important role in the life-extending and anti-aging actions of DR, it is not a view held by most biological gerontologists. There are several possible reasons for this disconnection between the database and the skepticism of its critics. Calebrese [95] points out that biologists in general have long discounted the importance or even the existence of hormesis. Thus, part of the negative attitude of biological gerontologists may stem from biologists’ disregard for hormesis. Calabrese feels that this attitude relates to several factors. First, in many cases, the hormetic effect of chemical agents occurs at extremely low doses, and it is thus often missed in dose-response studies. Second, the hormetic effect is often small, leading one to doubt that it is real. Third, hormesis presents difficulties for those in regulatory agencies charged with determining safe limits for toxic agents, which they prefer to avoid. Fourth, hormesis is often confused with homeopathy, an approach to medicine that has been in disrepute for some time. Biological gerontologists have encountered similar problems when they attempt to explore hormesis. For example, Michalski et al. [96] found that if 3-day-old C. elegans undergo heat stress for less than 2 h, their longevity is increased; heating for one half-hour had the greatest effect. However, the magnitude of the effect decreased with longer heating periods and disappeared when the heating period exceeded 2 h. Another example is the D. melanogaster heat stress study of Le Bourg et al. [97]. They found that heating young flies for a 5-min

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period for 5 consecutive days increased longevity, but heating them for 10, 20, 30 min or longer had either no effect or decreased longevity. It is clear that in such studies, hormesis can easily be missed or interpreted as noise rather than real. However, in my opinion, the major reason that hormesis is discounted relates to the fact that stressors often cause damage and accelerate aging. Indeed, McEwen [98] has promoted the concept of allostatic load, which he defines as the cumulative physiological toll over time by the organism’s efforts to adapt to stressors. Certainly allostatic load is an important gerontological concept, but it should not overshadow the fact that stressors can also have beneficial actions. Clearly, whether chronic stressors are detrimental or beneficial depends on the nature of the stressor and its intensity. Returning to DR, it is clear that a significant reduction in food intake is a beneficial stressor. Also, the hormetic component of this chronic stressor is not likely to be missed, because it has beneficial actions over such a wide range of restrictions, with benefits documented for levels of food restriction ranging from 10 to 50% of the ad libitum intake [2]. Moreover, by focusing on the retardation of the accumulation of molecular and cellular damage, the hormesis hypothesis unifies many of the other hypotheses proposed to explain the antiaging and life-extending actions of DR. For example, in the attenuation of oxidative stress hypothesis, the retardation of oxidative damage to cellular structures is viewed as the basis of the anti-aging actions of DR; the hormesis hypothesis encompasses such protection. In the attenuation of the glycation and glycoxidation hypothesis, the focus is on the damage caused by non-enzymatic glycation and glycoxidation; again, the hormesis hypothesis encompasses protection against such damage. However, it should be pointed out that although hormesis is an important component of the anti-aging actions of DR, it is not likely to be the only one. Hormesis can explain all the protective components of DR, but in addition, it is likely that DR has a different general action, namely that of decreasing the generation of damaging agents. For example, in regard to the attenuation of oxidative stress hypothesis, there is evidence that DR decreases the generation of harmful reactive oxygen molecular species; this action should decrease the formation of oxidatively damaged cellular macromolecules [99]. However, Merry [100] points out that a caveat is in order since the effect of DR on the generation of reactive molecules has been found to occur in in vitro preparations such as isolated mitochondria, but it has yet to be shown in the intact organism. Another example relates to the attenuation of glycation and glycoxidation hypothesis; it has been shown that DR causes the blood glucose level to be significantly less throughout the lifetime of rats on DR compared to those fed ad libitum [101]. And this reduction in glycemia, which should result in a decreased glycation and glycoxidation of macromolecules, is also not likely to have a hormetic link.

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Ideally the hormesis hypothesis of DR action should be tested by studies that can falsify it. Given the current state of knowledge, such experiments are difficult, if not impossible, to design for the following reasons. First, hormesis is only one of the two general mechanisms proposed for DR action, which makes interpretation of findings equivocal. Second, more than one hormetic pathway is likely to be involved in DR’s actions, and the number and nature of such pathways have yet to be defined. The study of Pashko and Schwartz [74] illustrates both of these problems. As mentioned above, these investigators tested the role of glucocorticoids in DR’s protection against cancer in mice (a major disease affecting longevity of this species), and they found that adrenalectomy abolishes this protective effect. This finding is in accordance with a role of elevated plasma glucocorticoid levels in DR’s anticancer action, which provides some support for the hormesis hypothesis. However, if adrenalectomy had not abolished the anticancer action of DR, that finding would not have falsified the hormesis hypothesis for two reasons: hormesis is not likely the only process underlying the life-extending action of DR and the pathway involving glucocorticoids is probably not the only hormetic pathway involved. Both of these problems require further research before meaningful studies can be designed that can falsify the hormesis hypothesis. It may be possible to address the first problem through analyses of the effect of DR on the lifetime characteristics of agespecific mortality (see Masoro [82] for a discussion of this possibility). The second problem – i.e. the hormesis pathway(s) involved – should initially be explored in young animals, e.g. the pathway(s) by which hormesis modulates the inflammatory response can be readily investigated in young animals. Armed with this information, the lengthy process of determining the pathway(s) underlying life extension can then be more efficiently explored. In summary, current evidence suggests that DR retards aging and extends life by two general processes. The first is the reduction in the generation of damaging agents. The second is the enhancement of protective and repair processes, and hormesis is the basis of this enhancement. The relative importance of DR’s two general processes probably depends on both genetic and environmental factors and their interaction. Indeed, there are interspecies and intraspecies differences in age-specific mortality characteristics underlying the life-extending action of DR [82] and this strongly indicates the involvement of more than one general process. References 1 2

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Pipkin JL, Hinson WG, Feuers RJ, Lyn-Cook LE, Burns ER, Duffy PH, Hart R, Casciano D: The temporal relationship of synthesis and phosporylation in stress proteins 70 and 90 in aged caloric restricted rats exposed to bleomycin. Aging Clin Exp Res 1994;6:125–132. Heydari AR, Conrad CC, Richardson A: Expression of heat shock genes in hepatocytes is affected by age and diet at the level of transcription. J Nutr 1995;125:410–418. Moore SA, Lopez A, Richardson A, Pahlavani MS: Effect of age and dietary restriction on the expression of heat shock protein 70 in rat alveolar macrophages. Mech Ageing Dev 1998;104:59–73. Heydari A, You S, Takahashi R, Gutsmann A, Sarge KD, Richardson A: Effect of dietary restriction on the expression of heat shock protein 70 and the activation of heat shock. Dev Genet 1996;18:114–124. Tatar M, Khazaeli AA, Curtsinger JW: Chaperoning extended life. Nature 1997;390:30. Garigan D, Hsu A-L, Fraser AG, Kamath J, Kenyon C: Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation. Genetics 2002;161:1101–1112. Hsu AL, Murphy CT, Kenyon C: Regulation of aging and age-related disease by DAF-16 and heatshock factor. Science 2003;300:1142–1145. Yokoyama K, Fukumoto K, Murakami T, Harada S, Hosono R, Wadhwa R, Mitsui Y, Ohkuma S: Extended longevity of Caenorhabditis elegans by knocking in extra copies of hsp70F, a homolog of mot-2 (mortalin)/mthsp70/Grp 75. FEBS Lett 2002;516:53–57. Morley JF, Morimoto RI: Regulation of longevity in Caenorhabditis elegans by heat shock factor and molecular chaperones. Mol Biol Cell 2004;15:657–664. Walker GA, Lithgow GJ: Lifespan extension of C. elegans by a molecular chaperone dependent upon insulin-like signals. Aging Cell 2003;2:131–140. Guo ZM, Heydari A, Richardson A: Nucleotide excision repair of actively transcribed versus nontranscribed DNA in rat hepatocytes: effect of age and dietary restriction. Exp Cell Res 1998;245:228–238. Guo ZM, Van Remmen H, Wu WT, Richardson A: Effect of cAMP-induced transcription on the repair of the phosphoenolpyruvate carboxykinase gene by hepatocytes isolated from young and old rats. Mutat Res 1998;409:37–48. Lewis SE, Goldspink DF, Phillips JG, Merry BJ, Holehan AM: The effects of aging and chronic dietary restriction on whole body growth and protein turnover in the rat. Exp Gerontol 1985;20:253–263. Goto S, Takahashi R, Araki S, Nakamoto H: Dietary restriction initiated in late adulthood can reverse age-related alterations of protein and protein metabolism. Ann NY Acad Sci 2002;959:50–56. Youngman LD, Park JY, Ames BN: Protein oxidation associated with aging is reduced by dietary restriction of protein or calories. Proc Natl Acad Sci USA 1991;89:9112–9116. Chen LH, Snyder DL: Effect of dietary restriction and germ-free environment on glutathionerelated enzymes in Lobund-Wistar rats. Arch Gerontol Geriatr 1992;14:17–26. Chung MH, Kin HJ, Nishimura S, Yu BP: Protection of DNA damage by dietary restriction. Free Radic Biol Med 1992;12:523–525. Sohal RS, Agarwal S, Candas M, Forster M, Lal H: Effect of age and dietary restriction on DNA oxidative damage in different tissues of C57BL/6 mice. Mech Ageing Dev 1994;76: 215–224. Sohal RS, Ku HH, Agarwal S, Forster MJ, Lal H: Oxidative damage, mitochondrial oxidant generation, and antioxidant defenses during aging and in response to food restriction in the mouse. Mech Ageing Dev 1994;74:121–133. Dubey A, Forster MJ, Lal H, Sohal RS: Effect of age and caloric intake on protein oxidation in different brain regions and on behavioral functions of the mouse. Arch Biochem Biophys 1996;333: 189–197. Aksenova MV, Aksenov MY, Carney JM, Rutterfield DA: Protein oxidation and enzyme activity decline in old Brown Norway rats are reduced by dietary restriction. Mech Ageing Dev 1998;100:157–168.

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Leakey JE, Chen S, Manjgaladze M, Turturro A, Duffy PH, Pipkin JL, Hart RW: Role of glucocorticoids and ‘caloric stress’ in modulating the effects of dietary restriction in rodents. Ann NY Acad Sci 1994;719:171–194. Pashko LL, Schwartz AG: Reversal of food restriction induced inhibition of mouse skin tumor promotion by adrenalectomy. Carcinogenesis 1992;10:1925–1928. Schwartz AG, Pashko LL: Role of adrenocortical steroids in mediating cancer-prevention and ageretarding effects of food restriction in laboratory rodents. J Gerontol Biol Sci 1994;49:B37–B41. Birt DF, Yatkins A, Duysen E: Glucocorticoid mediation of dietary energy restriction inhibition of mouse skin carcinogenesis. J Nutr 1999;129(suppl):571S–586S. Neafsey PJ: Longevity hormesis: a review. Mech Ageing Dev 1990;51:1–31. Pletcher SD, Khaezaeli AA, Curtsinger JA, Why do life spans differ? Partitioning mean longevity differences in terms of age-specific mortality parameters. J Gerontol Biol Sci 2000;55A: B381–B389. Pugh TD, Oberly TD, Weindruch R: Caloric restriction but not dehydroepiandrosterone sulfate increase lifespan and lifetime cancer incidence in mice. Cancer Res 1999;59:1642–1648. Mair W, Goymer P, Pletcher SD, Partridge L: Demography of dietary restriction and death in Drosophila. Science 2003;301:1731–1733. Driver C: A further comment on why the gompertzian plot does not measure aging. Biogerontology 2003;4:325–327. Masoro EJ: Caloric restriction and aging: controversial issues. J Gerontol A Biol Sci Med Sci 2006;61:14–19. Harrison DE, Archer JR: Effects of food restriction on aging mice. J Nutr 1987;117:376–382. Reiser K, McGee C, Rucker R, McDonald R: Effects of aging and dietary restriction on extracellular matrix biosynthesis in a model of injury repair in rats. J Gerontol Biol Sci 1995;50A: B40–B47. Reed MJ, Penn PE, Li Y, Birnbaum R, Vernon RB, Johnson TS, Pendergrass WR, Sage EH, Abrass IB, Wolf NS: Enhanced cell proliferation and biosynthesis mediate improved wound repair in refed, calorie-restricted mice. Mech Ageing Dev 1996;89:21–41. Campbell BA, Richardson R: Effect of chronic undernutrition on susceptibility to cold stress in young adult and aged rats. Mech Ageing Dev 1988;44:193–202. Gerbase-Delima M, Liu RK, Cheney KE, Mickey R, Walford RL: Immune function and survival in the long-lived mouse strain subjected to undernutrition. Gerontologia 1975;21:184–193. Pahlavani MA: Intervention in the aging of the immune system: influence of dietary restriction, dehydroepiandrosterone, melatonin, and exercise. Age 1998;21:153–173. Roecker EB, Kemnitz JW, Ershler WB, Weindruch R: Reduced immune responses in rhesus monkeys subjected to dietary restriction. J Gerontol Biol Sci 1996;51A:B276–B279. Weindruch R, Lane MA, Ingram DK, Ershler WB, Roth GS: Dietary restriction in rhesus monkeys: lymphopenia and reduced mitogen-induced proliferation in peripheral blood mononuclear cells. Aging Clin Exp Res 1997;9:304–308. Sun D, Muthukumar AR, Lawrence RA, Fernandes G: Effects of calorie restriction on polymicrobial peritonitis induced by cecum ligation and puncture in young C57BL/6 mice. Clin Diag Lab Immunol 2001;8:1003–1008. Effros RB, Walford RL, Weindruch R, Mitcheltree C: Influence of dietary restriction on immunity to influenza in aged mice. J Gerontol Biol Sci 1991;46:B142–B147. Gardner EM: Caloric restriction decreases survival of aged mice in response to primary influenza infection. J Gerontol Biol Sci 2005;60A:688–694. Sapolsky RM: Glucocorticoids, stress, and their adverse neurological effects: relevance to aging. Exp Gerontol 1999;35:721–732. Calebrese EJ: Changing view of dose-response, a personal account of the history and current status. Mutat Res 2002;511:181–189. Michalski AI, Johnson TE, Cypser JR, Yoshin AI: Heating stress patterns in Caenorhabditis elegans longevity and survivorship. Biogerontology 2001;2:35–44. Le Bourg E, Valenti P, Lucchetta P, Payre F: Effects of mild heat shock at young age on aging and longevity of Drosophila melanogaster. Biogerontology 2001;2:155–164.

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98 McEwen BS: Protective and damaging effects of stress mediators. N Engl J Med 1998;338: 171–179. 99 Gredilla P, Sanz A, Lopez-Torres M, Barja G: Caloric restriction decreases mitochondial free radical generation at complex 1 and lowers oxidative damage to mitochondrial DNA. FASEB J 2001;15:1589–1591. 100 Merry BJ: Oxidative stress and mitochondrial function with aging – The effect of calorie restriction. Aging Cell 2004;3:7–12. 101 Masoro EJ, McCarter RJM, Katz MS, McMahan CA: Dietary restriction alters the characteristics of glucose fuel use. J Gerontol Biol Sci 1992;47:B202–B208.

Edward J. Masoro, PhD Professor emeritus of Physiology 211/2 Legare Street Charleston, SC 29401 (USA) Tel. ⫹1 843 853 3445, E-Mail [email protected]

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Mobbs CV, Yen K, Hof PR (eds): Mechanisms of Dietary Restriction in Aging and Disease. Interdiscipl Top Gerontol. Basel, Karger, 2007, vol 35, pp 18–38

Metabolic Reprogramming in Dietary Restriction Rozalyn M. Anderson, Richard Weindruch Department of Medicine, University of Wisconsin, Madison, Wisc., USA

Abstract It is widely accepted that energy intake restriction without essential nutrient deficiency delays the onset of aging and extends life span. The mechanism underlying this phenomenon is still unknown though a number of different, nonmutually exclusive explanations have been proposed. In each of these, different facets of physiology play the more significant role in the mechanism of aging retardation. Some examples include the altered lipid composition model, the immune response model and models describing changes in endocrine function. In this paper we propose the hypothesis that metabolic reprogramming is the key event in the mechanism of dietary restriction, and the physiological effects at the cellular, tissue and organismal level may be understood in terms of this initial event. Copyright © 2007 S. Karger AG, Basel

Dietary restriction (DR) is the most successful intervention tested to date in mammals which greatly extends maximum life span and keeps animals ‘younger longer’ [1–3]. Consequently, any hypothesis about the etiology of aging must reconcile the effects of DR on aging. With increased knowledge of the mechanism of DR, we stand to gain a considerable insight into the process of aging. We propose that a change in the regulation of energy metabolism in response to DR is the primary step in the retardation of aging (fig. 1). First we describe the evidence in support of metabolic reprogramming, a switch to an altered metabolic state, by DR in mice. Next we consider evidence for metabolic shifts in other model organisms where life span is extended by DR or by genetic manipulation. Then we focus on changes in mitochondrial energy metabolism with age and DR in mammals. Next we will explore the effects of altered mitochondrial function in the context of reactive oxygen species (ROS) generation and oxidative stress. Finally we describe the metabolic and morphological

Control

DR

Master regulators

Normal metabolism

Altered energy metabolism

Effectors Normal aging cellular damage dysfunction and loss

Morbidity and mortality

Reduced rate of aging

Longevity

Fig. 1. Master regulators respond to DR and induce shifts in metabolism in the restricted organism. These regulators may include the transcriptional coactivator PGC-1␣ and members of the nuclear receptor family PPAR-␣ and PPAR-␥. Effectors that respond to the altered metabolic state are involved in tissue-specific changes that ultimately lead to changes at the organismal level, delaying aging and promoting longevity.

changes in white adipose tissue that we believe are a result of altered mitochondrial function. We propose that the activation of adipose tissue through metabolic reprogramming is critical to the mechanism of DR and that it leads to the changes in the animal physiology that are described in the models indicated above.

Metabolic Reprogramming in Tissues from Dietary-Restricted Animals

The inverse linear relationship between calorie intake and life span in mice [4] suggests that genes central to energy metabolism may be critical in the underlying mechanism of DR in mammals. We have examined transcriptional changes with age and with DR in multiple tissues and find shifts in the expression of genes encoding proteins involved in energy metabolism to be a prominent feature of DR. We propose that these shifts directly contribute to the longevity of the animal. Our studies have primarily focused on postmitotic tissues because these are most vulnerable to the effects of age. Analysis of the

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transcriptional response to DR in these tissues is likely to reveal clues to the mechanism of aging retardation. In skeletal muscle, a decrease in metabolic activity with age is suggested through a reduction in the expression of genes involved in energy metabolism [5]. This extends to genes associated with mitochondrial function as well as genes involved in glycolysis and glycogen synthesis, and large reductions in expression were also observed for genes involved in fatty acid biosynthesis. We observed a striking attenuation of these age-related changes in gene expression in skeletal muscle from DR animals compared to age-matched controls. In particular, we observed a transcriptional shift toward increased energy metabolism and increased biosynthesis. The expression of genes involved in glycolysis and gluconeogenesis was increased, as was the expression of transcripts associated with fatty acid metabolism. The increased expression of peroxisome proliferatoractivated receptor ␥ (PPAR-␥) may contribute to the increased insulin sensitivity in skeletal muscle of the DR animals [6]. In the heart, lipid metabolism and fatty acid oxidation (FAO) are the major energy source in adults [7]. In old age, genes involved in lipid transport, lipolysis and FAO are downregulated and genes involved in carbohydrate metabolism are upregulated, resulting in an overall shift in metabolism [8]. These metabolic alterations, which are also observed in pathological heart conditions [9], are completely or partially prevented by DR [8]. In addition, we observed a significant change with DR in the expression of key genes that are not affected with age. It is important to emphasize that this latter type of DR effect is distinct from the prevention of age-associated transcriptional changes. Genes that shift in expression with DR but do not change with age may provide clues to the mechanism of aging retardation by DR and may lead to the identification of primary regulators. In the heart, this group includes many nuclear genes encoding components of the electron transport system (ETS) that show a striking and coordinated upregulation with DR [unpubl. data]. In our earlier experiments we sought to characterize the tissue-specific transcriptional changes with age and examine the effect of DR. We identified two groups of genes that are regulated by DR: one group is regulated by age at the transcriptional level and DR either partially or completely compensates for the age-induced changes; the other group is regulated specifically by DR and does not show age-dependent changes in gene expression. Clearly the genes that are regulated specifically by DR and not affected with age are reasonable candidates in the mechanism of life span extension. The potential involvement of genes that are regulated by age in the mechanism of DR is less easily deduced. In analyzing these data it became clear that in order to dissect out the DR-specific transcriptional changes we would be better served looking at young to mid-age mice where the age-related changes in transcription would be

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less significant. Specifically, we examined the transcriptional changes with fasting, short-term DR (23 days) or long-term DR (9 months) in epididymal white adipose tissue from 10-month-old mice [10]. Here again we identified metabolic reprogramming as a prominent feature. White adipose tissue is remarkably refractory to both fasting and short-term DR but undergoes a dramatic transformation in response to long-term DR. This is in contrast to similar experiments in the liver where many of the DR-induced changes were observed with short-term DR [11]. In white adipose tissue, long-term DR increases the expression of genes involved in the glycolytic pathway, the lipolytic pathway, amino acid metabolism and mitochondrial energy metabolism in young mice [10] suggesting an activation of energy metabolism. Again, these shifts in gene expression are not compensatory in the delay of aging and may be involved in the mechanism of aging retardation. One of the more striking findings was the concerted increase in expression of 26 nuclear genes encoding mitochondrial ETS proteins. We also identified a dramatic decrease in the expression of genes encoding inflammatory molecules (56 genes); these alterations may play an important role in the protection against inflammation derived from white adipose tissue and in life span extension by DR [12]. The transcriptional shifts observed in each of these tissues are indicative of metabolic reprogramming which we believe is a key component of the mechanism of aging retardation by DR (fig. 1). The coordinated increase in expression of genes encoding components of mitochondrial ETS in both heart and adipose tissue is striking. Several aging studies in yeast, worms and flies support a role for metabolic regulation in longevity. We discuss the evidence below, with a particular focus on mitochondrial energy metabolism. Studies in transgenic and wild-type mice further support our hypothesis that shifts in energy metabolism can affect a broad spectrum of phenotypes and support our proposal that metabolic shifts are key elements in the mechanism of DR.

Evidence for Metabolic Reprogramming in Organisms with Extended Life Span

In Saccharomyces cerevisiae, life span extension by DR induces an active regulated response [13] and there is a shift in metabolism in the restricted organisms toward increased respiration [14]. The increase in respiration is associated with a decrease in ROS production and this is thought to be indicative of increased mitochondrial uncoupling [15]. Although yeasts are facultative anaerobes, the influence of mitochondrial perturbations on life span under aerobic conditions indicates that manipulation of mitochondrial function directly

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influences longevity. The retrograde response pathway in yeast provides a mechanism for communication of changes in mitochondrial function to the nucleus [16]. This pathway has been linked to adaptive regulation of metabolism and the stress response [17] and its activation induces the expression of cytoplasmic, mitochondrial and peroxisomal metabolic genes [18] and life span extension [19]. These studies demonstrate that changes in mitochondrial function are transmitted to the nucleus and induce pathways that will provide a compensatory metabolic change. The fact that changes in mitochondrial efficiency can and do exert large-scale changes in gene expression and metabolic regulation supports the idea that a program as complex as life span extension by DR could conceivably be initiated in such a manner. Interestingly, mitochondrial signaling seems to converge with the nutrient-sensing TOR (target of rapamycin) pathway in yeast, where TOR inhibition activates the transcription factors involved in the retrograde response [20]. There is evidence that this cross talk may be conserved in mammalian systems where mitochondrial deficiency stabilizes the interaction between TOR and the inhibitory regulatory associated protein of mTOR (Raptor) protein [21]. These findings demonstrate that there is an open line of communication between mitochondrial efficiency and the nutrient-sensing TOR pathway, allowing for an integration of signaling pathways and a coordinated metabolic response. Inhibition of TOR signaling extends life span in yeast [22, 23], worms [24] and flies [25]. Reduced TOR signaling in mice by knockout of the TOR effector S6K1 increases FAO and negatively regulates insulin signaling [26]. It will be interesting to see what role TOR signaling plays in life span extension by DR. In Caenorhabditis elegans, a systematic RNA interference screen to identify gene alterations that affect life span has uncovered a complex relationship between mitochondrial function and longevity [27], and inhibition of mitochondrial function early in development extends life span in this organism [28]. More recent studies in worms have clearly identified a role for metabolic regulators in longevity [29, 30]. These studies involve RNA interference knockdown of specific gene products starting from the egg hatching stage. This is a situation where the animal must survive in the absence or depletion of the requisite pathway and while novel factors that influence life span have been discovered, no information about any metabolic compensatory mechanisms in response to this inhibition in the targeted animal has been gleaned. It is possible that the inhibition of mitochondrial oxidative phosphorylation at an early stage induces alternative energy metabolism pathways in these animals, resetting the longevity of the animal [31]. This may explain why inhibiting respiratory chain components in the adult animal does not affect life span [28]. DR does not appear to alter the metabolic rate in C. elegans [32] and genetic analysis indicates that life span extension by DR is independent of

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DAF-16, the forkhead transcription factor involved in the insulin/IGF pathway [33]. A number of components downstream of DAF-16 have been shown to influence metabolism and life span. These include the DAF-12 nuclear hormone receptor [34] and its coregulator DIN-1 [35], and the DAF-15 regulator of TOR (homologue of mammalian Raptor) [36]. These factors conceivably represent points of convergence of signaling by glucose/insulin, lipophilic factors and amino acid limitation-sensing factors. Such cross talk between pathways would permit regulation of the appropriate coordinated metabolic response to the nutritional status of the animal. Several studies in Drosophila have also demonstrated that life span may be extended by genes involved in metabolic regulation. Flies with a mutation in INDY (‘I’m not dead yet’), a Krebs cycle intermediate transporter, are longlived [37]. These animals do not show a change in metabolic rate [38], reminiscent of the results of some DR studies [39], although the presumed changes in metabolism have yet to be characterized. Disruption of ecdysone steroid hormone signaling in Drosophila also extends life span [40]. In the absence of this hormone, the ecdysone receptor complex interacts with transcriptional repressors Rpd3 and Sin3 [41]. What is interesting about this, from our point of view, is that reduction of Rpd3 extends life span in flies [42] and reduction in Sin3 causes upregulation of genes involved in the oxidative metabolism of fatty acid to acetyl-CoA and genes involved in mitochondrial oxidative phosphorylation [43]. The similarity between the ecdysone receptor complex and the nuclear hormone complexes in mammalian systems has led to speculation that the mammalian counterparts may also participate in the regulation of aging [44]. As in worms, life span is extended in flies by reduction of insulin signaling [45, 46]. Here again the mechanism of life span extension in insulin-signalingdeficient animals is not thought to be the same as that for DR, but the evidence suggests that there are common elements [47], perhaps downstream of the forkhead transcription factor dFOXO. The reduction of blood glucose and enhanced insulin sensitivity in DR mammals hints at the involvement of altered insulin signaling in the mechanism of aging retardation. We do not dispute this but suggest that the changes in insulin signaling and sensitivity are secondary to the metabolic shift in these animals. Studies on long-lived transgenic mouse models indicate that the effects of DR are not fully explained by reduced growth hormone/IGF-1 axis activity [48–51]. Overexpression dFOXO in the fat body of flies extends life span [52, 53]. Forkhead transcription factors are downstream of the insulin signaling pathway and in worms regulate metabolism and the stress response [54, 55]. These findings point to the importance of the fat body in whole-body regulation of metabolism and longevity and indicate that secreted factors are involved in the

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mechanism of life span extension in these transgenic animals. Even though there is evidence to suggest that DR acts independently of FOXO transcription factors [33], factors downstream of FOXO appear to be common to both insulin/IGF longevity pathways and life span extension by DR, in particular members of the nuclear receptor family and possibly factors influencing the TOR nutrient signaling pathway. There is now mounting evidence that signals from white adipose tissue in mammals can influence whole-body metabolism and life span. These quantitative and qualitative changes in adipose tissue may be critically involved in the mechanism of aging retardation by DR (see below). Taken together, the evidence presented here confirms that life span may be influenced by regulation of metabolism, that mitochondrial efficiency influences the metabolic state and that a communication network exists to coordinate changes in mitochondrial function with regulation of metabolism. These data support the concept that metabolic reprogramming could be an initial event in the mechanism of life span extension by DR and that many genetic manipulations that extend life span may also be viewed in this way.

Mitochondria in Aging and Dietary Restriction

Mitochondria are the key organelle in substrate utilization and energy production. DR directly affects mitochondrial function, increasing the expression of components of the ETS as well as genes involved in fatty acid transport and ␤-oxidation [8, 10] and there is a clear reduction in the production of ROS [56–61]. DR enhances mitochondrial oxidative capacity in liver and skeletal muscle in rats [62]. ROS are generated continuously as part of normal mitochondrial function [63]. One inbuilt mechanism to combat ROS accumulation is through uncoupling of the mitochondrial membrane potential by proton leak [64]. However, studies with mitochondria isolated from liver and skeletal muscle from age-matched control and restricted rats demonstrate that ROS production in mitochondria is reduced even though there is no change in proton leak [65, 66]. These studies touch on the role of mitochondrial uncoupling proteins UCP2 and UCP3 and raise questions as to how mitochondrial function is altered by DR. The role of ROS in aging and DR will be discussed in the following section. In heart, skeletal muscle and white adipose tissue, expression of the mitochondrial uncoupling protein UCP3 is elevated by DR [8, 10, 66]. Studies in mice overexpressing UCP3 support a role for this protein in energy balance and lipid metabolism [67]. Although the physiological role of UCP3 is controversial [68–70], increased UCP3 expression augments FAO and decreases ROS production without uncoupling respiration [71] and enhances the capacity for fatty

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acid transport and FAO in skeletal muscle [72]. These data argue that UCP3 is not merely a mitochondrial uncoupling protein involved in the regulation of the proton leak. Elevated free fatty acids induce UCP3 expression consistent with UCP3 playing a role in the use of free fatty acid as a fuel [73]. In one model [68], UCP3 works with the carnitine palmitoyl transferases, CPT-1 and CPT-2, to cycle fatty acid anions through the mitochondria. The increase in CPT-1 expression in restricted tissues supports a role for UCP3 in fatty acid transport, providing increased capacity for fatty acid metabolism. Studies in type 2 diabetes have implicated mitochondrial dysfunction in this disorder [74, 75] providing a link between mitochondrial function and whole-body endocrine signaling. Mitochondrial abnormalities lead to neuromuscular disorders known as mitochondrial myopathies and encephalomyopathies [76], as well as heart disease [77]. Mitochondrial function declines with age in humans [78]; however, the extent of the contribution of mitochondrial function to the onset of age-related pathologies like diabetes and heart disease is not yet clear. Tissue-specific disruption of the respiratory chain in mouse hearts causes a switch from fatty acid to glucose metabolism that precedes the inevitable heart failure in these animals [79]. This demonstrates that changes in mitochondrial function are sufficient to implement large-scale metabolic changes in mice in vivo. Recent studies have demonstrated that mice with a mitochondrial mutator phenotype develop several age-associated disorders providing strong support for a model in which mitochondrial function is a determinant of aging [80, 81]. Finally, analyses of individual mice have revealed a positive association between metabolic intensity and life span [82]. One issue that complicates studies involving isolated mitochondria is that the experimental setup measures the maximum capacity of the isolated mitochondria in an environment that is experimentally determined, but cannot reveal the actual in vivo differences in mitochondrial function where the intracellular environment may not be equivalent in the organism as a whole. Nevertheless, together these data support a key role for mitochondrial energy metabolism in the control of life span.

Stress, Oxidative Stress and Longevity

Mice [83], rats [84] and monkeys [85] subjected to DR demonstrate decreased body temperatures indicative of altered energy balance. Reduction in oxidative stress is a feature of DR in rodents [57, 86] and may be a direct result of this metabolic reprogramming. DR attenuates the age-associated increase in rates of mitochondrial ROS generation in multiple tissues and reduces the accrual of oxidative damage [58–61, 87, 88]. Mitochondrial function is

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preserved with age in DR animals, and the loss of mitochondrial membrane fluidity is delayed [89] compared to control animals. Reduction of mitochondrial H2O2 production and oxidative damage to mtDNA in rat gastrocnemius muscle with DR has been described [90] and we have reported that DR in monkeys lowers oxidative damage in skeletal muscle [91]. More recently, the role of ROS has broadened to encompass the control of normal cellular functions (e.g. transcriptional control, signal transduction) and cell death pathways [92]. These data reveal a potential role of ROS in aging that is independent of damage induction. It has been proposed that DR, by lowering ROS, attenuates ageassociated increases in the binding activities of redox-sensitive transcriptional factors (e.g. HIF-1, NF-␬B, AP-1) [93]. These factors may be important in the mechanism of aging retardation by DR where reduced ROS production prevents these signaling molecules from implementing the changes we see in the transcriptional profiles of aging animals. In the course of our analysis of mouse microarray databases, a number of interesting candidates were identified including PPAR coactivator 1a (PGC-1␣, and the redox-sensitive transcription factors HIF-1␣ and NF-␬B. Surprisingly, RT-PCR analysis demonstrated that DR has little effect on genes from the sirtuin and forkhead transcription factor families, genes that have been associated with longevity in lower organisms [unpubl. data]. Instead, SIRT1 and FOXO3 are regulated post-transcriptionally by DR [94] [unpubl. data]. SIRT1 has subsequently been associated with DR in cell culture models [94] and activation of SIRT1 is thought to be a key feature in the mechanism of DR, although this has yet to be conclusively shown in mice. FOXO3 is a homologue of the worm longevity factor DAF-16, a component of the insulin signaling pathway. FOXO3 has been linked to cell survival and the stress response in mice and is associated with both SIRT1 and p53 [95, 96]. The regulation and activation of factors associated with the stress response has led us to ask if other elements associated with the stress response might also play a role in DR’s action. We performed a screen to identify kinases activated by DR in the mouse heart. We used tissue from 10-month-old animals to eliminate the influence of age-dependent changes. Interestingly, we identified a number of kinases that are regulated by DR both in terms of total protein levels and degree of modification. Among these are JNK and GSK3␤, which are respectively regulated by DR in multiple tissues [unpubl. data]. JNK signaling enhances resistance to oxidative stress and extends life span in worms and flies [97, 98]. In mice, JNK plays a role in insulin signaling and obesity [99, 100], and affects insulin resistance in the liver and insulin production in the pancreas [101]. Factors downstream of JNK include FOXO [97, 98], which is required for JNK-dependent life span extension in worms and flies, and PPAR-␥ [102]. Interestingly, activation of JNK under conditions of oxidative

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stress is initiated in the mitochondria [103] suggesting that there is direct communication between this organelle and effectors of the stress response. These findings prompt further investigation into a role for JNK in the mechanism of life span extension by DR. It is noted that JNK-dependent life span extension in worms requires DAF-16/FOXO [98] and that life span extension by DR does not [33]; however, constitutive activation of JNK is not a genetic mimic of DR. Regulation of JNK by DR potentially provides a link between stress resistance pathways and longevity, perhaps by influencing factors downstream of FOXO. The mitogen-activated protein kinase p38 is responsive to numerous stimuli, including environmental stress and cytokine signaling [104]. Activation of p38 increases insulin sensitivity in skeletal muscle in a manner that is independent of contractile induced insulin sensitivity [105]. p38 activates the transcriptional coactivator PGC-1␣ by phosphorylation, thereby regulating the induction of mitochondrial respiration in muscle [106, 107] where PGC-1␣ plays a role in fiber type switching [108]. The yeast homologue of GSK3␤ is involved in nutrient sensing and the stress response [109]. In mammals, GSK3␤ is a negative regulator of JNK [110] and is involved in insulin sensitivity in skeletal muscle [111]. Interestingly both JNK and p38 are activated by ROS signaling from the mitochondria [112]. It is unclear how kinases usually associated with the stress response are activated by DR; however, longevity and stress resistance have been linked in most genetic studies performed to date.

The Role of Adipose Tissue in Aging Retardation by Dietary Restriction

Recent studies have highlighted the importance of white adipose tissue in overall metabolic regulation and data from our laboratory and others suggest that the changes in white adipose tissue observed in animals on DR are of particular significance. In mice, long-term DR induces morphological and transcriptional alterations. The mass of epididymal white adipose tissue is reduced by 75%, which appears to be due to a reduction in cell size [10]. DR suppresses the expression of over 50 genes in inflammation and promotes structural remodeling of the cytoskeleton, extracellular matrix and vasculature [12]. It is probable that reductions in systemic inflammatory tone caused by DR may underlie its ability to oppose a broad spectrum of age-associated diseases including cancers and cardiovascular disease. We contend that a key consequence of the metabolic reprogramming induced by DR is the alteration in adipose tissue physiology and metabolism. Aging is associated with alterations in body fat distribution, obesity and insulin resistance [113, 114]. High levels of leptin are observed with obesity in

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humans and rodents [115, 116]. DR reduces plasma insulin and leptin levels [117, 118], and opposes the development of age-related insulin and leptin resistance [119, 120]. Transgenic mice lacking the insulin receptor in adipose tissue have reduced adiposity and display a modestly extended longevity compared to DR [121]. These data argue that disruption of IGF signaling in adipose tissue alone is sufficient to extend life span and mirrors the experiments in worms and flies where fat-body-specific knockdown or overexpression of components of the insulin signaling pathway affect life span [52, 53, 122]. The concept of adipose tissue as an endocrine organ has come into focus recently [123]. Elevated serum levels of adipose tissue secretory products have been associated with numerous pathologies including cardiovascular disease, insulin resistance and diabetes. Resistin and adiponectin are adipocyte secretory proteins that negatively and positively regulate insulin sensitivity, respectively [124–126]. Resistin expression was upregulated by DR [10] and adiponectin was not significantly altered. While the significance of the DRinduced changes is not yet clear, the fact that adipocyte-derived signaling molecules are directly affected by DR lends support to the idea that changes in adipose tissue by DR can be transmitted throughout the organism. DR-induced transcriptional alterations in white adipose tissue included increased expression of genes involved in adipocyte differentiation. Both PPAR-␥ and SIRT1 have previously been implicated in this process [127] but it is as yet unclear if either is playing a role in DR-induced changes observed in white adipose tissue. Histological examination of white adipose tissue from mice on DR confirmed the presence of multilocular adipocytes which may represent an intermediate phenotype between white and brown adipocytes [10]. The metabolic shifts observed are consistent with this, including the increased expression of the ␤3-adrenergic receptor and UCP3. In white adipose tissue, activation of the ␤-adrenergic receptors leads to mobilization of fat stores and regulates the release of several adipokines [128]. In brown fat, ␤-adrenergic receptor activation leads to increased expression of the thermogenic uncoupler UCP1 via p38 and the transcriptional coactivator PGC-1␣ [129]. Ordinarily, PGC-1␣ protein levels are barely detectable in white adipose tissue but are increased in white adipose tissue from DR animals [unpubl. data]. The increase in PGC-1␣ may be critical in the activation of adipose tissue by DR. Adenovirus-driven expression of PGC-1␣ increased the expression of ETS components and FAO enzymes in human adipocytes, and transcription profiling indicated a metabolic activation of the fat cells [130]. Adenovirus-induced hyperleptinemia reduces fat stores in normal rats and increased the capacity for fat oxidation [131]. In these animals, expression of PGC-1␣ was dramatically increased, as was the expression of gene targets of PGC-1␣, and electron microscopy revealed changes in mitochondrial number and morphology. These

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data describe a striking similarity between the effect of DR and the effect of upregulation of PGC-1␣ on adipose tissue. We believe that the changes observed in white adipose tissue are fundamental to the mechanism of life span extension by DR. Age-related changes in adiposity correlate with systemic oxidative stress in humans and mice, and in cultured adipocytes, elevation of fatty acids increased oxidative stress and caused dysregulated production of adipokines [132]. The pharmacological induction of ␤-oxidation is currently being explored as a treatment for obesity and diabetes [133]; both of these disorders are prevented by DR. We suggest that the DR-induced shift in metabolism in white adipose tissue provides an increased capacity for FAO and permits the mobilization of fat stores without increasing oxidative damage through altered mitochondrial function and the induction of UCP3. The activation of white adipose tissue in this manner influences whole-body physiology in a manner that promotes longevity: in our model we predict that changes in levels of adipokines and other adipose secretory factors systemically influence metabolism, endrocine and immune function and that quantitative and qualitative changes in serum lipids affect nuclear receptor signaling in multiple tissues and influence lipid composition throughout the organism.

␣ is a Candidate Factor in the Mechanism PGC-1␣ of Aging Retardation by Dietary Restriction

We have presented evidence that DR induces metabolic shifts in multiple tissues and that the influence of metabolism on longevity is conserved across species. Based on these observations and because mitochondrial function has been linked to aging and life span extension by DR [134], we examined our heart microarray data set and looked for regulators of mitochondrial function as potential effectors of DR in mice. PGC-1␣ is a critical transcriptional coactivator of mitochondrial function that is responsive to changes in energy demands [135, 136]. It induces mitochondrial biogenesis and the expression of genes involved in multiple mitochondrial pathways. Overexpression of PGC-1␣ stimulates the mitochondrial antioxidant defense system in vascular endothelial cells [137]. PGC-1␣ has been associated with glucose regulation, the insulin signaling pathway and has been implicated in diabetes [138, 139] and obesity [140], conditions that are prevented by DR. Microarray analysis demonstrates that expression of PGC-1␣ is increased in hearts from DR mice and that there is a coordinated increase in expression of targets of PGC-1␣ activity [unpubl. data]. In both heart [8] and adipose tissue [10], we observe a clear trend of upregulation of nuclear genes encoding

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components of the ETS, many of which are targets of PGC-1␣. Expression of PGC-1␣ is also elevated in epididymal white adipose tissue of young/mid-age DR animals [unpubl. data], indicating that induction of PGC-1␣ is part of a regulated metabolic response to DR. Apart from its role in mitochondrial regulation, PGC-1␣ acts as a transcriptional coactivator of the PPAR nuclear receptor family. The PPARs have been linked to obesity and metabolic regulation and play a central role in the cross talk between glucose and lipid homeostasis [141]. Metabolic integration of FAO, carbohydrate metabolism, energy uncoupling and whole-body insulin sensitivity is attained through the coordinated activity of PPAR-␣, PPAR-␥ and PPAR-␦ in adipose and liver tissues where PGC-1␣ levels are elevated by DR. Transcriptional analysis of the effect of DR in wild-type and PPAR-␣ knockout mice has revealed that 19% of the transcriptional changes in the liver are dependent on PPAR-␣ [142] stressing the importance of this nuclear receptor in the mechanism of DR. Studies in PGC-1␣ null mice confirm the role of PGC-1␣ in adaptive energy metabolism [143, 144]. In the liver, PGC-1␣ is associated with FOXO1, one of the mammalian DAF-16/dFOXO homologues, and is involved in hepatic insulin signaling [145]. FOXO1 is involved in PPAR-␥ regulation in adipocytes, and there is a complex interplay between these factors in adipocyte differentiation [146]. PPAR-␥ is also regulated by mTOR, and PPAR-␥ activity is dependent on amino acid sufficiency [147]. Another factor that appears to provide a connection between PGC-1␣, FOXO and PPARs is SIRT1. SIRT1 regulates PPAR-␥ in adipocytes [148] and is involved in PGC-1␣ activation in the insulin signaling pathway in the liver [149, 150]. Control of gene expression in this manner, where transcriptional coactivators and repressor factors are the targets for numerous signaling pathways, provides a strategy that permits the functional integration of multiple distinct biological programs [151].

Conclusion

The central role of energy metabolism in longevity has been a unifying feature in our work and in aging research. Here we propose a model for the mechanism of DR where metabolic reprogramming, the coordinate induction of an altered metabolic state, is an early event in the mechanism of life span extension by DR. We predict that tissue-specific changes in energy metabolism occur through PGC-1␣ and the PPAR nuclear receptor family. These shifts in energy metabolism induce a move from fat storage to fat mobilization, influence stress pathway signaling and ROS production. Activation of adipose tissue is a critical event in the mechanism of life span extension and leads to altered adipokine

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and lipid signaling and reduced systemic inflammation. The influence of metabolic reprogramming on endocrine and immune function leads to a reduced rate of aging. It is clear from the data described here that this model is highly simplified. Many if not all of the pathways and factors described here have been shown to be interconnected and are influenced through multiple inputs. The key to our model is the initial event, which is the shift in how energy is generated and how fuel is utilized, and that this occurs through small changes in activity of metabolic regulators to influence the balance of fuel utilization without deregulating nutrient homeostasis in the animal as a whole. In thinking of the mechanism of DR in this way, it is possible to extrapolate and understand some transgenic models of longevity in the context of metabolic regulation and also to see where the effect of nonphysiological genetic manipulations on life span could be misleading. References 1 2 3 4 5 6 7 8

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Rozalyn M. Anderson, PhD GRECC, D5209 VA Hospital, 2500 Overlook Terrace Madison, WI 53705 (USA) Tel. ⫹1 608 256 1901 (ext. 11591), Fax ⫹1 608 280 7291, E-Mail [email protected]

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Mobbs CV, Yen K, Hof PR (eds): Mechanisms of Dietary Restriction in Aging and Disease. Interdiscipl Top Gerontol. Basel, Karger, 2007, vol 35, pp 39–68

Secrets of the lac Operon Glucose Hysteresis as a Mechanism in Dietary Restriction, Aging and Disease

Charles V. Mobbs, Jason W. Mastaitis, Minhua Zhang, Fumiko Isoda, Hui Cheng, Kelvin Yen Departments of Neuroscience and Geriatrics, Mount Sinai School of Medicine, New York, N.Y., USA

Abstract Elevated blood glucose associated with diabetes produces progressive and apparently irreversible damage to many cell types. Conversely, reduction of glucose extends life span in yeast, and dietary restriction reduces blood glucose. Therefore it has been hypothesized that cumulative toxic effects of glucose drive at least some aspects of the aging process and, conversely, that protective effects of dietary restriction are mediated by a reduction in exposure to glucose. The mechanisms mediating cumulative toxic effects of glucose are suggested by two general principles of metabolic processes, illustrated by the lac operon but also observed with glucose-induced gene expression. First, metabolites induce the machinery of their own metabolism. Second, induction of gene expression by metabolites can entail a form of molecular memory called hysteresis. When applied to glucose-regulated gene expression, these two principles suggest a mechanism whereby repetitive exposure to postprandial excursions of glucose leads to an age-related increase in glycolytic capacity (and reduction in ␤-oxidation of free fatty acids), which in turn leads to an increased generation of oxidative damage and a decreased capacity to respond to oxidative damage, independent of metabolic rate. According to this mechanism, dietary restriction increases life span and reduces pathology by reducing exposure to glucose and therefore delaying the development of glucose-induced glycolytic capacity. Copyright © 2007 S. Karger AG, Basel

Masoro et al. [1] proposed that ‘dietary restriction retards the aging processes by altering the characteristics of fuel use’. Similarly, on the basis of a large-scale analysis of gene expression, Lee et al. [2] concluded that ‘aging was associated with transcriptional alterations consistent with a metabolic shift from fatty acid to carbohydrate metabolism’ and that dietary restriction

‘resulted in alterations in gene expression consistent with preserved fatty acid metabolism’ through ‘transcriptional reprogramming’ (see also Anderson and Weindruch in this volume). Indeed, life span in yeast is increased simply by reducing glucose concentrations which, interestingly, actually increases metabolic rate [3]. In the present review we extend these concepts and propose a specific mechanism by which a cumulative toxic effect of glucose drives at least some aspects of the aging process, reduction in which mediates protective effects of dietary restriction.

Glucose Increases Glycolysis and Inhibits Alternative Metabolic Pathways, Including ␤-Oxidation of Free Fatty Acids

A general feature of metabolic regulation is that substrates typically induce the metabolic machinery necessary for their own metabolism. The classic example of this phenomenon is the lac operon, in which lactose induces both the activity and gene expression of ␤-galactosidase, the rate-limiting enzyme for the degradation of lactose [4]. In yeast, whose natural history entails cyclic utilization of glucose followed by ethanol as energy sources, glucose depletion inhibits glycolysis and activates the pathways for ethanol metabolism [5]. In mammals, glucose induces the coordinated expression of glycolytic enzymes including phosphofructokinase, the rate-limiting enzyme for glycolysis [6]. Conversely, glucose inhibits the expression of pyruvate dehydrogenase kinase 4 (PDK-4), which is an inhibitor of pyruvate dehydrogenase [7]. Thus glucose induces the utilization of glucose carbons in both glycolysis and in the tricarboxylic acid cycle. In insulin-sensitive tissues, glucose acts in concert with insulin to increase glycolytic activity [8]. Cognate induction of its own metabolism is particularly salient for glucose, probably because, as the unique product of photosynthesis and the ultimate source of biological energy, metabolism of glucose has been particularly optimized through evolution. Thus, although lactose induces the lac operon, the lac operon is robustly inhibited by a similar concentration of glucose. Similar glucose repression of alternative metabolic pathways is observed in yeast [5, 9] and fungi [10]. As with the lac operon, induction of glycolytic capacity by glucose is accompanied by suppression of the utilization of alternate fuels such as oxidation of fatty acids [11]. It should be noted that fatty acids conversely induce the metabolic machinery for their own metabolism, especially through the regulation of members of the peroxisomal proliferator receptor family of transcription factors, which by inducing PDK-4 [7] can conversely reduce pyruvate dehydrogenase activity and therefore glucose metabolism. Thus in mammals glucose and free fatty acids are constantly engaged in substrate competition.

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Metabolic Regulation of Gene Expression Exhibits Hysteresis

Of particular relevance to aging, the effects of metabolites on their own metabolic fate may not be entirely reversible, i.e. metabolic reprogramming may exhibit hysteresis. Hysteresis refers to a phenomenon by which a system exhibits memory. For example, a classic example of hysteresis is the Schmidt trigger switch encountered in electrical engineering. If the switch is off and the voltage increases from 0 to 5 mV, for example, the switch is not activated until reaching 5 mV. However, if the trigger begins in the activated position at 5 mV and voltage decreases from 5 to 0 mV, the switch does not turn off until the voltage reaches 0 mV. Therefore at 3 mV, the trigger will be either on or off, depending on the history of the circuit. Although not as widely appreciated, gene expression also exhibits hysteresis, also sometimes called ‘gene memory’ or ‘priming’ [12–16]. For example, the first time the ovalbumin gene is exposed to estrogen the induction is sluggish and subtle, but subsequent exposures to estrogen produce much more rapid and robust inductions, a phenomenon associated with permanent changes in chromatin structure produced by the initial exposure to estrogen [17]. Indeed, ‘cellular memory’ has been invoked to describe the phenomenon of permanent change in gene expression after transient exposure to inducers during development [18]. Metabolic regulation of gene expression also exhibits hysteresis. For example, induction of the lac operon exhibits a history-dependent dose dependency that is precisely analogous to a Schmidt trigger [19, 20]. Thus if the system begins with the lac operon inactive at low levels of inducer, the operon does not become active until exposed to high levels of inducer. On the other hand, if the system begins with the lac operon active at high levels of inducer, it stays active until exposed to very low levels of inducer. Thus, depending on the history of the system, the operon would be either on or off at modest levels of inducer. In fact, this behavior has led investigators to refer to the lac operon as a multistable ‘switch’ [19, 20]. Accumulating evidence suggests that the regulation of gene expression by glucose and other factors also exhibits hysteresis [15]. For example, glucoseinduced expression of fibronectin appears to be remarkably persistent even after reduction of glucose concentration [21]. Furthermore, it is increasingly clear that deleterious effects of elevated glucose persist for years after correction of blood glucose in diabetes [22]. As described below, based on our analysis of glucose-regulated gene expression and studies of the mechanism of neuroendocrine glucose sensing, we propose that hysteresis of glucose-regulated gene expression entails persistent self-induction of cytoplasmic NADH, the signature metabolite of glycolysis.

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Mitochondrial Complex II Promotes Longevity, Other Mitochondrial Complexes Reduce Longevity

The fact that diabetes accelerates many age-related pathologies, especially cardiovascular pathologies, suggests that diabetes and aging may share common pathological mechanisms. Certainly this is clear for yeast, in which reducing glucose concentration is sufficient to increase life span [3]. A role for glucose metabolism in determining life span is also suggested by examination of the role of specific complexes of the mitochondrial electron transport chain (ETC) in determining longevity. Genome-wide screening studies have demonstrated that genes coding for mitochondrial functions constitute possibly the most conspicuous single class of ‘senescence assurance genes’, ablation of which increases life span [23, 24]. Almost all of these life-span-limiting mitochondrial genes code for proteins in mitochondrial complexes I, III, IV or V [23–26]. For example, of 23 genes discovered in an exhaustive genome-wide screen whose inhibition increased life span [26], 12 were genes coding for proteins in mitochondrial (ETC) complexes I, III, IV or V, and one gene coded for a key enzyme in glycolysis, glucose-6-phosphate isomerase. An independent screen from another laboratory obtained very similar results, though discovering a different glycolytic enzyme whose inhibition increases life span [25]. Strikingly absent from these screens were genes for proteins in mitochondrial complex II [25, 26]. Indeed, classic genetic screens had already identified that mutations causing impairments in complex II reduce life span [27]. Thus proteins in mitochondrial complexes I, III, IV and V and at least some glycolytic enzymes function to limit life span, whereas genes for proteins in mitochondrial complex II function to increase life span. As described below, this pattern also implicates glucose in regulating life span, because glucose catabolism preferentially utilizes complex I, whereas lipid catabolism preferentially utilizes complex II. Complex II Produces Fewer Reactive Oxygen Species than Other Complexes

The effects of mitochondrial impairments to increase life span are not due to reduced production of ATP (oxidative phosphorylation) since mutations in complex II function to reduce respiratory function [28], as do impairments in complexes I, III, IV and V [23]. Conversely, other studies demonstrate that simply reducing production of ATP is not sufficient to increase life span [23]. In fact, in yeast, reducing exposure to glucose increased metabolic rate while also increasing longevity [3]. The most likely mechanism for the striking dichotomy of mitochondrial complex function with respect to life span is that reactive

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oxygen species are normally produced in complex I, III, IV and V, but much less so in complex II [29–31]. This basic fact about mitochondrial function explains why inactivating mutations in complex II produce profound sensitivity to oxidative damage as well as reduced life span [28, 32]. They also suggest the hypothesis that disruption of mitochondrial complexes I, III, IV and V increase life span by producing a relative increase in the utilization of complex II over the other complexes. While such a pattern of fuel use might or might not reduce ATP synthesis, the actual mechanism extending life span would be, according to this hypothesis, reduced production of reactive oxygen species due to relatively increased utilization of complex II. Reduced Complex I Activity Is Associated with Increased Life Span in Worms, Mice and Humans

As indicated above, genome-wide screening revealed that RNA-interferencemediated reduction in complex I activity increases life span in Caenorhabditis elegans [25, 26]. Furthermore, classic genetic screens had previously identified that mutations in the clk-1 gene [33], which also influences mitochondrial function [34], increase life span. This gene codes for a protein that converts Q8 ubiquinone, supplied by bacteria, to Q9 and other ubiquinone species, used in oxidative phosphorylation [34]. Although it is not precisely yet clear how mutations in the clk-1 gene increase life span, the increased life span does not appear to be due to reduced respiration [35, 36] or increased superoxide dismutase (SOD) or catalase [36]. However, it has now been demonstrated that the clk-1 mutation depresses complex I activity while sparing complex II activity [37], thus favoring the utilization of complex II at the expense of complex I. Similarly, in mice, ablation of the p66shc gene increases life span and reduces the production of reactive oxygen species, apparently by reducing NADH levels and thus the utilization of complex I [38]. Interestingly, a mutation in NADH dehydrogenase, a key enzyme in complex I, is also associated with increased longevity in humans [39]. Dietary Restriction May Increase Life Span by Reducing Oxidative Stress through a clk-1-Like Mechanism

Dietary restriction may increase life span through a clk-1-like mechanism, since effects of dietary restriction and the clk-1 mutants on life span are reported to be nonadditive [40]. This hypothetical common mechanism does not appear to require a reduction in metabolic rate, since neither the clk-1 mutation [35, 36] nor dietary restriction [41, 42] reduce mass-specific oxygen

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consumption in C. elegans. Similarly, the mechanism by which dietary restriction increases life span in yeast [3] and mammals [1, 43] appears not to require a reduction in mass-specific oxygen consumption. On the other hand, several lines of evidence suggest that a final common mechanism by which life-extending mutations [44, 45] and dietary restriction [46, 47] increase life span entails reducing oxidative damage. Furthermore, dietary restriction reduces the activity of complexes I, III and IV [48]; ATP production is presumably maintained by elevation of the relative complex II function. Similarly, dietary restriction decreases the production of reactive oxygen species in complex I without reducing mitochondrial oxygen consumption or reducing the production of reactive oxygen species in complex II [49–51]. The authors of these elegant studies concluded that ‘caloric restricted mitochondria released less reactive oxygen species per unit electron flow, due to a decrease in the reduction degree of the complex I generator’ [50]. Since mitochondrial oxygen consumption did not change, this mechanism likely involves an increase in the relative utilization of complex II. These data suggest that dietary restriction, possibly like the clk-1 mutation, may increase life span by causing a relative reduction in the utilization of complex I and a relative enhancement in the utilization of complex II, without necessarily reducing the overall metabolic rate.

Dietary Restriction Reduces Glycolysis and Increases Oxidation of Lipids and Amino Acids; Aging Produces the Opposite Profile

The mechanism by which dietary restriction might produce this relative shift toward the utilization of complex II is suggested by studies examining effects of dietary restriction on the expression of metabolic genes and levels of metabolic products. Dietary restriction in most tissues produces a metabolic profile indicating a striking shift away from glycolysis and toward lipid metabolism, whereas aging produces the opposite profile relative to the young ad libitum profile [2]. The metabolic shift away from glycolysis in dietary restriction is illustrated by the effect of dietary restriction to increase PDK-4 [52]. PDK-4 is a key inhibitor of pyruvate dehydrogenase, which controls the rate-limiting step in the recruitment of glucose-derived carbons for complete oxidation in the Krebs cycle [53]. Conversely, the metabolic shift toward lipid metabolism is illustrated by the effect of dietary restriction to increase expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme for the transfer of lipids to the mitochondria [2, 54]. Similarly, direct analysis of glycolytic enzyme activities and metabolic products in young and old liver from ad libitum fed and dietaryrestricted mice elegantly demonstrated the same phenomenon, that chronic

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dietary restriction strikingly reduces glycolysis while apparently increasing reliance on lipid (and possibly amino acid) mitochondrial oxidation [55]. While reducing glycolysis, dietary restriction apparently increases the metabolism of glucose through the alternative pentose pathway, as evidenced by the induction of transketolase [2]. Thus dietary restriction produces a shift away from glucose metabolism in glycolysis, but increased metabolism in the pentose pathway, and toward lipid and amino acid oxidation, whereas aging produces the opposite effect. The significance of this metabolic profile for the regulation of life span is suggested by the observation that antioxidants produced many of the same effects as did dietary restriction, but failed to prevent the age-related shift toward glycolysis and also failed to increase life span [56]. As indicated above and as also described below, the effect of dietary restriction on metabolic gene expression is highly similar to the effect of hypoglycemia, i.e. the opposite of the effect of elevated glucose. This similarity further supports that in mammals effects of dietary restriction on life span are mediated by a reduction in exposure to glucose [1], as is the case in yeast [3]. It should be noted of course that dietary restriction also reduces insulin secretion, and genetic ablation of insulin-like pathways increases life span in worms [57] (see Houthoofd et al. in this volume), mice [58] (see Bartke et al. in this volume) and flies [59] (see chapter by Tatar in this volume). Furthermore, insulin produces metabolic effects (e.g. increased glycolysis and reduced ␤-oxidation) that are similar to, and thus potentially similarly as toxic as, those produced by glucose. These observations initially suggested that dietary restriction might increase life span by reducing insulin secretion. However, subsequently it has been shown that dietary restriction and ablation of the insulin-like pathway produce additive effects in worms [60] and mice [61], leading many investigators to conclude that dietary restriction increases life span through a mechanism other than reducing insulin secretion. This conclusion was questioned by elegant studies in flies [62], but as discussed in the chapter by Tatar in this volume, even those studies are not definitive. On the other hand, it should be noted that not all tissues are equally insulin sensitive, and in fact about half of the glucose disposal under normal conditions is not regulated by insulin. Therefore we hypothesize that aging is driven by the proglycolytic gene profile produced by both insulin and glucose, but that dietary restriction increases life span only by reducing glucose exposure because the reduction in insulin secretion is balanced by increased insulin sensitivity. Thus dietary restriction is additive with ablation of the insulin-like pathway because further reduction of glycolysis in insulin-sensitive tissues is additive with the reduction in glycolysis produced by dietary restriction. In short, we propose that the glucose switch with hysteresis is a final common pathway between dietary restriction and the insulin-like pathways.

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Because diabetes accelerates many age-related pathologies, especially cardiovascular diseases, hyperglycemia has long been considered a model for aging, thus leading to the hypothesis that exposure to glucose drives the aging process [63, 64]. Among many commonalities between diabetes and aging is that both conditions entail mitochondrial impairments that probably drive many of the pathologies associated with those conditions [65, 66]. An interesting model of aging has been developed in which mitochondrial function is impaired by genetic deletion of a mitochondrial transcription factor in specific tissues [67]. This model is characterized by progressive failure of function and progressive reduction in ATP synthesis in a variety of tissues, and, as has been suggested for aging, the reduced metabolic capacity was initially thought to be the cause of the progressive pathology. However, detailed examination of gene expression demonstrated that before significant pathology developed, genes that stimulated glycolysis were induced and genes that stimulated ␤-oxidation were inhibited [67]. The authors concluded that ‘at least some of the secondary gene expression alterations in mitochondrial cardiomyopathy do not compensate but rather directly contribute to heart failure progression’ [67]. In view of the evidence discussed above, it is clear why this profile of gene expression would produce pathologies and supports the hypothesis that the impairments in mitochondrial function observed during aging in many studies and tissues [66] could be secondary to the switch in metabolism toward enhanced glycolysis and reduction in ␤-oxidation.

Glucose Oxidation Favors Complex I, Lipid/Amino Acid Oxidation Favors Complex II

The significance of the shift in source of carbon atoms for oxidation produced by dietary restriction may be that the oxidation of lipids and amino acids depends much more on mitochondrial complex II than on (free-radical generating) complex I, whereas glucose oxidation depends much more on complex I than on complex II. When glucose is broken down by glycolysis, the only reducing equivalents it makes are in the form of NADH. When the final carbon product of glucose, pyruvate, is metabolized in the Krebs cycle, almost all the reducing equivalents are produced in the form of NADH, except for one step at complex II (succinate dehydrogenase) that makes (then oxidizes) FADH2. Ultimately the metabolism of one molecule of glucose produces an NADH: FADH2 ratio of 5:1 [53, p. 20]. In contrast, when lipids are broken down by ␤-oxidation (fatty acid counterpart to glycolysis), an equal number of NADH and FADH2 molecules are formed. When the lipid-derived carbons are metabolized in the Krebs cycle, reducing equivalents are produced in the ratio of

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3 NADH molecules per FADH2 molecule. Therefore ultimately lipid metabolism yields an NADH:FADH2 ratio of about 2:1 [53, p. 38] or even less if the fatty acid contains enough carbon atoms. For example, when one molecule of palmitate is oxidized, it produces 15 molecules of FADH2 and 31 molecules of NADH, which are ultimately oxidized to produce a net total of 129 ATP molecules. In contrast, production of the same number of ATP molecules from glucose would entail producing then oxidizing 8.66 FADH2 and 43.3 NADH molecules. Amino acid oxidation also proceeds by a similar 2-step mechanism yielding an NADH:FADH2 ratio between that of lipids and that of glucose, the precise number depending on the specific amino acid. The significance of this shift in the NADH:FADH2 ratio is that NADH is oxidized only at mitochondrial complex I, whereas FADH2 is oxidized only at complex II [53, p. 17]. Thus palmitate oxidation entails utilizing complex II at roughly twice the (FADH2dependent) rate as glucose oxidation entails. Therefore shifting away from glucose utilization toward lipid and amino acid utilization would be expected to substantially reduce the production of reactive oxygen species, without necessarily reducing ATP production. As described below, other beneficial effects also occur as a result of this altered pattern of glucose fuel use, including a shift toward producing antioxidizing NADPH and increased protein and lipid turnover, which reduces the accumulation of oxidized protein and lipids.

Potential Cumulative Toxic Effect of Glucose on Neuroendocrine Neurons Regulating Metabolic Function

A guiding hypothesis for our research program for almost 20 years has been that longevity is governed in part by a cumulative toxic effect of glucose, particularly on glucose-sensitive neuroendocrine cells in the hypothalamus and pancreas, a phenomenon we referred to as glucose hysteresis [13, 14]. Similarly Masoro et al. [1] proposed the hypothesis that ‘dietary restriction retards the aging processes by altering the characteristics of (glucose) fuel use’. Our ‘glucose hysteresis’ hypothesis and Masoro’s ‘glucose fuel use’ hypothesis are potentially complementary and could represent two aspects of the same overall mechanism, but exactly how these mechanisms are related has been unclear until recently (see below). Our hypothesis suggested that key peptides expressed in glucose-stimulated hypothalamic neurons would: (1) be reduced during aging; (2) be reduced by fasting and in genetic obesity; (3) be reduced by treatment with the glucose toxin gold thioglucose; (4) be stimulated by glucose and other nutritional factors; (5) exert catabolic neuroendocrine effects, and impairment in the function of such peptides would exert anabolic effects; (6) be irreversibly impaired by

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prolonged exposure to elevated glucose. In a series of studies we have found that hypothalamic ␣-melanocyte-stimulating hormone, which is produced from the proopiomelanocortin (POMC) precursor and is preferentially reduced during aging [68–73], is reduced by fasting and in genetic obesity [74], and is reduced by gold thioglucose [75]. POMC neurons are stimulated by glucose and other nutritional factors [76], and transgenic correction of reduced hypothalamic POMC corrects impairments in glucose homeostasis in obese mice [77]. Thus age-related impairments in hypothalamic POMC could plausibly contribute to age-related obesity and metabolic impairments. However, although we hypothesized that cumulative toxic effects of glucose cause the age-related reduction in hypothalamic POMC, the mechanism by which glucose would cause such a toxic effect has until recently been elusive.

Glucose Metabolism Is Necessary for Effects of Glucose on Glucose-Regulated Neurons: Key Role for the Production of NADH

To further assess the hypothesis that cumulative exposure to glucose may drive hypothalamic impairments during aging, it was necessary to assess in more detail the mechanisms by which these neurons sense glucose. Such studies have been useful for two reasons. First, determination of key elements of the glucose-sensing apparatus has provided useful markers to test the generality that glucose-sensitive neurons are specifically sensitive to aging and to protective effects of dietary restriction. Second, assessment of the sensing mechanisms suggested mechanisms, described below, mediating the vulnerability of such neurons to glucose toxicity. Our working hypothesis was that hypothalamic neurons sense glucose through a mechanism similar to that utilized by pancreatic ␤-cells. It is well established that the pancreatic form of the enzyme glucokinase constitutes a key component of the glucose-sensing mechanism in pancreatic ␤-cells [78–82]. We observed that the pancreatic form of glucokinase is expressed in hypothalamic neurons, but not significantly in the rest of the brain [83], consistent with results by Jetton et al. [84] and corroborated in detail by Lynch et al. [85]. Based on these results, we examined in greater detail the extent to which hypothalamic neurons sense glucose through mechanisms similar to those of pancreatic ␤-cells. Extensive examination with both metabolic inhibitors and glycolytic intermediates demonstrated that, like pancreatic ␤-cells, hypothalamic neurons sense glucose through glucose metabolism [83]. In particular, inhibitors of glucokinase blocked the response of hypothalamic neurons. However, surprisingly a key step was the conversion of NAD⫹ to NADH and not, as expected, the

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production of ATP [83]. The significance of this observation became clear when it was found that the NADH shuttle system plays a key role in mediating effects of pancreatic ␤-cells to glucose [86]. Of even greater potential significance, these studies suggest that glucose metabolism may drive specific neuroendocrine age-related impairments, through the conversion of NAD⫹ to NADH, a mechanism similar to that reported in yeast [87].

Glucose Regulates Its Own Metabolic Fate: The Glucose Switch Gene Profile

As indicated above, several lines of evidence suggested that protective effects of dietary restriction could be mediated by reduction of glucose [13] leading to changes in glucose utilization [1] through ‘metabolic reprogramming’ [2], but the mechanism mediating these effects has remained unclear. To address this question we sought to discover genes regulated by glucose using DNA microarray analysis. In our studies of the regulation of POMC by leptin, we had found that gene regulation often occurs more robustly to ablation of a signal than to enhancement of a signal [74]. Therefore to discover genes regulated by glucose, we examined molecular responses to low glucose (hypoglycemia) compared to normal glucose (euglycemia) [88]. We examined responses in the hypothalamus since we hypothesized that neuroendocrine responses to glucose would be particularly important in mediating effects of glucose on life span [13]. Hypoglycemia was produced by injecting mice that had been food deprived for 48 h with insulin; therefore initially we could not determine if the regulation was due to fasting or hypoglycemia; however, we subsequently demonstrated with RT-PCR that almost all genes regulated by fasting alone were also regulated similarly by hypoglycemia alone [88]. In our initial study, using a small cDNA microarray of our own fabrication, we observed only a fairly small number of genes induced in association with hypoglycemia [88]. Among these genes were the glucose transporter GLUT-1, and the transcription factor CITED-1, also known as p300/CBP-interacting protein. The induction of GLUT-1 by hypoglycemia suggested that reduced glucose would produce a compensatory increase in glucose utilization, which would limit the importance of this mechanism in mediating effects of dietary restriction. However, it seemed unlikely that this analysis provided a comprehensive view of the molecular effects of glucose, since only about 1,000 genes were represented on this cDNA microarray. We therefore used the much more extensive U74 microarray chip to analyze the same RNA samples as were analyzed in the cDNA microarray study. Focusing specifically on genes coding for intermediary metabolism function (representing several hundred genes), only 26 met

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criteria that we had established as highly predictive of true regulation verifiable by RT-PCR [89]. Examination of this select group of genes provided a strikingly clear mechanism activated by hypoglycemia, a mechanism we call the ‘glucose switch’. Specifically, hypoglycemia was associated with reduced expression of genes that stimulate glycolysis (e.g. phosphofructokinase) and mitochondrial utilization of carbons derived from glucose (e.g. by inducing PDK-4, which inhibits pyruvate dehydrogenase). Nevertheless, this study confirmed that GLUT-1 was induced by hypoglycemia, implying that glucose carbons were being utilized in nonglycolytic pathways. Several genes whose products stimulate the pentose pathway were also induced, suggesting that hypoglycemia leads to a shunting of glucose carbons away from glycolysis towards the pentose pathway. In contrast, genes coding for peroxisomal proteins and genes involved in protein degradation and utilization of amino acids were induced by hypoglycemia. Furthermore, a rate-limiting enzyme for the tricarboxylic acid cycle, NADP-dependent isocitrate dehydrogenase, was also induced by hypoglycemia, suggesting that overall respiration might actually be increased, not decreased, by hypoglycemia. It should be noted that, as indicated above, this general metabolic profile, away from glycolysis and toward the pentose pathway, ␤-oxidation and protein turnover is very similar to that produced by chronic dietary restriction [2, 54, 55, 90]. This profile of gene expression implies a strikingly clear response to low glucose that could serve as the mechanistic basis of Masoro’s ‘alternate fuel use’ and the ‘reprogramming’ hypothesis (see Anderson and Weindruch in this volume) to explain effects of dietary restriction on life span. This response to low glucose involves extensive rerouting of glucose and other carbons without reducing (indeed, possibly elevating) the respiration rate. Hence we refer to this response as the ‘glucose switch’ profile. In this response to low glucose, glucose carbon atoms are shunted away from production of NADH through glycolysis and toward synthesis of NADPH through the pentose pathway and by the NADP-dependent form of isocitrate dehydrogenase. Since NADPH is the only major source of reducing equivalents for antioxidant defense, this profile would be expected to enhance antioxidant defenses. For example, elevation of glucose-6-phosphate dehydrogenase, the rate-limiting step in the pentose pathway, produces dramatic resistance to oxidative damage without changing levels of catalase or SOD [91, 92], whereas reduction of this enzyme greatly enhances cellular sensitivity to oxidative stress [93]. The pentose pathway absolutely requires carbons derived from glucose, so for this essential source of cytoplasmic NADPH to function in the presence of low glucose, alternative metabolic pathways for glucose carbons must be inhibited by low glucose, which they robustly are at several rate-limiting steps. Indeed, polymorphisms in the gene

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for glucose-6-phosphate dehydrogenase are strikingly correlated with life span across strains of Drosophila: the longest-lived strain exhibited a 64% higher activity of glucose-6-phosphate dehydrogenase than the shortest-lived strain [94]. Similarly, the NADP-dependent form of isocitrate dehydrogenase is a major source of NADPH in mitochondria, and elevation of this enzyme protects against oxidative stress in vitro [95] and even increases replicative life span [95]. Furthermore, expression of this enzyme decreases with age [96] and variants in NADP-dependent isocitrate dehydrogenase were associated with life span in Drosophila [97]. Interestingly, however, this ‘alternate pattern of fuel use’ produces two other possibly coincidental antioxidative effects. First, as indicated above, ␤oxidation of lipids, the lipid equivalent of glycolysis to prepare lipid carbons of oxidative phosphorylation in the Krebs cycle, produces reducing equivalents in the form of FADH2, rather than the NADH produced by glycolysis. FADH2 is oxidized at mitochondrial complex II, whereas NADH is oxidized at mitochondrial complex I. As reviewed above, reactive oxygen species are produced at much greater rates in complex I than in complex II, and indeed mutations in complex I extend life span whereas mutations in complex II reduce life span. Therefore a shift toward lipid oxidation (and to a lesser extent amino acid oxidation) would be expected to produce a lower rate of reactive oxygen species compared to deriving energy from glucose. Second, by enhancing the turnover rate of proteins and lipids, the average half-life of these macromolecules will be reduced, thus reducing the cellular burden of oxidatively damaged macromolecules. A final effect of this ‘alternate fuel use’ would be to dramatically shift the redox state of the NAD system away from NADH toward NAD⫹, though the overall redox state of the cell would be shifted toward a reduced state by elevation of NADPH and FADH2. It has been reported that the effect of dietary restriction to increase life span (in yeast) requires the activity of the silencing protein SIR2 which is dependent on (oxidized) NAD⫹ [87]. Although the precise role of NAD⫹ in regulating SIR2 activity has been disputed, considerable evidence supports that the NAD⫹:NADH ratio serves as a key signal for the metabolic state of the cell, as we showed in our own studies [83]. The significance of this shift has therefore to be fully elucidated. It should be noted that this antioxidant profile is potentially independent of the classic antioxidant system involving SOD and catalase, although we did find that hypoglycemia induced several isoforms of SOD, glutathione peroxidase and glutathione reductase (though not catalase), consistent with effects of dietary restriction [98, 99]. In fact, we have found that hypoglycemia decreases, and hyperglycemia increases, expression of other isoforms of SOD and that ablation of specific isoforms of SOD has no effect on life span in C. elegans [Yen et al., unpubl. data], consistent with studies in mice [100]. Furthermore,

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expression levels of SOD either did not correlate with life span or even correlated negatively with life span across 5 strains of mice (see below). On the other hand, even to the extent that classic antioxidant defenses play a role in the aging process, it should be noted that glutathione, a key metabolite in antioxidant defenses, ultimately derives its reducing potential from NADPH and thus mainly from glucose-6-phosphate dehydrogenase and NADP-dependent isocitrate dehydrogenase. Based on these observations, we propose that glucose regulates its own metabolic fate, promoting glycolysis and reducing the relative activity of the pentose pathway, ␤-oxidation and amino acid oxidation. Thus glucose produces a metabolic profile that produces NADH at the expense of NADPH and FADH2. The net effect of elevated glucose would therefore be to decrease antioxidant capacity (by decreasing NADPH), decrease the activity of Sir-type histone acetyltransferases (by converting NAD⫹ to NADH), increase the production of free radicals (by promoting the oxidation of NADH at complex I, which is the main site of free radical production, at the expense FADH2 oxidation at complex II, which is a minor site of free radical generation) and decrease the turnover of oxidized lipids and proteins (by decreasing ␤- and amino acid oxidation). While the ‘glucose switch’ hypothesis clearly suggests a mechanism for diabetic complications, which are currently thought to be due to oxidative damage [101], by itself it would be insufficient to explain the aging process, since glucose levels in general do not increase with age. Even for diabetic complications, however, the glucose switch mechanism by itself does not explain why, as with aging, impairments develop progressively and are apparently irreversible. To explain the progressive nature of aging and diabetic complications, we propose that the glucose switch transcriptional machinery exhibits hysteresis, as demonstrated with the highly analogous lac operon [19]. As described above, the lac operon exhibits hysteresis, in that sensitivity to the inducer depends on the history of exposure: if previously exposed to a high concentration of inducer, the operon is highly sensitive to inducer, whereas if previously exposed to a low concentration of inducer, the operon is relatively insensitive to inducer. Also as described above, there is evidence that the regulation of gene expression by glucose also exhibits hysteresis [21, 22]. Indeed, to the extent that transcriptional effects of glucose are mediated through NADH, the glucose switch mechanism directly predicts glucose hysteresis: since glucose induces the machinery to produce NADH, prior exposure to elevated glucose would subsequently lead to more NADH production per glucose molecule, i.e. greater sensitivity to glucose. Thus two features of the lac operon lead to a comprehensive mechanism that accounts for key features of aging, dietary restriction and diabetic complications: that substrates induce the machinery of their own metabolism (in the

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case of glucose especially, at the expense of alternative pathways), and that this self-induction entails hysteresis. Therefore we propose that postprandial excursions of glucose produce a glucose switch response, inducing glycolysis at the expense of ␤-oxidation, a state that would tend to persist even when glucose levels return to their preprandial levels. It should be noted that hysteresis in the lac operon is observed stochastically across cells so that the lac operon is either completely on or completely off. Thus we propose that the effect of successive exposures to (postprandial) glucose would be to produce progressively more cells in the proglycolytic glucose switch position. It should be noted, however, that as glycolysis increases and produces monotonic increases in oxidative damage, especially in mitochondria, and a greater reliance on glycolysis, this could eventually lead to a reduction in mitochondrial metabolic capacity in the late phases of senescence, which could be a final precipitating event leading to mortality, as appears to be the case with mitochondrial impairments produced by genetic modification [67]. The elevation of insulin secretion early during the prediabetic phase of type 2 diabetes, followed by a reduction in insulin secretion as the precipitating event of diabetes itself, might be considered an analogous process, and indeed may well be produced by exactly the same mechanism (increased glycolysis leading to increased secretion, followed by oxidativestress-induced ‘burnout’, followed by hyposecretion of insulin and diabetes).

Genetic Correlation between Gene Expression and Life Span

Other than two studies linking life span to expression levels of glucose-6phosphate dehydrogenase [94] or to variants in NADP-dependent isocitrate dehydrogenase [97], very few studies have directly linked variations in levels of glucose-sensitive genes to life span. We therefore examined the correlation between hypothalamic expression of glucose-sensitive genes and average life span across 5 strains of mice. This analysis was validated by the observation that, in our analysis, hypothalamic expression of sirtuin 2, the mammalian homolog of yeast SIR2, was positively correlated with life span (r2 ⫽ 74%) and inhibited by 10 versus 2 mM glucose (p ⬍ 0.01) as well as leptin, thus behaving quite similarly to yeast Sirt-2 [87]. Over 90% of the genes whose expression correlated positively with life span (and with an r2 greater than 70%) were inhibited by glucose in vitro. Most of these genes were transcription factors or involved in neuroendocrine signaling. For example, expression of polycomb group ring finger 5 correlated positively with life span (r2 ⫽ 75%) and was significantly and independently inhibited by glucose, leptin and insulin (p ⬍ 0.05 for all three). Interestingly, polycomb transcription factors primarily function to inhibit gene

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expression [102], as is the case for genes in the sirtuin family. Conversely, genes that correlated negatively with life pan were generally induced by glucose. For example, the transcription factor hypoxia-induced factor 1␣ is negatively correlated with life span (r2 ⫽ 83%) and induced by glucose (p ⬍ 0.01). The significance of the relationship between hypoxia-induced factor 1␣ and life span may be that this gene is an important stimulator of glycolysis [103]. Indeed, pyruvate dehydrogenase expression is negatively correlated with life span and stimulated by glucose. Also of some interest in view of the hypothesis of Andersen and Weindruch (in this volume), the expression of the peroxisome proliferationactivated receptor ␥ coactivator 1␣ is also highly and positively correlated with life span (r2 ⫽ 83%). Similarly, hypothalamic expression of the gene for stearoyl coenzyme A desaturase 1 is positively correlated with life span (r2 ⫽ 83%). The significance of these latter two genes is that they are induced by free fatty acids (which of course are elevated during fasting and dietary restriction for prolonged periods of time) and act primarily to increase fat metabolism. Thus across 5 strains of mice, elevated expression of glucose-stimulated genes predicts a reduced life span and elevated expression of glucose-inhibited genes predicts an increased life span; expression of genes induced by free fatty acids also predicts a longer life span.

Problems

The glucose hysteresis hypothesis suggests, though does not require, that the respiratory quotient (RQ) should increase with age, reflecting more carbohydrate utilization and less ␤-oxidation, whereas dietary restriction should produce the opposite effect. While some studies have reported an increase in RQ with age [104], others have not observed this effect. Similarly, while one study reported that dietary restriction reduces 24-hour RQ [43] (though not specifically indicating the significance of this effect), in a different study, although the same effect of dietary restriction to reduce RQ was observed, the effect was not significant [1]. In our own studies (unpublished) we observed that chronic dietary restriction in young mice significantly reduced 24-hour RQ by about the same amount as reported by McCarter et al. [43] in rats. Although the effect was small, it was about the same magnitude as observed for the effect of a highfat diet. We therefore conclude that while measurements of RQ are not uniformly supportive of the glucose switch hypothesis, neither are they uniformly contradictory. Further studies should clarify this issue. Nevertheless, even if the reduction of total 24-hour RQ by dietary restriction is not robust, we would suggest that the robust alteration in temporal pattern, in which lipid is the dominant fuel for a much longer part of the day with dietary restriction than with ad

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libitum food intake, would still produce a protective effect by reducing the total time glucose metabolism is the main source of energy. The glucose hysteresis hypothesis also predicts that glycolysis relative to alternate sources of fuel should increase with age, which is observed in the heart [2], brain [54], and liver [55], but in mouse muscle at least some genes for glycolysis as well as many other genes coding for mitochondrial function were reported to decrease with age [105]. On the other hand, this decrease in glycolytic gene expression was not observed in muscle tissue from nonhuman primates [52]. Therefore we hypothesize that even though expression of some glycolytic genes may decrease with age in mouse muscle, the utilization of other fuels (e.g. ␤-oxidation) decreases more so that the net effect is still a shift toward glycolysis. Similarly, the hypothesis predicts that dietary restriction should decrease glycolysis relative to the use of other fuels, which is observed in yeast [5], flies [106], the heart [2], liver [55] and brain [54], but in adipose tissue, long-term dietary restriction was reported to increase the expression of glycolytic genes [107]. However, dietary restriction also robustly increased many other metabolic pathways in adipose tissue, including ␤-oxidation and the synthesis of fatty acids. As with mouse muscle in aging, therefore, it is difficult to determine simply from gene expression whether glycolysis is actually increased or decreased relative to other metabolic pathways. A possibly more serious problem with the emphasis on glucose metabolism is that restriction of methionine only is reported to increase life span in rats [108] and mice [109]. Furthermore, restriction of yeast is reported to extend life span in flies more robustly than restriction of glucose [110]. On the other hand, dietary restriction without reduction in protein extends life span as well as dietary restriction with reduction of protein [111]. Furthermore, reduction of lipids only without reduction of caloric intake failed to increase life span [112]. Therefore while the effect of (extreme) methionine restriction is indeed interesting, it is unlikely to mediate the effects of dietary restriction on life span, at least in rats. With regard to the methionine effect in mice, it is of some interest that methionine restriction reduced plasma glucose, raising the possibility that restriction of methionine actually increases life span by reducing blood glucose, as we propose for dietary restriction [109]. Regarding the role of glucose versus yeast in flies, there are a number of complexities in the design of that study which precludes definitive interpretation, as described by Tatar in the present volume. Perhaps the best position to take at the moment is that across species it is not clear how much of the effect of dietary restriction is due to reduced exposure to glucose. To the extent that the effect is due to reduction in exposure to glucose, we propose that glucose hysteresis is a mechanism that could explain these toxic effects of glucose during aging. We further propose that at least with respect to one major disease of aging, diabetes, glucose hysteresis constitutes

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the major mechanism mediating the effects of hyperglycemia to induce diabetic complications.

Implications: Oxidative Stress and Tumor Burden

As described above, glucose hysteresis is a relatively simple unitary mechanism that potentially explains the following apparently otherwise unrelated phenomena: (i) glycolysis increases with age (in at least some tissues); (ii) oxidative damage increases with age; (iii) reducing glycolysis or the activity of ETC complexes I, III, IV and V increases life span, whereas reducing the activity of ETC complex II reduces life span; (iv) dietary restriction increases the relative utilization of complex II; (v) intermittent dietary restriction that does not reduce the average caloric intake nevertheless produces beneficial effects of dietary restriction, associated with reduced plasma glucose [113]. Thus glucose hysteresis can plausibly account for the majority of age-related impairments arising from oxidative stress and their attenuation by dietary restriction. However, the attenuation by dietary restriction of at least one major agerelated pathology probably does not arise by decreasing oxidative stress: tumor burden. As described by Klebanov in this volume, dietary restriction primarily if not exclusively reduces tumor burden by inhibiting the promotion phase of tumor growth. However, it is difficult to envision a mechanism by which reducing oxidative damage would inhibit the promotion phase. If anything, it would be the initiation phase that would probably be sensitive to oxidative stress. These considerations would seem to suggest that dietary restriction reduces oxidative stress and tumor burden by two distinct mechanisms and, conversely, that aging promotes these two pathological processes through distinct mechanisms. Nevertheless, the glucose switch hypothesis suggests a unifying mechanism: increased glycolysis during aging. Tumor cells are characterized by a unique dependency on glycolysis, a phenomenon known as the Warburg effect [114, 115]. This unique dependency on glycolysis possibly arises from mitochondrial damage, but has the effect of allowing tumor cells to thrive at relatively low oxygen tensions typically observed in tumors. Of particular interest, this unique dependency on glycolysis makes tumor cells highly sensitive to the toxic effects of glycolysis inhibitors, which have therefore been proposed as a promising class of antitumor agents [116]. It is therefore plausible that tumor cells are particularly sensitive to the low levels of glucose, and the resulting shift away from glycolysis, that are at least transiently produced by dietary restriction. Indeed, even a transient (3-hour) reduction in ATP produces robust apoptosis in transformed cells, 48 h later [117]. Furthermore, glycolytic inhibitors have been shown to produce many of the protective effects of dietary restriction [118]. Thus

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reduction in glycolysis reduces both oxidative stress and tumor burden, thereby potentially accounting for most or all beneficial effects of dietary restriction. A final implication of glucose hysteresis is its relevance to the genetic control of longevity. A key aspect of the theory is that it is largely independent of the classic antioxidant enzymes catalase and SOD, and focuses instead on the production of reactive oxygen species in complex I, as well as the role of increased protein and lipid turnover. However, it is by now well established that across species, ‘the longer the life span, the lower the rate of mitochondrial oxygen radical production. This is true even in animal groups that do not conform to the rate of living theory of aging, such as birds’ [119–121]. Similarly, as described above, genes involved in the glucose switch mechanism constitute the main set of genes that limit life span, according to nonbiased genome-wide screening [26]. In contrast, activities of the classic antioxidant defense enzymes do not correlate with life span across species [122]. Furthermore, it is now clear that impairments in classic antioxidant defenses do not reduce life span, even when such impairments increase oxidative damage [100]. Thus we propose that genetic influences on the regulation of gene expression by glucose could constitute key genetic influences that regulate life span. On the other hand, there are at least two relevant potentially distinct genetic influences, those that influence acute control of gene expression by glucose and those that influence the development of hysteretic effects. We suggest that genetic effects on both of these mechanisms must play a role in determining life span, since even at a young age species differ in their rate of production of reactive oxygen species [119] (possibly reflecting at least in part the acute effects of glucose on complex I activity), but on the other hand, the age-related increase in oxidative damage, reflecting hysteresis, scales with life span [123–125]. Thus we suggest that the rate of hysteresis may also be higher in short-lived than in long-lived species [119].

Context

It is of some interest to place the mechanism of glucose hysteresis into the context of current approaches being developed in the field of dietary restriction, as reflected in the present volume. First, Masoro has elegantly developed the concept that dietary restriction acts through hormesis, a protective mechanism activated by low-level stressors. Our proposal is quite consistent with this hypothesis, since hypoglycemia is a classic inducer of the stress response. Thus, for example, reduction of blood glucose stimulates secretion of glucocorticoids (a classic stress response) in mice [126], rats [127] and humans [128]. Similarly, reduction of blood glucose activates the sympathetic nervous system, also a classic stress response, in mice [129], rats [127] and humans [128]. Indeed,

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hypoglycemia even produces homotypic densitization, a classic feature of stress responses, in mice [129], rats [127] and humans [130]. Anderson and Weindruch in the present volume extend the concept, already articulated by Weindruch and colleagues previously [2], that protective effects of dietary restriction may be mediated through ‘transcriptional reprogramming’. Clearly glucose hysteresis represents precisely an example of transcriptional reprogramming, and indeed the work of Weindruch and colleagues represents some of the most important evidence for the mechanism. Bartke et al. in the present volume examine the allied concept that effects of the insulin-like pathway and dietary restriction are mediated by a common set of genes. While so far the evidence does not support this hypothesis, as argued above, it may well be the case that the proglycolytic effects of insulin drive senescence in insulin-sensitive tissues, whereas the proglycolytic effects of glucose drive senescence in insulininsensitive tissues. Morgan et al. in the present volume develop an elegant argument that many age-related pathologies arise from age-related increases in inflammatory processes and that dietary restriction attenuates age-related impairments in part by reducing inflammation. Morgan et al. also speculate about mechanisms mediating effects of dietary restriction to reduce inflammation, including a role for glucose acting through the receptor for advanced glycation end products. However, while this is a plausible explanation for the effect of dietary restriction, this mechanism fails to explain why inflammation increases with age, since in general, certainly in rodents, plasma glucose does not increase with age. Furthermore, elevated glucose induces cytokines very rapidly (within a few hours) through a mechanism requiring glucose metabolism [131], whereas advanced glycation end products develop far too slowly to account for such rapid effects. However, as emphasized by Morgan et al., at least some cytokines are induced by reactive oxygen species. We therefore suggest that inflammation increases with age because of increased reactive oxygen species, secondary to glucose hysteresis, and that dietary restriction retards this age-related increase in inflammation by reducing exposure to glucose. Houthoofd et al. in the present volume describe the value of C. elegans as a model organism. We completely agree that C. elegans constitutes possibly the most powerful system to study mechanisms of aging, and indeed much of the most compelling support for the mechanism of glucose hysteresis comes from studies in C. elegans. Houthoofd et al. also argue strongly against the rate of living theory and indicate that dietary restriction increases life span without reducing metabolic rate, a conclusion also drawn by investigators studying rodents [1, 43]. In our view one of the most compelling aspects of the glucose hysteresis model is that, by invoking substrate competition, it can explain how the oxidative damage can decrease even without reducing ATP production.

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Interestingly, Tatar in the present volume reviews the use of Drosophila in aging research and carefully analyzes difficulties in interpreting studies that suggest a minimal role for glucose mediating the effects of dietary restriction [110]. As discussed in some detail elsewhere [132], a number of results suggest that mechanisms mediating effects of dietary restriction in Drosophila, and indeed mechanisms of senescence themselves, may be distinct to some extent from those in other species. It should be noted that of the many organisms and experimental paradigms described in the present review in support of the glucose hysteresis hypothesis, none have involved Drosophila. This corroborates the importance of studying these mechanisms in a wide variety of organisms to probe for the generality of those mechanisms. On the other hand, as described by Mattison et al. in the present volume, so far dietary restriction appears to produce similar results in nonhuman primates, including a reduction in mean blood glucose. We take this as evidence that dietary restriction is therefore likely to actually increase life span in nonhuman primates. In the final two chapters of the present volume, effects of dietary restriction on specific disease processes, Alzheimer’s disease and cancer, are discussed. We should note that we have no plausible mechanism through which the specific effects of dietary restriction on ␤-amyloid peptide generation described by Pasinetti et al. in the present volume can be accounted for by glucose hysteresis, and agree with them that insulin, rather than glucose, probably plays a key role. On the other hand, in view of the critical role of oxidative stress in mediating ␤-amyloid peptide toxicity in particular [133] and neurodegeneration in Alzheimer’s disease in general [134], it is plausible that glucose hysteresis plays a role in the age-related neurodegeneration associated with Alzheimer’s disease. Finally, in the last chapter Klebanov makes a strong case that dietary restriction reduces cancer burden specifically by interfering with the promotion phase of tumor growth, not the initiation phase. As noted above, the promotion phase is unlikely to be dependent on oxidative stress, in contrast to the initiation phase. However, also as indicated above, tumor cells become increasingly dependent on glycolysis as they progress, due to the Warburg effect [114]. Therefore, the effect of dietary restriction to interfere with the promotion phase is plausibly due directly to the reduction of glycolytic capacity, rather than the reduction of oxidative stress.

Other Age-Related Diseases

One of the most challenging problems in gerontology is to account for the age dependency of diseases whose incidence increases with age. As indicated above, at least for Alzheimer’s disease, the age dependency can be explained at

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least in part by the increase in oxidative stress with age, which in our view can in turn be accounted for by glucose hysteresis. In fact, it is plausible to hypothesize that the dependence of most age-related diseases (except, as indicated above, possibly cancer) can be accounted for by an age-related increase in oxidative stress. For example, Huntington’s disease appears to be due to a reduction in the utilization of ETC complex II [135]. Furthermore, dietary restriction substantially ameliorates symptoms in a mouse transgenic model of Huntington’s disease [136]. We therefore propose that the increased penetrance of the Huntington’s disease gene with age is due to a gradual reduction in the utilization of complex II in neurons during aging, secondary to glucose hysteresis, which in patients with Huntington’s disease becomes lethal due to a preexisting impairment in complex II. With regard to metabolic diseases, while it is clear how glucose hysteresis would account for age-related increases in diabetic complications, it is not so evident how this mechanism would account for the increased incidence in (type 2) diabetes with age. Type 2 diabetes is caused by the development of insulin resistance, usually accompanied by increased insulin secretion, eventually followed by pancreatic decompensation [12]. Oxidative stress plays a causal role in multiple forms of insulin resistance, many of which can be ameliorated by antioxidant treatments [138]. Similarly oxidative stress is implicated in pancreatic ␤-cell impairments [139]. Thus it is clear that increased oxidative stress during aging, secondary to glucose hysteresis, could plausibly account for the increased incidence of type II diabetes.

Testing the Hypothesis: Reversibility of Senescence

Although a substantial array of evidence supports that glucose hysteresis mediates at least part of the effects of dietary restriction, the hypothesis is nevertheless still subject to falsifiability. For example, the hypothesis predicts that factors which mediate effects of glucose on gene expression also mediate at least some of the effects of dietary restriction on gene expression. The hypothesis further predicts that manipulations that block effects of hypoglycemia on gene expression would also block at least some effects of dietary restriction on gene expression, and, more importantly, would attenuate beneficial effects of dietary restriction. For example, it may be possible to block effects of hypoglycemia on gene expression by ablating specific transcriptional factors or cofactors [140]. If blocking hypoglycemia-induced gene expression fails to block beneficial effects of dietary restriction, this would effectively falsify the glucose switch hypothesis. Another obvious prediction is that reduction of plasma glucose by, for example, transgenic expression of a glucose transporter, to the same extent as is

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observed with dietary restriction, should mimic effects of dietary restriction without caloric restriction. While this would indeed constitute a test of the hypothesis, there would be two caveats in interpreting these results. First, as indicated above, considerable evidence suggests that glucose signaling as well as the toxic effects of glucose are mediated by the production of NADH. If reduction of plasma glucose is accompanied by an increase in lactate, and lactate is used as an alternative fuel for glucose, lactate would plausibly produce the same levels of NADH as glucose, and therefore might prevent the beneficial effects of reduced glucose. This could be assessed by examining the expression of genes regulated by glucose: if hypoglycemia-induced genes are not induced by a reduction in plasma glucose, this would suggest that some compensatory mechanism, likely lactate, is preventing the beneficial effects of lower plasma glucose. Second, as also described above, some evidence suggests that the total 24-hour RQ is not reduced by dietary restriction and, if so, we would argue that the protective effect arises from reducing the total amount of time that tissues are predominantly using glucose as a fuel. If so, simply reducing total exposure to glucose may not be adequate to reproduce the extended period of time that cells are protected by predominant ␤-oxidation. If so, a test of the theory would require reproducing the pattern of RQ produced by dietary restriction, possibly using inducible promoters. Another implication of the hypothesis is that it may be possible to reverse age-related impairments by producing carefully controlled hypoglycemia at levels lower than can be produced by optimum dietary restriction. Dietary restriction at 50% ad libitum levels, about the most restriction compatible with extended life, only reduces plasma glucose to around 4.5 mM. However, counterregulatory and other protective responses are not activated until lower glucose levels of 3.6 mM or possibly even lower are reached, whereas cognitive impairments are not observed until much lower levels yet, around 2.6 mM. Therefore there is a potentially safe range of plasma glucose that would allow the production of even more potent beneficial effects than are produced by dietary restriction. The hysteretic behavior of the lac operon suggests that even transient repetitive reductions in glucose could reverse the proglycolytic glucose switch transcriptional state, thereby reversing the increase in glycolysis, oxidative damage and tumor burden. Studies to assess this hypothesis are currently under way.

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Charles V. Mobbs, PhD Departments of Neuroscience and Geriatrics Mount Sinai School of Medicine, Box 1639, 1 Gustave L. Levy Place New York, NY 10029 (USA) Tel. ⫹1 212 659 5929, Fax ⫹1 212 849 2510, E-Mail [email protected]

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Mobbs CV, Yen K, Hof PR (eds): Mechanisms of Dietary Restriction in Aging and Disease. Interdiscipl Top Gerontol. Basel, Karger, 2007, vol 35, pp 69–82

Effects of Dietary Restriction on the Expression of Insulin-Signaling-Related Genes in Long-Lived Mutant Mice Andrzej Bartke, Michal M. Masternak, Khalid A. Al-Regaiey, Michael S. Bonkowski Geriatrics Research, Departments of Internal Medicine and Physiology, Southern Illinois University School of Medicine, Springfield, Ill., USA

Abstract Hypopituitary Ames dwarf mice and growth-hormone-resistant (growth hormone receptor knockout, GHRKO) mice have reduced plasma levels of insulin-like growth factor 1 and insulin, enhanced insulin sensitivity and a remarkably increased life span. This resembles the phenotypic characteristics of genetically normal animals subjected to dietary restriction (DR). Interestingly, DR leads to further increases in insulin sensitivity and longevity in Ames dwarfs but not in GHRKO mice. It was therefore of interest to examine the effects of DR on the expression of insulin-related genes in these two types of long-lived mutant mice. The effects of DR partially overlapped but did not duplicate the effects of Ames dwarfism or GHR deletion on the expression of genes related to insulin signaling and cell responsiveness to insulin. Moreover, the effects of DR on the expression of the examined genes in different insulin target organs were not identical. Some of the insulin-related genes were similarly affected by DR in both GHRKO and normal mice, some were affected only in GHRKO mice and some only in normal animals. This last category is of particular interest since genes affected in normal but not GHRKO mice may be related to mechanisms by which DR extends longevity. Copyright © 2007 S. Karger AG, Basel

Many of an animal’s physiological responses to nutritional signals are mediated through insulin, insulin-like growth factor 1 (IGF-1) or homologous pathways. There is considerable evidence that dietary restriction (DR) reduces circulating levels of both IGF-1 and insulin, and diminished IGF-1/insulin signaling is among the mechanisms believed to link DR with delayed aging and extended longevity [1, 2]. In laboratory stocks of house mice (Mus musculus),

Table 1. Mice with altered somatotropic signaling and prolonged longevity Genotype

Endocrine effect

Reference

Ames dwarf Prop-1df Snell dwarf Pit-1dw

GH deficiency (combined with PRL and TSH deficiency)

[3] [4]

Little GHRHRlit

GH deficiency

[4]

GHR/GHBP –/–

GH resistance

[5]

IGFIR ⫹/–

partial IGF-1 resistance

[6]

␣MUPA

reduced IGF-1 (reduced food intake)

[7]

GHBP ⫽ Growth hormone-binding protein; GHR ⫽ growth hormone receptor; GHRHRlit ⫽ growth hormone-releasing hormone receptor, little; IGFIR ⫽ IGF/ insulin receptor; Pit-1dw ⫽ pituitary factor-1, Snell dwarf; PRL ⫽ prolactin; Prop-1df ⫽ prophet of pit-1, Ames dwarf; ␣MUPA ⫽ urokinase-type plasminogen activator; TSH ⫽ thyroid-stimulating hormone.

several spontaneous or experimentally produced mutations were shown to increase longevity. Most of these ‘longevity genes’ cause a disruption of somatotropic signaling, leading to reduced peripheral IGF-1 levels or partial IGF-1 resistance (table 1). In at least 3 of these long-lived mutants, reduced somatotropic signaling is associated with secondary alterations in insulin release and responsiveness to insulin actions [8–10]. It was therefore of interest to examine the interaction of DR with murine longevity genes. In this paper, we will discuss the effects of DR on the expression of insulin-related genes in 2 types of long-lived mutant mice: Ames dwarfs and growth hormone receptor knockout (GHRKO) mice.

Ames Dwarf Mice

Ames dwarfism is due to a loss-of-function mutation at the Prop-1 locus (df; Prop-1df) that leads to a congenital absence of cells producing growth hormone (GH), prolactin (PRL) and thyroid-stimulating hormone in the anterior pituitary [11–13]. Consequently, Ames dwarf mice are deficient in GH, PRL and thyroid-stimulating hormone, small and hypothyroid. Females are infertile due to PRL deficiency and the resulting luteal failure [14, 15]. In 1996 we reported that Ames dwarf mice live 45–60% longer than normal animals from

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90 80

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70 60 50 40 30 20 10 0 0

5

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25 Months

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Fig. 1. Survival plot for Ames dwarf (Prop-1df) mice.

the same line [3] (fig. 1). This observation was confirmed in subsequent studies [16]. The increased longevity of Ames dwarf mice is associated with retention of cognitive functions and other indices of delayed aging [17]. Characteristics of Ames dwarf mice which may contribute to their prolonged longevity include increased activity and/or level of antioxidant enzymes [18, 19], reduced oxidative damage [19], reduced plasma glucose and insulin levels [17], increased responsiveness to insulin [20], reduced body temperature [21], hypothyroidism [11, 13, 17] and reduced adult body size [22].

Growth Hormone Receptor/Binding Protein Knockout Mice

Targeted disruption of the GHR/BP gene in GHRKO mice leads to an absence of GHRs, GH resistance, and reduced plasma IGF-1, postnatal growth and adult body size in spite of elevated GH levels [23]. Both sexes of GHRKO mice can reproduce in spite of quantitative deficits in sexual maturation and fertility [23–25]. GHRKO ⫺/⫺ animals live significantly longer than normal (⫹/⫹ or ⫹/⫺) mice [5]. This has been shown in two laboratories and on three different genetic backgrounds [26, 27]. The extension of life span in GHRKO animals is striking and statistically significant, although apparently somewhat smaller than the extension of life in

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Table 2. Effects of murine ‘longevity genes’ resemble effects of DR

Body weight Body temperature Plasma glucose Plasma insulin Fertility Life span

Ames and Snell dwarfs

GHRKO mice

DR

↓↓ ↓↓ ↓ ↓ ↓↓ ↑↑

↓↓ ↓ ↓ ↓↓ ↓ ↑↑

↓ ↓↓ ↓ ↓ ↓↓ ↑↑

Arrows denote statistically significant effects; double arrows mean proportionately larger effects. Arrows pointing up indicate increases; arrows pointing down indicate decreases.

Ames dwarfs. The reported increases in the average longevity of GHRKO as compared to normal mice in different studies ranged from 26 to 55%, depending on genetic background and gender, while in Ames dwarfs they were between 35 and 70%, depending on gender and diet. The characteristics of GHRKO mice that might be contributing to their longevity include reduction in plasma insulin and glucose levels with increased responsiveness to insulin [28], IGF-1 deficiency and reduced body size [23, 24] and mild hypothyroidism with slight reduction in body temperature [29].

Comparison of the Effects of Dietary Restriction, Dwarfism and GHRKO

A comparison of the physiological consequences of DR with the characteristics of Ames dwarfs and GHRKO animals (table 2) reveals numerous similarities, as well as many quantitative and some qualitative differences. Reductions in body size and temperature, and lower levels of IGF-1, insulin, glucose and thyroid hormones are observed in each of the 3 ‘models’ of delayed aging, but the magnitude of these effects differs, often drastically. For example, plasma insulin levels are lower in GHRKO than in DR or dwarf mice, while thyroid hormones and body temperature are lower in Ames dwarfs than in GHRKO mice. Moreover, Ames dwarfs are completely PRL deficient [15], and DR mice are hypoprolactinemic [30], while in GHRKO mice, PRL levels are significantly elevated [24]. Food consumption per gram of body weight is significantly greater in Ames dwarfs [16] and GHRKO mice [unpubl. data] than in the corresponding

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N-AL

90

N-DR Df-AL

80

Df-DR

Survival (%)

70 60 50 40 30 20 10 0 0

5

10

15

20

25 Months

30

35

40

45

50

Fig. 2. Effects of DR in Ames dwarf mice. N-AL ⫽ Normal mice fed ad libitum; N-DR ⫽ normal mice fed under DR; Df-AL ⫽ dwarf mice fed ad libitum; Df-DR ⫽ dwarf mice fed under DR.

normal controls. This contrasts with the situation in DR animals in which reduced food consumption is imposed and eventually becomes approximately normal, i.e. appropriate for the animal’s body weight. Lastly, middle-aged and elderly dwarf and GHRKO mice often become obese, while genetically normal (wild-type) DR animals do not. Thus, it can be concluded that neither hypopituitary dwarf mice nor GHRKO mice are DR mimetics, although they share many characteristics.

Effects of Dietary Restriction on Longevity of Ames Dwarf and GHRKO Mice

In our studies, the DR protocol consisted of feeding the animals every day 70% of the amount consumed daily by mice of the same sex, age and genotype that were given unlimited access to food (ad libitum). This regimen was introduced gradually, starting at approximately 2 months of age and continued until the animals died of natural causes or were killed either for experiments or because of severe illness or apparently imminent death. In Ames dwarf mice, DR significantly increased the average and the maximal life span resembling the effects of DR in normal animals from the same stock [31] (fig. 2). This result

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was unexpected because Ames dwarfs fed ad libitum exhibit many characteristics of normal animals subjected to DR, and their exceptionally long life span would seem to make further increases in longevity very unlikely. Results of our recent study of the effects of identical regimens of DR on the longevity of GHRKO mice indicate that DR has no effect on the median or average life span of these animals [51], although it produces the expected robust increase in longevity of normal animals from the same line. Microarray analysis of wide profiles of hepatic gene expression in Ames dwarf, GHRKO and normal mice fed ad libitum or subjected to long-term 30% DR [32, 33] confirmed our earlier suggestion [31] that the effects of these ‘longevity assurance genes’ (LAGs) and DR are overlapping but not identical. From a suite of genes included in the Affymetrix U74Av2 mouse array, the expression of 212 genes was affected by Ames dwarfism only, 77 genes were affected by DR only, and 100 genes were affected by both dwarfism and DR in either an additive (n ⫽ 95) or interactive (n ⫽ 5) fashion [33]. These marked differences between the effects of a LAG and DR on gene expression were in excellent agreement with the conclusion from an earlier study in which separate and interactive effects of a different LAG (GHRKO) and DR were analyzed using Clontech microarrays [32]. However, comparison of these two studies also revealed interesting differences. For instance, in comparison to DR, Ames dwarfism affected expression levels of a greater number of genes, while GHRKO affected much fewer genes. Moreover, for genes affected by DR in both normal and GHRKO mice, the effects of DR on the expression level were proportionally smaller in the mutants [32].

Quantitative Analysis of Expression Levels of Insulin- and IGF-1-Related Genes

In view of the findings summarized above and our recent evidence that effects of DR on the longevity of Ames dwarfs and GHRKO mice are substantially different, we became interested in quantitative (real-time PCR) analysis of the effects of LAGs and DR (singly and combined) on the expression of selected genes. In these studies we are examining the expression of insulin- and IGF-1-related genes because of their suspected involvement in the control of longevity. We are also interested in the comparison of the alterations of gene expression in different organs, including major targets of insulin action, namely liver, skeletal muscle and fat. In a series of studies conducted in collaboration with Drs. Turyn, Dominici and their colleagues, we have obtained evidence that effects of LAGs on early steps of insulin signaling can be different and in some

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cases opposite in the liver as compared to the skeletal muscle [34, 35]. Moreover, the dependence of IGF-1 expression on prevailing GH levels differs widely between different tissues; therefore the consequences of GH deficiency (in Ames dwarf mice) or GH resistance (in GHRKO mice) on local IGF-1 biosynthesis are organ specific [36, 37]. The major results of our studies on the expression of insulin- and IGF-1related genes are briefly described here. In these experiments, mRNA levels were quantitated by real-time PCR and levels of the corresponding protein products were assessed by Western blots. (1) Effects of Ames dwarfism and GHRKO on the expression of the examined genes are not identical. Hepatic expression of peroxisome proliferatoractivated receptor (PPAR) ␥ was increased in both Ames dwarf and GHRKO mice in comparison to the normal animals from the same stock, while expression of IGF-1 was reduced in both mutants, as expected. However, expression of insulin receptor (IR), insulin receptor substrate (IRS) 2 and PPAR-a was increased in GHRKO but not in Ames dwarf mice [38–40]. (2) Effects of DR on gene expression differed between the Ames dwarf and the GHRKO mice. The levels of PPAR-␣ mRNA and protein in the liver were increased by DR in GHRKO mice but were not affected in Ames dwarfs. Suppression of IRS-1 message levels by DR was also observed in GHRKO but not in Ames dwarf mice [38, 40, 41]. Further work will be necessary to relate differences between the effects of DR on gene expression to the differential effects of DR on longevity in these 2 mutants. (3) Subjecting normal animals to DR does not reproduce the effects of Ames dwarfism or GHRKO on gene expression. Several of the alterations in hepatic gene expression in long-lived mutants resembled the changes measured in normal animals subjected to DR. These included increase in IR protein in dwarf mice and increases in IRS-2 message, PPAR-␣ message and PPAR-␣ protein in GHRKO mice. However, increases in IR message, IRS-1 protein, or PPAR-␥ message and protein in one or both mutants were not reproduced by DR in normal mice. Moreover, IGF-1 expression was profoundly suppressed in both mutants, but was either unaltered or increased by DR in normal mice from the examined stocks [38, 40, 41]. Studies of hepatic expression of a different set of insulin- and IGF-1related genes in GHRKO and normal mice fed ad libitum or subjected to DR revealed numerous differences [40, 42]. DR failed to reproduce stimulatory effects of GHRKO on the expression of protein kinase B (Akt-1, Akt-2), forkhead box class O (Foxo-1), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), superoxide dysmutase-2 (SOD-2), PPAR-␥, peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC-1␣) or PPARs binding partners retinoid X receptor (RxR)-a, -g or -b/d. In the same

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studies, both DR and GHRKO increased the expression of PPAR-␣, reduced the levels of phosphorylated Akt protein, and increased the levels of PGC-1␣ protein, phosphorylated p38 mitogen-activated protein kinase (MAPK) and phosphorylated cAMP response element-binding protein (pCREB). Sirt-1 protein levels were not affected by GHRKO but were increased by DR in both normal and GHRKO mice. (4) Expression of insulin- and IGF-1-related genes in different organs is differentially affected by LAGs as well as by DR. Results of ongoing studies of gene expression in the skeletal muscle, heart and adipose tissue revealed numerous differences from the results obtained in the liver. For example, IR and IRS-2 message levels were increased in the liver of GHRKO mice but not in their skeletal muscle, although IGF-1 expression was reduced in both organs. The response to DR also differed between the organs in both normal and mutant mice. In GHRKO mice and in normal animals from the same stock, DR did not affect the expression of IR, IRS-1 or IGF-1 in the liver but reduced expression of these genes in the skeletal muscle. In normal mice, levels of IRS-1 message were increased by DR in the liver but reduced in the muscle [40]. (5) Effects of DR on the expression of insulin and IGF-1-related genes differ between GHRKO and normal mice. Some of the changes produced by DR were similar in normal and GHRKO animals and thus are unlikely to be related to differential effects of DR in their longevity. These changes included increases in the levels of messages for PPAR-␣, PGC-1␣ and Sirt-1 and in the level of pCREB protein in the liver, reduced levels of mRNAs for IR, IRS-1, IRS-2, Foxo-1 and Foxo-4, reduced levels of c-Jun N-terminal kinase (pJNK)-2 and increased levels of adenosine monophosphate-activated protein kinase (pAMPK) in the skeletal muscle, and increased levels of IRS-1 and Glucose transporter-4 (Glut-4) mRNAs in the heart [40, 42, 43; unpubl. observations]. However, GHRKO mice exhibited multiple DR-induced alterations in gene expression that were not observed in normal animals. These changes could be viewed as potential causes for the failure of these animals to respond to DR with increased longevity. Among the changes we detected were increased expression of genes related to gluconeogenesis (PEPCK and G6Pase) in the liver, reduced levels of Akt-2, PGC-1␣, mRNAs and pAkt in the muscle, and increased levels of PPAR-␥, PPAR-␣, PPAR-␤/␦ and Glut-4 proteins in the heart. Of particular interest were alterations that were induced by DR in normal mice but not in GHRKO animals and thus correlated with the effects of DR on longevity. These included increased IRS-2 mRNA and reduced pAkt in the liver, reduced hepatic levels of PPAR-␤/␦ mRNA and protein, increased levels of Akt-2 and PGC-1␣ and reduced levels of JNK-1 in the skeletal muscle, and increased levels of IGF-1 and IR messages in the heart [40–43].

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Table 3. Effects of DR on the levels of proteins and phosphorylated proteins related to insulin and IGF-1 signaling in the liver and skeletal muscle in GHRKO as compared to normal mice Affected in both GHRKO and normal mice

Affected only in normal mice

Liver

PPAR-␣ ↑ pCREB ↑

pAkt ↓ p38 MAPK ↑ PPAR-␤/␦ ↓

Muscle

pAMPK ↑ pJNK-2 ↑

Akt-2 ↑ pJNK-1 ↓ PGC-1␣ ↑

Affected only in GHRKO mice

pAkt ↓

Arrows pointing up indicate increases; arrows pointing down indicate decreases. pCREB ⫽ Phosphorylated cAMP response element-binding protein.

Effects of DR on the levels of proteins and phosphorylated proteins related to insulin and IGF-1 signaling in GHRKO and normal mice are compared in table 3.

Discussion

In mammals, DR reduces plasma insulin levels, enhances insulin sensitivity and exerts complex, age-related effects on somatotropic signaling. In rats subjected to DR, GH secretion is initially suppressed but subsequently the difference in GH levels between ad libitum and DR animals becomes reversed as a result of DR delaying or reducing age-related decline in GH secretion [44]. Plasma IGF-1 levels are suppressed in DR animals, although hepatic levels of IGF-1 mRNA in DR mice may be elevated rather than reduced [38]. Studies in long-lived mutant and gene knockout mice provide very strong evidence for the role of somatotropic and insulin signaling in the control of mammalian aging and longevity and indirectly support the role of these signaling pathways in mediating the effects of DR. Results obtained in these mutants also emphasize very important differences between the consequences of partial versus complete inhibition of the release or actions of IGF-1 and insulin. Mice with the absence of IGF-1 signaling due to disruption of IGF-1 or IGF-1R genes rarely survive to adulthood [36]. In contrast, IGF-1R ⫹/⫺ mice, with approximately 50% reduction in the levels of IGF-1 receptors, and Ames, Snell and GHRKO mice, with an organ-specific deficiency of GH-dependent IGF-1

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expression, live significantly longer than their normal siblings [4, 6, 8–10]. Similarly, absence of insulin or its actions in mice or humans leads to diabetes and death, while reduced levels of insulin in dwarf and GHRKO mice and adipocyte-specific absence of IRs in FIRKO mice are associated with extended longevity [7–10]. Following this evidence, it appears that longevity benefits are related to a modest or organ-limited deficiency of IGF-1 and insulin signaling, while severe or complete suppression of the same signaling pathways may be detrimental or lethal. This conclusion helps to address a conundrum of qualitatively similar endocrine conditions that lead to serious disease in humans but delay aging and prolong life in mice. A similar conclusion derived from multiple studies – that reduced somatotropic and insulin signaling is involved in mediating the effects of DR on aging – can now be sharpened by suggesting that DR produces a favorable combination of modest, likely organ-specific reduction of IGF-1 expression, reduced insulin release and enhanced sensitivity to insulin actions. Examining the interaction of mutations affecting somatotropic and/or insulin signaling with DR may facilitate the identification of mechanisms that link altered endocrine function with delayed aging and extended longevity. Considerable overlap of phenotypic characteristics of long-lived Ames dwarf and GHRKO mice with the characteristics of normal mice subjected to DR would seem to suggest that these mutants are unlikely to benefit from DR. Indeed, one may assume that the additive effects of these mutations and DR on growth and glucose homeostasis might be incompatible with survival of these animals. Surprisingly, the mutants seem unimpaired; both Ames dwarf and GHRKO mice readily tolerate 30% DR. Moreover, Ames dwarfs respond to 30% DR by an additional extension of their life span [31]. Interestingly, our recent results indicate that an identical regimen of DR has very little effect on the longevity of GHRKO mice; the median life span was not affected and the extension of the estimated maximal life span was small and limited by gender. These unexpected observations create some novel opportunities for identifying those effects of DR that are likely to be causally related to extended longevity. For example, reduction in hepatic levels of phosphorylated Akt and PPAR-␤/␦ protein, along with an increase in phosphorylated p38 MAPK and a reduction in the levels of phosphorylated pJNK-1 in the skeletal muscle were produced by DR in normal but not in GHRKO mice. Although association does not imply causality, it is noteworthy that these DR effects were seen in animals in which DR extended life span but not those in which it did not. Comparison of the effects of DR in normal and GHRKO mice also suggests that some of the changes that would seem likely to be of functional importance may have no (or a limited) role in mediating the effects of DR on life span. For example, body weight was reduced and hepatic expression of

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PGC-1␣, Sirt-1 and PPAR-␣ were increased by DR in both normal mice that lived longer and in GHRKO animals that did not. Thus, it could be suggested that the well-documented induction of PGC-1␣ and Sirt-1 under conditions of reduced food availability is either not sufficient to increase longevity or is not effective in animals in which GH action is blocked. Similar suggestions can be made about the alterations in the expression of a number of insulin-related genes in the skeletal muscle and the heart, identified earlier in this review. Further study will be necessary to elucidate the interaction of DR with various mammalian longevity genes in the control of aging. An exploration of the responses of long-lived mouse mutants to both milder and more severe regimens of DR would also be of considerable interest, as suggested by the findings of Clancy et al. [45] in long-lived chico mutant Drosophila. The effects of genes related to an insulin/IGF-1-like signaling pathway on the life spans of worms and flies were studied in considerable detail [reviewed in 46–49]. These genes exhibit considerable homology to IGF-1- and insulinrelated genes in mammals [46, 48]. In the worm Caenorhabditis elegans, the life-extending effects of gene mutations in this pathway are generally additive to the effects of DR, while in a fly (Drosophila melanogaster), the additive effects of DR and insulin-related longevity genes have been difficult to demonstrate [48]. Making direct comparisons between the effects of DR in these species and in the mouse is challenging, because there are profound differences in life cycles, physiology (heterothermic vs. homothermic) and in the ways in which DR is implemented. In worms and flies, DR usually consists of reducing the caloric and/or nutritional value of food by dilution or changes in composition, while in laboratory rodents DR involves ingestion of reduced amounts of a complete, nutritionally balanced diet or a similar diet supplemented with a mixture of micronutrients. In future studies we will utilize differential responses to DR in normal, Ames dwarf and GHRKO mice to identify those organ-specific alterations in IGF-1 and insulin signaling that are associated with an increased life span [50]. The downstream targets of these genetic pathways should offer important clues to the mechanisms of delayed aging and extended longevity in both long-lived mutants and in normal animals subjected to DR. Acknowledgement This work was supported by the National Institutes of Health (NIH/NIA 1U19 AG023122–01A and RO1 AG19899), the Ellison Medical Foundation and the Southern Illinois University Geriatrics Research Initiative. We thank Steve Sandstrom for his help in preparing this manuscript and our colleagues who contributed to the studies discussed in this article, especially Dr. John Kopchick, who provided us with GHRKO breeder mice to start

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our colony of these animals, and to Drs. Daniel Turyn and Fernando Dominici, who demonstrated alterations in insulin signaling in Ames dwarf and GHRKO mice.

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activated receptor superfamily in muscle of normal and long-lived growth hormone receptor/ binding protein knockout mice. J Gerontol A Biol Sci Med Sci 2005;60:1238–1245. Al-Regaiey KA, Masternak MM, Bonkowski M, Sun L, Bartke A: Long-lived growth hormone receptor knockout mice: interaction of reduced insulin-like growth factor 1/insulin signaling and dietary restriction. Endocrinology 2005;146:851–860. Al-Regaiey KA: Long-Lived Growth Hormone Receptor Knockout Mice: Interaction of Reduced IGF-1/Insulin Signaling and Dietary Restriction; PhD dissertation, Department of Physiology, Southern Illinois University, Carbondale, 2005, p 103. Sonntag WE, Cefalu WT, Ingram RL, Bennett SA, Lynch CD, Cooney PT, Thornton PL, Khan AS: Pleiotropic effects of growth hormone and insulin-like growth factor (IGF) on biological aging: inferences from moderate caloric restricted animals. J Gerontol 1999;54A:B521–B538. Clancy DJ, Gems D, Harshman LG, Oldham S, Stocker H, Hafen E, Leevers SJ, Partridge L: Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein. Science 2001;292:104–106. Tatar M, Bartke A, Antebi A: The endocrine regulation of aging by insulin-like signals. Science 2003;299:1346–1351. Longo VD, Finch CE: Evolutionary medicine: from dwarf model systems to healthy centenarians? Science 2003;299:1342–1346. Kenyon C: The plasticity of aging: insights from long-lived mutants. Cell 2005;120:449–460. Kimura KD, Tissenbaum HA, Liu Y, Ruvkun G: daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 1997;277:942–946. Blüher M, Kahn B, Kahn CR: Extended longevity in mice lacking the insulin receptor in adipose tissue. Science 2003;299:572. Bonkowski MS, Rocha JS, Masternak MM, Al-Regaiey KA, Bartke A: Targeted disruption of growth hormone receptor interferes with the beneficial actions of calorie restriction. Proc Natl Acad Sci USA 2006;103:7901–7905.

Andrzej Bartke, PhD Geriatrics Research, Departments of Internal Medicine and Physiology Southern Illinois University School of Medicine PO Box 19628, Springfield, IL 62794–9628 (USA) Tel. ⫹1 217 545 7962, Fax ⫹1 217 545 8006, E-Mail [email protected]

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Anti-Inflammatory Mechanisms of Dietary Restriction in Slowing Aging Processes T.E. Morgan, A.M. Wong, C.E. Finch Leonard Davis School of Gerontology and USC College, University of Southern California, Los Angeles, Calif., USA

Abstract Dietary restriction (DR) remains the most powerful and general environmental manipulation of aging processes in laboratory animals with strong beneficial effects on most agerelated degenerative changes throughout the body. Underlying the beneficial effects of DR is the attenuation of system-wide inflammatory processes including those occurring within the central nervous system. During normal aging a progressive neuroinflammatory state builds in the brain involving astrocytes and microglia, the primary cellular components of neuroinflammation. DR attenuates the age-related activation of astrocytes and microglia with concomitant beneficial effects on neurodegeneration and cognition. Increasing evidence suggests that common pathways are emerging that link many normal aging inflammatory processes with agerelated diseases such as Alzheimer, cancer, diabetes and cardiovascular disease. Copyright © 2007 S. Karger AG, Basel

Dietary restriction (DR) remains the most powerful and general manipulation of aging processes in laboratory animals. Evidence is now overwhelming that DR increases life span by slowing the Gompertz mortality rate acceleration. The Gompertz analysis of DR was first made by Berg [1] in 1976. This fundamental effect of DR has been amply verified [2, p. 508; 3]. Corresponding to slowed mortality rates, most spontaneous degenerative changes in aging are attenuated. In rodents, the age-related increases in tumor and organ-specific pathology are delayed by DR, according to the genotype [2, 4–7]. In the widely used F344 rats, for example, chronic renal disease, which may be the major cause of morbidity, is strikingly reduced [8]. Cardiomyopathy of F344 rats is also strongly associated with the severity of kidney degeneration, but the mechanisms may be different [9]. Nonetheless, we must confront the

puzzle in F344 rats that about 25% of old DR rats have no gross organ pathology at death [8]. We suggest the possibility of metabolic instability during DR below in lesion-free aging rodents.

Dietary Restriction Attenuates Neuroinflammatory Aspects of Aging

The first indication that DR is neuroprotective for aging came from a 1985 report [10]. In some rodent colonies, hind limb paralysis becomes increasingly common during aging in association with degeneration of spinal motor neurons (radiculoneuropathy) [10, 11]. The degeneration of myelin sheaths in spinal roots arises after sporadic axonal atrophy and is associated with segmental demyelination and local ballooning [12–14]. Hind limb paralysis was markedly attenuated by DR [10, 11]. Hind limb paralysis varies widely between colonies and is unfamiliar to current researchers of aging. The greatest incidence reported, 100%, was observed in colonies before the era of modern husbandry (specific-pathogen free). In the NIA contract colony at Charles River Laboratory, in 1978–1983, rats (9 genotypes, both sexes) had a 25% incidence, with a mean age at lesion of 31 months; the incidence in mice (12 genotypes) was ⬍0.1% [15]. These major differences are puzzling and not easily attributed to improved husbandry and health. Early rodent colonies before 1970 often carried a much higher load of infections than the present specific-pathogen-free colonies. While most attention has been given to the biochemical, metabolic and genomic effects of DR, evidence is growing for the importance of system-wide anti-inflammatory effects of DR in attenuating aging [16–18]. Our laboratory is focused on neuroinflammatory changes of aging in rodents, primates and humans. These generalized aging changes arise in the absence of specific neurodegeneration [16, 17]. In aging rodents, the main brain aging changes are glial activation (microglia and astrocytes)1 [17] and synaptic atrophy [19–24]. These changes are progressive during middle age into old age and arise in the absence of disease. The type and extent of change are selective and differ extensively between even closely connected brain systems. The opposing glial and synaptic changes span a range of about 50% but are much larger than changes in cell number. In fact, several exacting studies have looked for but did not detect age changes in the total numbers of neurons [25] or glia [26]. Thus, in

1

Microglia are bone-marrow-derived monocytes which are constantly repopulated in adult brains. Astrocytes are of neural crest origin and share the same stem cell precursors as neurons.

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a

b

c

Fig. 1. The age-related increase in macrosialin (CD68) expression is attenuated by DR. Macrosialin immunoreactivity in the corpus callosum and corticostriatal bundles (insets) of 4-month ad libitum (a), 24-month ad libitum (b) and 24-month calorie-restricted (c) C57BL/6NNia mice. The arrow identifies macrosialin immunostaining at the periphery of a corticostriatal bundle in 24-month ad libitum mice. Bars ⫽ 100 ␮m for micrographs, 30 ␮m for insets. Reprinted from Wong et al. [35], with permission from Elsevier.

aging rodents and perhaps in humans, the main brain aging changes represent a type of plasticity that remodels cell cytoarchitectonic relationships without cell death. DR has a remarkable ability to attenuate these changes. White matter myelinated tracts are a robust example of the plasticity of neuroinflammatory aging. Age-Related Microglial Activation: White Matter Degeneration Macroscopically, magnetic resonance imaging studies on aging humans and monkeys show subtle structural changes in the corpus callosum, striatum and other white-matter-rich tracts [27–29]. These changes may be caused by the focal degeneration of myelin sheaths and differ by brain region. The latermyelinated regions are more susceptible to demyelination during normal aging and Alzheimer disease (AD) [30]. White matter aging is accompanied by increased microglial activation [31, 32], but cause and effect are unclear. Aging rodent models show robust increases in markers of microglial activation, e.g. CR3 (complement receptor) and MHC class II antigens (antigen presentation by macrophages) [33, 34]. These changes are attenuated by DR [33]. Most recently, we found that the scavenger receptor macrosialin (CD68), a member of the lysosomal/endosomal-associated membrane glycoprotein family, shows the greatest age-related increase in the corpus callosum of C57BL/6NNia mice; again, this is attenuated by DR (fig. 1) [35]. Because macrosialin is increased in peripheral macrophages by oxidized lipids (low-density lipoproteins) [36] and because oxidized lipids generally promote inflammation [37, 38], we hypothesize that the oxidation of white matter lipids is a factor in microglial activation. In fact, we showed that oxidized low-density

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lipoproteins induced CD68 in BV-2 microglial cells [35]. Moreover, CD68 is induced by inflammatory stimuli (lipopolysaccharide plus ␥-interferon) in BV-2 cells [35]. Therefore, CD68 serves as an inflammatory marker as well as an indicator of oxidative damage during normal brain aging. Because DR clearly attenuates age-related increases in inflammatory genes such as CD68, CR3 and MHC class II antigens, we hypothesize that DR will protect against age-related demyelinating events. An ongoing study of DR in rhesus monkeys has not given definitive information for technical reasons. After 11–13 years of DR, middle-aged (⬍24 years old) and old monkeys (⬎24 years old) had smaller putamen volumes than ad libitum fed animals [39]. However, there were no initial magnetic resonance imaging data to establish the baseline (before or at the beginning of DR). Thus, it is unresolved if the smaller putamen volumes in DR animals resulted from DR, or if the volume differences were present at the beginning of the study. Age-Related Astrocytic Activation: Glial Fibrillary Acidic Protein Astrocytes are an important source of neurotrophic factors, axonal guidance molecules and extracellular matrix molecules crucial for neuron survival and sprouting. In response to injury or disease, astrocytes take on an activated phenotype that is characterized by cell hypertrophy and upregulation of the intermediate filament proteins, glial fibrillary acidic protein (GFAP) and vimentin, as well as inflammatory mediators and extracellular matrix molecules [40, 41]. However, during normal aging astrocytes become activated with concomitant increases in GFAP and vimentin in the absence of overt pathology [42–44]. This age-related astrocytic activation [45, 46] contributes to agerelated increased inflammatory and oxidative damage [44, 47], decreased neurogenesis [48] and synaptic atrophy [19]. We are investigating the hypothesis that the increase in GFAP expression is a primary cause in synaptic atrophy and impaired synaptogenesis during normal aging [46]. We have developed a heterochronic cell culture model to test this hypothesis. In brief, test neurons (E18 cortex) are seeded on monolayers of primary cultures of astrocytes from young adult or aging rat cerebral cortex. The old-rat-derived astrocytes retain the high GFAP per cell [49] as observed in vivo [33]. Moreover, the E18 neurites outgrow poorly on old-rat-derived astrocytes. These age impairments in neurotrophic support are rapidly reversed by downregulating GFAP by small interfering RNA [46]. The mechanism involves an inverse relationship between GFAP expression and secretion of laminin, a critical component of the extracellular matrix that guides neurite outgrowth. Additional support for the critical role of GFAP comes from studies on mice lacking both GFAP and vimentin which have improved synaptic regeneration and increased neurogenesis [50, 51].

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Just as age-related microglial activation is reduced by DR (discussed above), DR is also effective at attenuating many of the genotypic and phenotypic changes that astrocytes undergo during aging. The age-related increase in GFAP is attenuated by DR [43, 44, 52] and this occurs at the transcriptional level [33, 53]. Microarray profiling confirmed the effects of DR on GFAP [17, 44]. Although neuropathologists have long used GFAP as a marker of neurodegeneration, our work clearly shows that the age increase in GFAP arises in the absence of neuron cell death and may be an upstream factor in synaptic atrophy during aging. Because of the concurrent activation of microglial inflammatory markers, we provisionally consider that GFAP is embedded in a neuroinflammatory network. The beneficial effect of DR on glial activation may underlie DR’s ability to attenuate age-related declines in synaptic plasticity and neurogenesis [54–58]. Ongoing studies are evaluating if DR improves the neurotrophic support of aging glia. Age-Related Neurodegenerative Disease: Experimental Rodent Models DR also protects against neurodegenerative processes in experimental rodent models. For example, DR protects neurons from many toxins, including methylphenyltetrahydropyridine [59], kainic acid [60], 3-nitropropionic acid and malonate [60, 61]. AD-like changes do not arise in aging rodents, possibly because the rodent ß-peptide has several amino acid substitutions that decrease its aggregation into fibrillar amyloids that are characteristic of AD [62]. However, mice carrying human transgenes for early-onset familial AD develop fibrillar amyloids and various other specific AD-like neuropathological changes during aging. We have recently shown that DR attenuated brain deposits of brain amyloid by 50% within the short time of several months [63] (fig. 2a). These changes were accompanied by a reduction in GFAP in astrocytes surrounding the plaque (fig. 2b). We demonstrated these beneficial effects of DR in two transgenic mouse models of AD, APPswe/ind and APP⫹PS1 [63]. These effects of DR also extend to a third genotype, Tg2576 [64]. Low-energy diets in humans are being considered as an approach to lowering AD risk, because in retrospective studies, AD victims tended to have higher calorie intake [65, 66]. Of course, it is much harder to establish causality of diet in humans, because individuals who adopt special diets also often pursue other health-promoting activities, such as exercise which may protect against cognitive declines in normal aging [67, 68]. Another example of DR providing age-related neuroprotective activities is in the experimental model of retinal ischemia/reperfusion [69]. As observed in cortical and hippocampal regions (see above), microglia and astrocytes become progressively activated in the aged retina [69]. Further glial activation occurs when the aged retina is subjected to ischemia/reperfusion with concomitant

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Fig. 2. DR reduces ␤-amyloid (A␤) number and ␤-amyloid-associated astrocyte activation relative to ad libitum (AL) feeding. a Plaque size and total ␤-amyloid plaques were reduced by DR in APPswe/ind (*p ⬍ 0.05, n ⫽ 7–8). b Sholl analysis of concentric rings around ␤-amyloid plaques (inset) showed reduced GFAP immunoreactivity nearest to plaques in DR versus ad libitum feeding (p ⬍ 0.05). Reprinted from Patel et al. [63], with permission from Elsevier.

neuronal damage. In this model of ischemia/reperfusion with individual eyes, DR attenuated retinal glial activation and neuronal damage [69]. In fact, these authors suggest that the beneficial effects of DR are directly related to its effect on glial activation supporting the hypothesis that the anti-inflammatory actions of DR on glia may mediate neuroprotection.

Dietary Restriction Attenuates Inflammatory Processes

Microarray Profiling Highlights Anti-Inflammatory Effects of Dietary Restriction The broad scope of inflammatory gene expression during brain aging has become clear through the numerous publications utilizing microarray gene expression profiling [44, 70, 71]. These studies showed that inflammation-related genes increased during aging. Importantly, DR attenuated the age-related increase in inflammatory genes [44]. In fact, DR prevented the age-related increased expression of 65% of those genes involved in the inflammatory response in the neocortex [44] suggesting that this is a primary mechanism underlying the beneficial effect DR has on brain aging processes. Suppression of Inflammation in Acute Dietary Restriction Inflammatory responses are attenuated by DR throughout the body [17, 72]. We begin with examples from the skin. In the classic pharmacological

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model of footpad edema induced by subcutaneous injection, DR shortened the inflammatory responses in young mice on DR for 8 weeks [73]. In clinical studies, dermatitis was also improved by 8 weeks on a low-energy diet with micronutrient supplements. All patients responded to some degree, with the reductions of edema, oozing and skin sloughing (excoriation) being correlated with weight loss [74]. Inhibition of keratinocyte proliferation, an observed effect of DR in young mice [75], may contribute to the reduced excoriation. In humans, serum C-reactive protein (CRP) was 80% lower in a selfselected group that had maintained DR for 8 years [76]. CRP is an acute-phase protein secreted by the liver in humans [77], which is an important host defense molecule by binding to Gram-negative bacteria and enhancing their clearance by phagocytosing macrophages. However, CRP also has major importance in vascular disease as a risk indicator and for its potential direct role in lipid accumulations by macrophages (foam cells) in atheromas. Serum CRP is elevated during obesity and, not surprisingly, short-term weight reduction decreased serum CRP by 30% [78, 79]. Here we confront the complexities of weight reduction. DR could enhance the host defense by lowering blood glucose [80–82], yet DR diminishes CRP and possibly other defenses. Changes in gene expression in the liver during short-term DR (3–30 weeks) have been profiled by microarrays in several studies [83–87]. Agreement is emerging, despite differences in the choice of rodent genotypes, duration of DR and microarray technologies. Short-term DR induces and represses many mRNAs in the liver that mediate increased gluconeogenesis, increased protein and fatty acid catabolism, and decreased synthesis of cholesterol, fatty acids and triglycerides [84, 87]. The Krebs cycle (tricarboxylic acid cycle) drives these changes, with increased shunting of pyruvate to oxaloacetate in the liver by increased activity of pyruvate carboxylase [88]. The increased oxaloacetate feeds into gluconeogenesis after conversion by malate dehydrogenase, which is also increased by DR. Besides transcriptional changes in these genes, levels of activity in some enzymes are allosterically regulated, e.g. pyruvate carboxylase is activated by acetyl-CoA, which is increased by the ß-oxidation of fatty acids liberated during lipolysis. Acute-phase response mRNAs are also decreased, including serum amyloid A4 and several complement system factors (mannose-binding lectin, C4-binding protein, C9) [84]. DNA repair is upregulated (Rad511), as are CYP450 family genes that mediate detoxification and decrease DNA damage. Again, there is impressive overlap of genes associated with lipid metabolism and vascular disease. Overall, these 50–100 mRNA changes are a small subset (⬍1%) of all the genes active in the liver. The race is on to find transcription factors that are shared key regulators of these gene subsets. The effects of DR on many diseases of aging with inflammatory components give a basis to look for transcription

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factors that could modulate inflammatory gene subsets implicated in AD, cancer, diabetes and vascular disease [17, 72]. Corton et al. [84] have shown in the liver that the transcription factors peroxisome proliferator-activated receptor (PPAR), liver X receptor and retinoid X receptor, which regulate many genes during DR, also have major roles in inflammation. Additional experimental models include PPAR knockout mice and drug antagonists, which induce mRNA changes that overlap with DR to some extent (see below).

Mechanisms Underlying Anti-Inflammatory Actions of Dietary Restriction

Glucocorticoids DR increases blood glucocorticoids by 20% or more [18, 73, 89]. The increased glucocorticoids are a homeostatic response to increase the catabolism of fatty acids for energy (gluconeogenesis), while decreasing the synthesis of fatty acids and cholesterol. If the energy deficit is prolonged, protein catabolism is also increased. DR also decreases the tissue content of oxidatively damaged proteins and lipids, which are always present with enough food intake and which accumulate during aging. Importantly, glucocorticoids have broad anti-inflammatory effects mediated by the direct interaction between the glucocorticoid receptor and the transactivation domain of NF-␬B which serves as a key transcription factor in the regulation of inflammation [90, 91]. Because chronically elevated glucocorticoids are also broadly associated with neuronal damage and neuronal death, it is paradoxical that DR is neuroprotective [18]. Glucose and Advanced Glycation End Products DR lowers blood glucose by about 10–15%. Blood glucose levels directly influence the formation of oxidation products, as was outlined two decades ago in Cerami’s hypothesis of glucose as a mediator of aging [92]. Glucose and other reducing sugars react spontaneously (nonenzymatically) with free amino groups of proteins (e.g. –NH2 of lysine) to form an initial ‘glycation’ product by the Amadori reaction, which is assayed as furosine. Then, Amadori glycation products become oxidized to ‘glycoxidation products’, assayed as pentosidine, which are also referred to as advanced glycation end products [93]. DR inhibits glycoxidation during aging in rodent skin, whereas diabetes and end-stage renal disease accelerate glycoxidation [93–95]. Advanced glycation end product adducts are recognized by a macrophage scavenger receptor, the RAGE (receptor for advanced glycation end products) of monocytes (macrophages, microglia) and other cells. RAGEs are also

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activated by the ␤-amyloid peptide and other stress-associated proteins (S100/calgranulins). A working hypothesis is that advanced glycation end products and RAGEs mediate feed-forward loops of oxidative stress and inflammation that increase bystander molecular damage in atherosclerosis, AD and other chronic inflammatory diseases [96, 97]. In turn, RAGE activation enhances proinflammatory pathways that release cytokines (e.g. interleukin 6) and leukocyte adhesion factors (e.g. monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1), and that induce the enzymatic synthesis of reactive oxygen species through NAD(P)H oxidases (e.g. gp91phox) and mitochondrial electron transport. Lastly, RAGE activation may stimulate feed-forward vicious cycles by autoinduction in the same cell [98–100]. RAGE downstream signaling pathways include phosphatidylinositol triphosphate kinase, NF-␬B and JAK/stat. Feedback loops include the induction of RAGE by tumor necrosis factor ␣ through production of reactive oxygen species, mediated by NF-␬B [101]. RAGE-dependent processes are also implicated in AD. The lower glucose may also be a risk factor in sudden death. Recall the puzzle that some DR rats died without evidence of gross pathology. We suggest the precedent of the sudden ‘dead-in-bed syndrome’ of humans. Transient hypoglycemia is implicated in sudden death from cardiac arrest in type 1 diabetics (insulin-deficient), who have 3-fold more unexpected death than healthy young individuals [102]. Peroxisome Proliferator-Activated Receptors As discussed earlier, the nuclear hormone superfamily of PPARs may play a critical role in mediating many of the transcriptional effects of DR in peripheral systems. Indeed, in the rat kidney PPAR mRNA, protein and DNA binding activities are decreased with age and these changes are attenuated with DR [103]. While the PPARs show wide distribution among glia and neurons in the brain [104], the effects of age or DR have not been documented. Although PPARs are best known for their precise transcriptional control of metabolic events, certain subtypes (in particular, PPAR-␥) mediate inflammatory processes [105–107]. Regarding the brain, PPAR stimulation reduces neuroinflammation, both in vivo [108, 109] and in vitro [105, 110, 111]. Thus, PPAR mediation of the anti-inflammatory effects of DR in the brain seems likely.

Conclusion

DR attenuates many age-related inflammatory events in the CNS and periphery of experimental animal models in concert with increasing life span. In the aging brain, DR suppresses the activation of microglia and astrocytes which are

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associated with demyelination, synaptic atrophy and neurodegeneration. These events are believed to be the underlying causes of age-related cognitive decline. Rodent models suggest that DR may also protect against age-related neurodegenerative diseases involving inflammation such as AD and ischemia/reperfusion. Even short-term DR can attenuate inflammation and affect metabolic and DNA repair pathways. Mechanisms by which DR suppresses peripheral inflammation include the elevation of glucocorticoids, lowering of glucose and activation of PPARs. Although the effects of DR are less understood in the brain, common pathways are emerging that link many normal aging inflammatory processes with agerelated diseases such as AD, cancer, diabetes and cardiovascular disease.

Acknowledgement Supported by grants to C.E.F. and T.E.M. (AG13499 and the Alzheimers Association Temple Award) and A.M.W. (AG00093).

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81 Butler SO, Btaiche IF, Alaniz C: Relationship between hyperglycemia and infection in critically ill patients. Pharmacotherapy 2005;25:963–976. 82 Swenne CL, Lindholm C, Borowiec J, Schnell AE, Carlsson M: Peri-operative glucose control and development of surgical wound infections in patients undergoing coronary artery bypass graft. J Hosp Infect 2005;61:201–212. 83 Miller RA, Chang Y, Galecki AT, Al-Regaiey K, Kopchick JJ, Bartke A: Gene expression patterns in calorically restricted mice: partial overlap with long-lived mutant mice. Mol Endocrinol 2002;16:2657–2666. 84 Corton JC, Apte U, Anderson SP, Limaye P, Yoon L, Latendresse J, Dunn C, Everitt JI, Voss KA, Swanson C, Kimbrough C, Wong JS, Gill SS, Chandraratna RA, Kwak MK, Kensler TW, Stulnig TM, Steffensen KR, Gustafsson JA, Mehendale HM: Mimetics of caloric restriction include agonists of lipid-activated nuclear receptors. J Biol Chem 2004;279:46204–46212. 85 Dhahbi JM, Mote PL, Wingo J, Tillman JB, Walford RL, Spindler SR: Calories and aging alter gene expression for gluconeogenic, glycolytic, and nitrogen-metabolizing enzyme. Am J Physiol 1999;277:E352–E360. 86 Dhahbi JM, Cao SX, Mote PL, Rowley BC, Wingo JE, Spindler SR: Postprandial induction of chaperone gene expression is rapid in mice. J Nutr 2002;132:31–37. 87 Dhahbi JM, Kim HJ, Mote PL, Beaver RJ, Spindler SR: Temporal linkage between the phenotypic and genomic responses to caloric restriction. Proc Natl Acad Sci USA 2004;101:5524–5529. 88 Hagopian K, Ramsey JJ, Weindruch R: Krebs cycle enzymes from livers of old mice are differentially regulated by caloric restriction. Exp Gerontol 2004;39:1145–1154. 89 Sabatino F, Masoro EJ, McMahan CA, Kuhn RW: Assessment of the role of the glucocorticoid system in aging processes and in the action of food restriction. J Gerontol 1991;46:B171–B179. 90 McKay LI, Cidlowski JA: Molecular control of immune/inflammatory responses: interactions between nuclear factor-␬B and steroid receptor-signaling pathways. Endocr Rev 1999;20: 435–459. 91 Smoak KA, Cidlowski JA: Mechanisms of glucocorticoid receptor signaling during inflammation. Mech Ageing Dev 2004;125:697–706. 92 Cerami A: Hypothesis: glucose as a mediator of aging. J Am Geriatr Soc 1985;33:626–634. 93 Ulrich P, Cerami A: Protein glycation, diabetes, and aging. Recent Prog Horm Res 2001;56:1–21. 94 Monnier VM, Kohn RR, Cerami A: Accelerated age-related browning of human collagen in diabetes mellitus. Proc Natl Acad Sci USA 1984;81:583–587. 95 Sell DR, Carlson EC, Monnier VM: Differential effects of type 2 (non-insulin-dependent) diabetes mellitus on pentosidine formation in skin and glomerular basement membrane. Diabetologia 1993;36:936–941. 96 Ramasamy R, Vannucci SJ, Yan SS, Herold K, Yan SF, Schmidt AM: Advanced glycation end products and RAGE: a common thread in aging, diabetes, neurodegeneration, and inflammation. Glycobiology 2005;15:16R–28R. 97 Lu C, He JC, Cai W, Liu H, Zhu L, Vlassara H: Advanced glycation endproduct (AGE) receptor 1 is a negative regulator of the inflammatory response to age in mesangial cells. Proc Natl Acad Sci USA 2004;101:11767–11772. 98 Basta G, Lazzerini G, Del Turco S, Ratto GM, Schmidt AM, De Caterina R: At least 2 distinct pathways generating reactive oxygen species mediate vascular cell adhesion molecule 1 induction by advanced glycation end products. Arterioscler Thromb Vasc Biol 2005;25:1401–1407. 99 Wautier MP, Chappey O, Corda S, Stern DM, Schmidt AM, Wautier JL: Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE. Am J Physiol Endocrinol Metab 2001;280:E685–E694. 100 Feng L, Matsumoto C, Schwartz A, Schmidt AM, Stern DM, Pile-Spellman J: Chronic vascular inflammation in patients with type 2 diabetes: endothelial biopsy and RT-PCR analysis. Diabetes Care 2005;28:379–384. 101 Mukherjee TK, Mukhopadhyay S, Hoidal JR: The role of reactive oxygen species in TNF␣-dependent expression of the receptor for advanced glycation end products in human umbilical vein endothelial cells. Biochim Biophys Acta 2005;1744:213–223. 102 Heller SR: Abnormalities of the electrocardiogram during hypoglycaemia: the cause of the dead in bed syndrome? Int J Clin Pract Suppl 2002;129:27–32.

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T.E. Morgan, PhD Leonard Davis School of Gerontology University of Southern California, 3715 McClintock Avenue Los Angeles, CA 90089–0191 (USA) Tel. ⫹1 213 740 4083, Fax ⫹1 213 740 0853, E-Mail [email protected]

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Mobbs CV, Yen K, Hof PR (eds): Mechanisms of Dietary Restriction in Aging and Disease. Interdiscipl Top Gerontol. Basel, Karger, 2007, vol 35, pp 98–114

Dietary Restriction in the Nematode Caenorhabditis elegans Koen Houthoofd a,b, David Gemsb, Thomas E. Johnsonc, Jacques R. Vanfleterena a

Department of Biology, Ghent University, Ghent, Belgium; bDepartment of Biology, UCL Centre for Research on Ageing, University College London, London, UK; c Department of Integrative Physiology, Institute for Behavioral Genetics, University of Colorado at Boulder, Boulder, Colo., USA

Abstract The nematode Caenorhabditis elegans has proved to be an excellent model organism for the study of development and aging. Many aging mutants have been discovered in the past two decades, and much has been discovered about the physiology of long-lived mutants. It therefore seems surprising that dietary restriction (DR) has not been extensively studied using C. elegans. The main reason for this is the lack of an ideal method to subject C. elegans to DR. However, several authors have tried to study the effect of DR on the metabolism and physiology of C. elegans, and epistasis-type interaction studies have been carried out in order to detect genes that might be involved in DR effects. These studies show that DR life extension is not caused by a reduced metabolic rate, consistent with results in other species. Moreover, the well-known insulin/IGF-1 pathway seems not to mediate life-extending effects. One possibility is that target of rapamycin signaling mediates the effects of DR on life span in C. elegans. Copyright © 2007 S. Karger AG, Basel

The beneficial effect of food restriction on life span was first described in rodents 70 years ago and later shown to occur in a wide range of vertebrate and invertebrate taxa [1–4]. Because of its wide occurrence it is believed that this effect enhances fitness and represents an evolutionary adaptation. The underlying reasoning is that there are trade-offs between longevity and reproduction. Fitness is strongly determined by reproductive success. It will therefore be advantageous to allocate energy resources to reproduction as long as investment

in maintenance is sufficient to support a longevity that is only limited by environmental hazards, e.g. predation. When food is scarce, reproductive success will decrease to some point where it becomes advantageous to invest as much as possible in somatic maintenance thereby delaying reproduction until food supply improves. This disposable soma theory of aging is very plausible and mathematical modeling suggests that it is applicable to rodents [5, 6]. The notion that life span extension by dietary restriction (DR) is a direct evolutionary adaptation suggests the existence of regulatory (signaling) pathways that sense nutrient availability and enhance investment in somatic maintenance accordingly to maximize life time fitness. However, nonadaptive explanations for the effect of DR remain possible. For example, DR might attenuate oxidative damage accumulation inflicted by reactive oxygen species (ROS) by lowering ROS production. Alternatively, DR could stimulate degradation of macromolecules for recycling when basic units for synthesis (e.g. amino acids, monosaccharides, purines, pyrimidines) are scarce. Enhanced recycling could assure rapid clearance of damaged macromolecules to the benefit of somatic maintenance. Simple invertebrate species including the nematode Caenorhabditis elegans and the fly Drososphila melanogaster are experimentally tractable model organisms for studying the interplay between environmental conditions and the genes and signaling pathways that mediate life extension. Experiments in C. elegans suggest that the insulin (Ins)/IGF-1 pathway is not involved in DR-induced life extension and point to a more important role of the target of rapamycin (TOR) pathway.

Studying Dietary Restriction in C. elegans

C. elegans is a free-living, microbivorous soil-dwelling nematode. Food is detected via olfactory and chemosensory perception by amphids, paired sense organs in the head region of the worm that contain the ciliated endings of sensory neurons allowing the worm to respond to changes in the environment. Food is taken up via peristaltic contractions of muscles in the pharyngeal region. The microbes (e.g. bacteria) are ground in the terminal bulb of the pharynx, and the remaining debris is passed into the intestine, which runs most of the body length. The intestine is a 1-cell-thick epithelial tube with microvilli on the luminal side. The intestinal cells are thought to absorb nutrients via pinocytosis while smaller molecules are probably taken up by specific receptors; however, the biology of nutrient uptake remains poorly characterized. Nutrients are then most probably secreted through the basal surface into the pseudocoelomic fluid, which contacts most tissues [7, 8].

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In the laboratory, C. elegans is usually cultured on a lawn of Escherichia coli bacteria on agar plates. Typically, the slow-growing OP50 strain, which is auxotrophic for uracil, is used because the thin bacterial lawn that it forms makes the microscopic study of C. elegans easier. A thin, live E. coli lawn is thus considered as the normal, nonrestricted diet of the worm. C. elegans can also be grown in liquid suspension cultures with E. coli as food source, but shaking vigorously is needed if the depth of the medium exceeds a few millimeters, to prevent hypoxic stress to the worms [9]. The ideal method for studying the importance of calories in the DR effect in C. elegans would be the use of a medium containing all essential nutrients needed for a maximal life span and a reduced amount of calories (e.g. in the form of E. coli). However, this is difficult to achieve for several reasons. The first problem is that the normal food source of C. elegans in the laboratory, E. coli, is slightly toxic to the worm. In old worms, E. coli cells frequently accumulate in and block the pharynx, the intestine and the uterus of the worm [10; own observations]. Feeding C. elegans with E. coli that was killed by UV irradiation or by antibiotics resulted in a 16–40% increase in mean life span [10, 11]. Treating the bacteria with a bacteriostatic agent also resulted in life extension, suggesting that something associated with the proliferation of bacteria reduces the life span of the worm [10]. This is consistent with previous claims that toxins that are produced by proliferating bacteria might be the causative agent of life span reduction [12, 13]. Reducing the E. coli intake thus not only lengthens the worm’s life span by reduced caloric intake, but also by reducing E. coli toxicity. A second difficulty in studying DR in the worm is that the beneficial effects of reduced caloric intake are possibly offset by malnutrition, since both calories and essential nutrients are provided by the same food source (E. coli cells). Thus, reducing the E. coli intake also reduces the availability of compounds that are necessary for maximizing the life span of the worm. Thirdly, wild-type C. elegans that are fed E. coli that has a deficiency in the synthesis of ubiquinone, live substantially longer [14]. Therefore, reducing the amount of bacterial uptake might lead to life span extension due to reduced ubiquinone uptake. It is therefore necessary to keep in mind that the beneficial effects of reduced bacterial food intake are not only due to reducing calories. Given that no evidence has ever been presented that it is the reduction of calories that is critical to DR effects in C. elegans, we use the term ‘dietary restriction’ rather than ‘caloric restriction’ (CR) throughout the text. Many different methods have been used to investigate the effects of DR on various parameters in C. elegans, but all suffer from at least one of the abovementioned potential problems. The methods used can be roughly grouped into three classes: (1) reducing the available amount of bacteria, (2) using synthetic

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media and (3) reducing levels of the receptors necessary for the uptake of molecules into the intestinal cells. In a pioneering study, Klass [15] decreased the bacterial concentration in suspension culture to impose DR on C. elegans. He found a mean life span extension of 60% when the bacterial density was decreased from 109 to 108 bacterial cells/ml (higher concentrations lead to a decreased life span and decreased reproductive capacity, probably because of hypoxic stress). Under these conditions, progeny production was decreased more than fourfold. Hosono et al. [16] reduced bacterial concentrations on agar plates by decreasing the amounts of bactopeptone. They also observed life extension, but no reduction of reproductive capacity or body volume was seen in this case. The advantage of restricting worms by reducing bacterial concentration is that this treatment can be applied in a quantitative manner, which is useful if one wants to test the interaction with other life-extending mechanisms [17]. Reduced bacterial uptake can also be obtained genetically by using mutants with a reduced pumping rate. Such Eat mutants have a starved appearance and were used by Lakowski and Hekimi [18] to study the genetics of DR. [Note that in C. elegans nomenclature, Eat refers to the phenotype, eat-1 (ad427) to the gene (allele) and EAT-1 to the protein encoded by eat-1.] These authors found that most Eat mutants were indeed long-lived, with a maximal life extension of about 50%. However, smaller or no effects on life span were found in some other laboratories [19]. A likely explanation is that these mutants experience DR depending on the environmental conditions such as the thickness of the bacterial lawn. For example, we saw life extension when the Eat mutants were grown in liquid culture, but not on plates [Houthoofd, unpubl. results]. Perhaps reduced bacterial intake of Eat mutants is not limiting when they are grown on plates with plenty of E. coli. Reducing the amount of E. coli is the method that shows the most similarity with DR studies in other species but suffers from the above-mentioned problems. C. elegans can also be grown in sterile axenic media (axenic: grown in the absence of any other species). One example of such a medium is Caenorhabditis briggsae maintenance medium, a defined medium containing 54 compounds [20]. A more frequently used, semidefined, axenic medium is composed of yeast extract and soy peptone [21, 22]. A sterol and heme source must be added to axenic media since C. elegans is not able to synthesize these compounds. The heme requirement was originally met by adding tissue extracts, e.g. liver extract or chicken embryo extract. Later it was found that pure hemoglobin is a suitable supplement [23]. Sufficient sterols are supplied as impurities in yeast extract, soy peptone and the heme source. When grown in axenic media, the life span of worms is about twice as long as in populations maintained on E. coli [22, 24]. Axenically cultured worms have a retarded development and severely reduced fertility compared to monoxenic culture conditions, which is also observed

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under other DR regimens. That axenic culture entails DR is also suggested by the observation that worms grown in axenic medium show several metabolic and stress defense alterations similar to those seen in worms that are restricted by eat mutation or by lowering bacterial food supply [22, 25]. However, axenic media are generally rich in nutrients and it is therefore puzzling that worms that are grown axenically seem to experience DR. Possible explanations are that axenic medium cannot be taken up by the worms efficiently, either because worms are filter feeders, spitting out most of the liquid medium, or because compounds are not taken up efficiently by the intestine. Another possibility is that some nutrients in the medium cannot be metabolized by the worm. Finally, this medium might simply not fully meet the nutritional needs of this worm, though this seems unlikely. Since axenic media are sterile, life extension is partially caused by the absence of pathogenic bacteria and possibly ubiquinone. And, clearly, axenic medium cannot be used as a means to partially reduce caloric intake: it is loaded with calories. Knocking down the activity of several transporters has also led to life extension in C. elegans. For example, RNAi (RNA-mediated interference) of nac-2 (transporter of di- and tricarboxylates) or nac-3 (transporter of dicarboxylates) leads to a life extension of 19 and 15%, respectively [26, 27]. Decreased NAC-2 activity also caused a reduction in body size and intestinal lipid content, phenotypes not seen in worms with lower NAC-3 activity. nac-2 is an orthologue of the Drosophila Indy (‘I’m not dead yet’) gene. As its name suggests, a mutation in Indy also causes life extension, but these flies have no lower brood size (when fed ad libitum) [28]. PEP-2 (formerly known as OPT-2) is a proton-dependent carrier responsible for the uptake of di- and tripeptides. Mutation in pep-2 leads to a smaller body size and reduced developmental rate and fertility but does not increase life span. However, mutation in pep-2 extends the life span of long-lived daf-2 mutants [29]. The reason for this is unclear, but it might be that the pep-2 mutation is too strong, causing malnutrition effects offsetting the positive effects of DR in wild-type worms, but not in daf-2 worms, which have more fat stores, possibly protecting them against starvation. The NHX-2 Na⫹/H⫹ exchanger is needed to prevent acidification of the cytoplasm [30]. RNAi of nhx-2 increases life span, among other DR phenotypes, probably because uptake of di- and tripeptides is inhibited when the cytoplasmic pH drops.

Possible Mechanisms of Dietary-Restriction-Mediated Life Extension

The mechanism by which DR extends life span is still unknown. Stochastic as well as regulated mechanisms have been proposed. Most early models

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attribute the action of DR to a reduction of ROS production, consistent with the free radical theory of aging [31]. Drawing on the discovery of several signaling pathways that regulate the aging rate of C. elegans, there is a growing belief that DR effects may be mediated by (one of) these pathways [19]. Reduced Metabolic Rate? Early in the 20th century, an inverse relationship between life span and mass-specific oxygen consumption was observed in a comparison of eight mammals [32]. Others found a similar inverse relationship between life span and environmental temperature in insects [33]. These observations were used by Pearl [34] as a basis for his rate-of-living theory (‘live fast, die young’). This theory revived strongly when Harman formulated the free radical theory of aging in 1956 [31]: a lower metabolic rate would, it was thought, lead to reduced mitochondrial ROS production. It seemed reasonable that DR could lead to a reduced metabolic rate since less fuel is available for driving the mitochondrial electron transport chain, and it was thus not surprising that one of the most commonly proposed mechanisms of DR-induced life extension was reduced metabolic rate. Since then it was realized however that a higher aerobic metabolism is not necessarily linked to increased free radical generation, since ROS production is dependent on the inner mitochondrial membrane potential. In fact, membrane potential and ROS production are inversely related with respiratory activity: high in resting mitochondria and low in actively respiring mitochondria [35–37]. Uncoupling proteins can also lower the membrane potential, again leading to a lowering of free radical generation. For instance, Speakman et al. [38] found that individuals with the highest metabolism, in a mouse population, had the highest mitochondrial uncoupling rate and the longest life span. A reduced ROS production rate could be caused by shifting to an anaerobic metabolism [39] or by increasing the efficiency of the electron transport chain [40]. In an attempt to study the interaction between dietary restriction and metabolic rate, Lakowski and Hekimi [18] constructed double mutants of eat-2 and clk-1, a mutant with a slowed behavior and long life span [41–43]. They found that mutation in the clk-1 gene could not further extend the life span of eat-2 mutants and hence concluded that both mutations lengthen the C. elegans life span via the same mechanism. Since it was believed that clk mutants were longlived due to a reduction of metabolic rate, it was concluded that DR postpones aging by lowering the metabolic rate. However, later studies showed that clk mutants had no reduced respiration or heat production rate [44, 45], undermining the proposed hypothesis. Houthoofd et al. [22, 25, 46] measured respiration and heat production rate directly in worms that were restricted by E. coli dilution, eat mutation or growth

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in axenic medium. Surprisingly, they found that DR leads to an increase in metabolic rate, at least when expressed per unit of body mass. They next determined the ATP content in these worms and found that DR causes lower ATP concentrations. These authors also found that DR led to a higher reductive capacity, measured as the maximal potential to reduce XTT in the presence of exogenous NAD(P)H. A possible reason for these observations is that the high metabolic rate is needed for the synthesis of molecules that are freely available in the diet of ad libitum fed worms, but absent or at a lower concentration in restricted worms. These anabolic reactions would require ATP and reductive reactions. Another possible energy-demanding process is increased protein turnover, as observed in mammals and yeast subjected to DR [47, 48]. In principle, this could retard aging by lowering levels of damaged proteins in the cell. The protein turnover rate has not been tested in C. elegans subjected to DR yet. An increased respiration rate as a response to DR is consistent with experiments in yeast. Lin et al. [49] have shown that DR causes a shift from fermentative towards respirative metabolism and that life extension caused by DR depends on the activity of the tricarboxylic acid cycle. Mitochondria from DR rodents show a lower membrane potential and produced less ROS [reviewed in 40, 50]. The ROS production rate in response to the nutritional regime of C. elegans awaits testing. Increased Stress Resistance? Life span extension in C. elegans is often correlated with increased resistance to various stresses [51–53], and an upregulation of the activity of ROSdetoxifying enzymes is often observed in long-lived mutant strains [52, 54, 55]. Houthoofd et al. [22, 24] determined the resistance to oxidative and heat stress in worms that were grown under axenic conditions. They found that restricted worms had a superior resistance to both stresses. Moreover, this was accompanied by higher activities of superoxide dismutase and catalase, two enzymes that are involved in the breakdown of ROS. pep-2 mutants have a higher heat tolerance and an increased resistance to oxidative stress [29], but are not longlived, as mentioned previously. An upregulation of stress resistance in response to DR could be the consequence of a hormetic response (i.e. stress-induced stress resistance) [56, 57]. By this view, DR is a low-intensity stressor, and animals subjected to DR react to this stressor by upregulating the stress defense system which also protects them against aging. Reduced Ins/IGF-1 Signaling? If increased stress defense is necessary for DR-induced life extension, the DR response is likely mediated by a signaling pathway that regulates the expression of a life-extending program in response to the nutritional status of

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the organism. Good candidates are the JNK [58] and the Ins/IGF-1-like signaling pathways. Both converge on the transcription factor DAF-16. The Ins/IGF1-like signaling pathway is an evolutionary conserved pathway similar to the Ins and IGF-1 mammalian pathways. This pathway regulates the aging rate in worms, flies and mice [reviewed in 59]. Inactivation of this pathway by mutation in the Ins/IGF-1 receptor daf-2 or in one of the downstream genes (e.g. age-1) in the worm results in a substantial life span extension that is dependent on the transcription factor DAF-16 [60]. DAF-16 accumulates in the nucleus of worms with reduced signaling activity, resulting in the increased expression of many genes that confer resistance to stress or detoxification, and enhance mean and maximum life span [61–65] (fig. 1). The similarity with the mammalian Ins pathway and several observations in the worm suggested a role for the Ins/IGF-1 pathway in life span extension caused by DR. First of all, the Ins/IGF-1 pathway plays an important role in the formation of dauers (a long-lived and stress-resistant larval stage) in C. elegans, and food availability is one of the regulating factors for dauer formation [67]. Secondly, several of the 39 Ins-like peptides are expressed in amphid neurons [68] and the longevity phenotype of mutants with defective olfactory perception is dependent on the DAF-16 transcription factor, suggesting the involvement of the Ins/IGF-1 pathway in the food-sensing process [69]. Also, it is reported by one group that DAF-16 activity in the intestinal cells, which are responsible for the uptake of nutrients and for the transport of nutrients to the pseudocoelomic fluid, is necessary and sufficient to regulate the worm’s life span [70]. The germline also influences Ins/IGF-1 signaling: worms lacking germline proliferation are long-lived, and this phenotype is also dependent on DAF-16 [71]. Since DR causes lower brood size, it is possible that germline proliferation is reduced. Finally, mutants with reduced Ins/IGF-1 signaling activity share the increased stress resistance phenotype with individuals subjected to DR. These results appear to show that Ins/IGF-1 signaling controls the allocation of energy usage from reproductive growth to somatic maintenance and longevity, depending on nutrient availability. C. elegans is a convenient model organism to test the role of Ins/IGF-1 signaling in the DR response. Many longevity mutants with impaired activity of the Ins/IGF-1 pathway are available and can be used to see whether Ins/IGF-1 signaling and DR act via common or different mechanisms. Several authors have used epistatic-like experiments to investigate the role of Ins/IGF-1 signaling in DR responses. For example, Johnson et al. [72] cultured the longevity mutant age-1 in liquid culture with different E. coli concentrations and found that the life span of age-1 could also be extended by DR, suggesting that the Ins/IGF-1 pathway is not needed for mediating DR life span effects. Lakowski and Hekimi [18] made double mutants of eat-2 and daf-2, and found additive

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Food Digestive system ? Food sensing system

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Fig. 1. Model describing the potential involvement of TOR and Ins/IGF-1 signaling in the response to food and regulation of life span in C. elegans. Food is detected via amphids, sensory organs in the head region of the worm. Specific neurons in this region produce insulin-like peptides (ILP), possibly in response to food. These can regulate the activity of the phosphorylation cascade Ins/IGF-1 pathway, a main regulator of longevity. Signals from the reproductive system also influence life span via this pathway. Nutrients are taken up by intestinal cells through specific transporters or via pinocytosis and transported to the pseudocoelomic fluid. Cells can then take up these nutrients via unknown mechanisms. In mammals, the TOR/Raptor pathway activity is regulated by nutrients, and by the AMP/ATP ratio (via AMP kinase). It seems therefore plausible that the life span extension observed in C. elegans worms grown under DR conditions is regulated via a similar mechanism. There is evidence for cross talk between Ins/IGF-1 and TOR signaling (not shown in the figure): pep-2 and daf-15 expression is under the control of DAF-16 [29, 63, 66]. In mammals, TOR activity is also regulated by phosphatidylinositol 3-kinase and AKT proteins. Houthoofd/Gems/Johnson/Vanfleteren

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life-extending effects, again an indication of different mechanisms of life span extension. Consistent with these results, Houthoofd et al. [24, 73, 74] cultured daf-2 mutants in axenic medium and found that daf-2 mutants live substantially longer in axenic medium. Axenic culture and reduced Ins/IGF-1 signaling had additive effects on the upregulation of stress defenses and altered metabolism [24, 46]. Meissner et al. [29] found that the life span of daf-2 mutants can be further extended by an additive mutation in pep-2 and that additive effects were also seen on heat tolerance of the double mutant. However, one difficulty with interpreting results of this trend is that the long-lived Ins/IGF-1 mutants are ‘reduction of function’ mutants and thus still have residual activity. One could therefore argue that a further life extension of Ins/IGF-1 mutants is simply the result of a further reduction of Ins/IGF-1 activity [17]. More definitive conclusions may been drawn from studies of daf-16 mutants. A variety of mutant alleles are available for this gene, including null and near-null mutants, and these mutations completely suppress the long life span of the long-lived Ins/IGF-1 mutants. If the life extension caused by DR is mediated by Ins/IGF-1 signaling, then mutation in daf-16 should suppress the long life span of DR worms. DR-mediated life span extension was not suppressed by daf-16 mutations when DR was imposed by using eat-2 mutants [18]. The elevated stress defense of pep-2 mutants was also unaffected by mutation in the daf-16 gene [29]. Finally, daf-16 failed to suppress the life extension, metabolism and stress resistance of worms that were cultured in axenic medium [24, 46]. These results are consistent with the cytosolic localization of DAF-16 in eat-2 mutants and in wild-type worms grown in axenic medium [24, 61]. The predicted role of Ins/IGF-1 signaling is therefore not supported by experimental testing. The dependence of JNK signaling on DAF-16 [58] similarly argues against its potential role in mediating the effect of DR on life span. However, it should be stressed that in the above-mentioned experiments, no null alleles of daf-16 were used. Although the used alleles completely suppress the daf-2 Age phenotype, it cannot be excluded that some residual activity causes the increased life span of DR-treated worms. Since the life extension of mutants with reduced food sensing is dependent on DAF-16, while DR is not, chemosensory perception of food and absorption of nutrients might differentially affect life span in C. elegans. Following similar reasoning, it can be concluded that the starvation response, which depends on DAF-16 [61], is different from responses to milder forms of DR. Flies carrying a mutation in the insulin receptor substrate gene chico respond to DR less efficiently; wild-type flies under DR show a maximal life span at a lower food concentration than mutant flies do [75]. The authors explain this by saying that Ins/IGF-1 signaling is involved in the DR response in flies. However, other reasons could underlie this effect. chico flies could feed

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less for example or could use other metabolic pathways for their energy supply, making them more vulnerable to nutritional deprivation. In rodents, reduced caloric intake leads to lower Ins, IGF-1 and growth hormone concentrations in the blood stream [4]. CR feeding also results in a decreased proton-motive force and ROS production, and these effects are reversed by subjecting CR animals to a short period of Ins treatment [50]. However, Bartke et al. [76] reported that CR further extends the life span of Ames dwarf mice, which produce less growth hormone and IGF-1. These authors therefore concluded that mutants with a reduced activity of the growth hormone/IGF-1 axis slow down the aging process by a mechanism that is different from CR. However, this conclusion was criticized by Clancy et al. [75] arguing that Ames dwarf mice have residual IGF-1 activity that could be further decreased by DR, resulting in a further life extension. Studying the effect of reduced FOXO activity on DR-treated mice and flies might give a more definitive proof of the potential role of Ins/IGF-1 signaling in the DR response in these species. Altered DNA Silencing? The life span of C. elegans can also be extended by extra copies of sir-2.1 [77] or (slightly) by resveratrol [78], once thought to be a stimulator of SIR proteins [79, 80]. sir-2.1 is a homologue of the yeast SIR-2 gene that encodes a histone deacetylase, overexpression of which increases the replicative life span of yeast mother cells [81, 82]. It was thought that CR increases the life span of yeast mother cells by regulating SIR-2 activity, because the life span of Sir-2 mutants does not respond to variations in caloric uptake [83]. However, this effect appears to be very strain dependent since other researchers, using different yeast strains, did not observe such an effect [84, 85]. This issue has recently been resolved by Lamming et al. [86] who detected that Hst-2, a Sir-2 homolog, mediates Sir-2-independent life span extension by CR. Longevity induced by increased SIR-2.1 activity in C. elegans is dependent on DAF-16 [77], and since DR promotes longevity independently from DAF-16 in the worm, SIR-2.1 activity seems unlikely to mediate the DR-mediated life span extension in C. elegans. However, the C. elegans genome contains 4 different sir-2 homologues, and it cannot be excluded that other sir-2 genes might regulate the DR response, independently of DAF-16. A direct test of the DR life span effect in C. elegans mutants with altered SIR-2 activity, either by mutation or induction by resveratrol, has not yet been reported. Reduced TOR Signaling? Another likely candidate for mediating a DR response is the TOR pathway. In mammals and fruit flies, TOR senses the cellular amino acid pool and regulates cell growth by a coordinated action on transcription, translation and protein

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degradation [reviewed in 87]. When amino acids are abundant, mammalian TOR (mTOR) phosphorylates, and thereby inactivates, the repressor of mRNA translation, initiation factor 4E binding protein, resulting in enhanced translation. Activated mTOR also phosphorylates and activates ribosomal S6 kinase, also favoring protein synthesis. Conversely, amino acid deprivation reduces mTOR signaling and leads to increased autophagy and protein degradation [87]. A mutation in let-363, the C. elegans TOR homologue, or in daf-15, the C. elegans homologue of Raptor (regulatory associated protein of TOR) causes arrest and death as dauer-like larvae [66]. Heterozygous daf-15 mutants are long-lived [66]. Knocking down let-363 by RNAi from the first day of adulthood also leads to increased life span, and this effect does not require DAF-16 [88]. Evidence for the role of TOR signaling in the DR response comes from Meissner et al. [29]. These authors found that RNAi against let-363 did not extend the life span of pep-2 mutants. This is consistent with pep-2 acting upstream of TOR. In mammals, TOR activity is stimulated by Ins (and other growth factors) via phosphatidylinositol 3-kinase and Akt [89] whereas in C. elegans, DAF-16 controls the expression of daf-15 [66] and pep-2 [29, 63]. Thus, Ins/IGF-1 and TOR signaling might cooperate in a complex metabolic control circuit that optimizes metabolism and life span as a function of nutrient availability (fig. 1). AMPK (AMP-activated protein kinase) activation leads to a decrease in mammalian TOR activity as measured by S6K phosphorylation [90]. AMPK proteins are potential candidates for the regulation of life span under DR conditions. The C. elegans genome contains two homologues of AMPK, aak-1 and aak-2. Overexpression of aak-2 extends life span after being activated by a high AMP/ATP ratio. Since reduced caloric intake is likely to increase this ratio, it seems plausible that aak-2 mediates DR-induced life extension [91]. This hypothesis has not been tested directly however. AAK-2 functions independently from DAF-16 to regulate life span. However, aak-2 is somehow regulated by daf-2 [91]. Thus, it seems plausible that TOR regulates the aging rate via stimulation by nutrients and by sensing the AMP/ATP ratio in the cell (fig. 1).

Conclusions

C. elegans has been proven to be a very useful model system to elucidate the role of specific genes on the aging process. Several methods have been used to study the effects of DR on the life span, stress resistance, metabolism and activity of signaling pathways in C. elegans. It seems that DR increases rather than decreases the metabolic rate of the worm, but the effect of DR on the ROS production is yet unknown. The increased life span that is seen in restricted

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worms is however accompanied by increased resistance to environmental stressors and elevated activity of stress defense enzymes, possibly leading to less molecular damage to macromolecules, and hence retarded aging effects. It would be interesting to know if DR influences repair capacity, protein turnover or both, but this issue has not been explored yet. Several signaling pathways have been proposed to be involved in the DR effects. Work has mostly focused on Ins/IGF-1 signaling as a potential regulator of DR-induced life extension, but experimental verification has refuted this hypothesis. TOR and AAK signaling seem to be likely candidates, but experiments to prove or disprove their role have not been performed yet. Performing epistasis-like experiments by combining nutritional restriction and reducing TOR expression via RNAi is not simple. RNAi is most commonly achieved by feeding worms with E. coli expressing double-stranded RNA for the target gene. Thus, implementing DR by reducing the E. coli concentration would inevitably reduce the amount of double-stranded RNA taken up by the worms resulting in a reduction of functional gene knockdown. Possibly this problem can be avoided by growing the worms under replete conditions up to the fourth juvenile or young adult stage harvest and clean the worms and continue growth on a restricted diet of the standard E. coli strain. Alternative approaches could be considered e.g. by adding double-stranded RNA to the axenic culture medium, but this approach is labor intensive, and as discussed previously it has not been proven that axenic culture extends life span uniquely by imposing DR. Nevertheless such experiments are urgently needed. Interestingly, the TOR pathway has been shown to link nutrient sensing with protein synthesis and degradation in mammals, and there is evidence of cross talk between TOR and Ins/IGF-1 signaling [89]. The possibility that TOR plays a role in controlling longevity in invertebrates and mammals is therefore plausible (fig. 1).

Acknowledgements K.H. is a postdoctoral fellow with the Fund for Scientific Research-Flanders, Belgium. D.G. acknowledges the financial support of the Wellcome Trust. J.R.V. is supported by the Fund for Scientific Research-Flanders (Project G.0002.02) and the European Union (Contract LSHM-CT-2004-512020).

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Prof. Jacques R. Vanfleteren Department of Biology Ghent University, K.L. Ledeganckstraat 35 BE–9000 Ghent (Belgium) Tel. ⫹32 9 264 52 12, Fax ⫹32 9 264 87 93, E-Mail [email protected]

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Mobbs CV, Yen K, Hof PR (eds): Mechanisms of Dietary Restriction in Aging and Disease. Interdiscipl Top Gerontol. Basel, Karger, 2007, vol 35, pp 115–136

Diet Restriction in Drosophila melanogaster Design and Analysis

Marc Tatar Division of Biology and Medicine, Brown University, Providence, R.I., USA

Abstract Diet restriction (DR) was first shown to extend adult survival in Drosophila only a bit longer than a dozen years ago. Limiting the amount of dietary yeast was sufficient to increase life span. In the short time since this initial observation, work with Drosophila has revealed several insights into the mechanisms of DR. It has also uncovered many unanticipated technical issues. This paper describes how resolving the way we study DR in Drosophila is a prerequisite to discover the way nutrition modulates aging. Key empirical problems include the necessity of measuring the impact of DR upon life span with multiple levels of diet, analysis of the demographic response to diet with mortality data and, in the context of reaction norms, methods of diet modification, and uncertainty as to how diet dilution translates to changes in actual nutrient uptake. We review the accumulated literature of DR in Drosophila from this methodological lens to distill four important results: yeast restriction alone is sufficient to increase survival; diet affects survival through two distinct physiological responses, starvation and longevity assurance; mortality has no memory of its past with respect to nutrition; the molecular operation of DR may involve processes of deacetylation via Sir-2 and Rpd-3. Finally, it remains unknown whether or not DR functions through insulin-related signaling. Copyright © 2007 S. Karger AG, Basel

Drosophila was introduced as a model organism at the turn of the last century. In the following decades, biologists extensively studied its diet to perfect methods of culture and to explore emerging concepts of animal nutrition. By 1930 Alpatov [1] could summarize this progress and contribute a novel observation: males lived longer when they were transferred to fresh food every 2 days rather than daily. If the action of transfer itself did not kill flies, this may be the first evidence of extended longevity upon diet restriction (DR) in

Drosophila melanogaster. Remarkably, 60 years passed before a report unambiguously documented DR in D. melanogaster [2]. Adults maintained on sugar medium with yeast lived longer and laid fewer eggs when yeast was scarce rather than abundant. Since attempts to document DR with Drosophila prior to this time found limited diets to reduce survival, Chapman and Partridge [3] resolved this contradiction by testing flies across a range of nutrient concentrations, from 1.25% w/v to 16% w/v of sugar and yeast (SY diet, see table 1 for a summary of diet compositions). The median life span was greatest upon a diet with 5% SY, more dilute diets reduced both survival and fecundity, and richer diets reduced survival but increased fecundity. To extend D. melanogaster life span by DR, nutrients must be reduced within a physiological range where trade-offs occur between reproduction and somatic survival. At lower levels of diet, starvation due to malnutrition impairs fitness by reducing both survival and fecundity. That DR can extend D. melanogaster survival is no longer in doubt. But whether DR does so because it retards senescence requires further analysis of the life table. Demographic studies with Drosophila measure the age at death of every individual in synchronous experimental and control cohorts. From the distribution of deaths we calculate median life span, survivorship (Lx, the proportion remaining alive at each age x), and, in some cases, a measure of ‘maximum life span’. These common life table statistics are useful summaries of the age at death distribution, yet they need not describe senescence. Life table data reveal senescence when the mortality rate progressively increases with age [4]. Mortality rates increase exponentially with age in D. melanogaster although this pattern is obscure at early ages when sample size is small and absent at late ages where mortality levels off [5, 6]. We judge that senescence is retarded (slowed, postponed, reduced) when the trajectory of age-dependent mortality is consistently lower in the treatment group relative to its concurrent control. Median life span and survivorship are increased under this condition, but the converse need not be true. Life span can differ among groups for reasons unrelated to aging, as when the proportion alive is diminished by accidental deaths that occur in one time period among young adults of one cohort [7, 8]. To accurately describe the impact of DR on senescence from life table data we must study the mortality pattern. Mortality analysis can be straightforward: plot mortality as a function of age. A useful approximation of the mortality rate mx is ⫺ln(1 ⫺ dx/Nx), where dx is the number of deaths in the census interval x to x ⫹ 1 and Nx is the number of individuals alive at age x [9]. Plot ln(mx) for each cohort (but do not interpolate across ages where dx ⫽ 0 since these mx are undefined, see Promislow et al. [5]). Across ages where ln(mx) increases progressively, evaluate if the trajectory for the DR group is consistently reduced relative to its control.

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The life table data of Chippindale et al. [2] are replotted as mortality rates to illustrate this approach (fig. 1a, b). DR consistently reduces mortality and we conclude that yeast restriction retards senescence – this increases the median life span and survivorship. Figure 1c and d illustrates a contrasting outcome for females maintained with and without yeast [10]. While survivorship is increased among females maintained without yeast, this difference arose from a temporary spike in mortality among young yeast-fed females (at approx. day 38); these data do not demonstrate that DR retards senescence [11]. Naturally, statistical tests are required to make strong inferences about mortality patterns. The nonparametric log rank test is simple but its interpretation requires some care since this evaluates mortality irrespective of consistency in age-dependent patterns. Proportional hazard and parametric (e.g. Gompertz function) methods can explicitly evaluate the temporal consistency of mortality differences and estimate their magnitude. Guidance on these tools can be found in standard references [9, 12, 13].

The Practice of Drosophila Diet Restriction

As with humans, there are many ways to put a fly on a diet. A common approach reduces the concentration of all nutrients within a fixed volume of agar-based medium. The base medium may or may not also contain cornmeal. An alternative design reduces only the concentration of nutrient yeast while holding the sugar concentration constant. Since both methods are able to extend life span (table 1), we might conclude that it is sufficient to limit a specific nutrient component from yeast rather than caloric intake per se to retard fly senescence. This simple idea proves difficult to verify. Because all these methods dilute nutrients rather than limit their absolute availability we do not know whether flies on DR media actually eat less yeast, calories or both. This basic problem has been addressed in several recent reports without reaching a common conclusion. Early work on feeding behavior explored how food intake changed in response to the concentration of specific dietary nutrients. Driver et al. [14] estimated food consumption by the rate of fecal deposition and concluded that feeding varied inversely with nutrient concentration. Edgecomb et al. [15] assessed mated, yeast-fed flies after they had been maintained for 3 days on sugar-only diets where sucrose varied from 0.5 to 17% w/v. Feeding was measured by the frequency of proboscis prints upon the food, and by the uptake and excretion of a soluble, indigestible dye. Especially among females, adults compensated for reduced sugar by feeding at higher rates. The diets of these behavioral inquiries were not designed to study aging. Therefore, to investigate how food intake varies under conditions where DR

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Table 1. Diet compositions in the literature Report

Diet components

Chippindale et al. [2]

Chapman and Partridge [3]

Relative longevity (DR/AL)

varied

constant

female

male

Y

C, S

1.24 (a)

1.28 (a)

reduced

Y

C, S

1.30 (a)

1.14 (a)

reduced

Y

C, S

1.14 (a)

1.06 (a)

reduced

Y Y⫹S

C, S

0.99 (a) n.s. 1.56

1.11 (a)

reduced reduced

Y⫹S

1.17

Clancy et al. [62]

Y⫹S

1.33

Rogina et al. [46]

Y⫹S

Pletcher et al. [72]

Y⫹S

1.82 (b)

Mair et al. [42]

Y⫹S

1.50 (c)

Wood et al. [26]

Y⫹S Y

Mair et al. [48]

1.41

C, S

1.89 1.19 (a)

1.29 1.07 (a)

Y⫹S

1.36

Y⫹S

1.53

Magwere et al. [73]

Y⫹S

1.64

1.32

Rogina and Helfand [64]

Y⫹S

1.23

1.26

Mair et al. [18]

Y⫹S

1.70 (a)

Y

S

1.53 (a)

S

Y

1.13 (a)

Min and Tatar [16]

Y

C, S

1.67

Carvalho et al. [17]

Y⫹S

C

1.57 (b)

van Herrewege [74]

casein

S

1.29 (b)

S

casein

1.16 (b)

casein

S

1.58

Min [75]

Fecundity (DR/AL)

1.50

1.54 (b)

1.69

reduced

Diets consisted of agar, water, antimicrobials and yeast (Y), sugars (S) and sometimes cornmeal (C), except when casein replaces yeast. Relative longevity calculated from median adult life expectancy as DR/ad libitum (AL) unless noted: a ⫽ ratio of replicate averages, b ⫽ ratio of mean life-span, c ⫽ ratio of maximum life-span, defined by reporting author. All cases significantly increased survival of DR adults, as reported by author, except where noted as not significant (n.s.).

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Yeast type

Mating conditions

Wild-type background

Diet notes

live yeast (paste)

male/female pairs

Ives B

live yeast (paste)

male/female pairs

Ives O

live yeast (paste)

male/female pairs

Ives D

live yeast (paste)

male/female pairs

Ives C

autolyzed yeast flakes autolyzed yeast flakes autolyzed yeast flakes autolyzed yeast autolyzed yeast flakes autolyzed yeast flakes autolyzed yeast autolyzed yeast (SAF™) autolyzed yeast flakes autolyzed yeast flakes autolyzed yeast flakes autolyzed yeast

males constant

Dahomey

males intermittent

Dahomey

virgin females

Dahomey

females once mated

Canton S Dahomey

1.5 mg/vial Y or 0.15 mg/vial Y on charcoal medium 1.5 mg/vial Y or 0.15 mg/vial Y on charcoal medium 1.5 mg/vial Y or 0.15 mg/vial Y on charcoal medium 1.5 mg/vial Y or 0.15 mg/vial Y on charcoal medium SY at 1.25, 2.5, 5, 10, 15% with agar constant; longevity maximal at 5% SY SY at 1.25, 2.5, 5, 10, 15% with agar constant; longevity maximal at 5% SY SY at 1.25, 3.0, 5, 6.5, 8, 10, 15% with agar constant; longevity maximal at 6.5% SY SY at 5 or 15%; agar-only base dilute medium; AL ⫽ 15% SY, DR ⫽ 5% SY

females once mated

Dahomey

dilute medium; AL ⫽ 15% SY, DR ⫽ 6.5% SY

mixed-sex vial mixed-sex cage

Canton S yw

males constant

Dahomey

AL 15% SY vs. DR 5% SY in agar-only base AL ⫽ 3% Y vs. DR ⫽ 2% Y in 10.5% S with cornmeal base dilute medium; AL ⫽ 15% SY, DR ⫽ 6.5% SY

females once mated

Dahomey

dilute medium; AL ⫽ 15% SY, DR ⫽ 6.5% SY

autolyzed yeast flakes autolyzed yeast flakes autolyzed yeast flakes autolyzed yeast (SAF™) yeast extract (Bacto™)

mated, then single-sex Dahomey bottles mixed-sex vials Canton S

SY at 2, 4, 6, 8, 10, 12, 14, 16%; agar from 12.5 to 20 g/l SY at 5 or 15%; agar-only base

females once mated

Dahomey

DR ⫽ 6.5% Y, 6.5% S vs. AL ⫽ 15% Y, 15% S

females once mated

Dahomey

DR ⫽ 6.5% Y, 15% S vs. AL ⫽ 15% Y, 15% S

females once mated

Dahomey

DR ⫽ 15% Y, 6.5% S vs. AL ⫽ 15% Y, 15% S

mixed-sex cage

Canton S

virgin females mixed-sex vials mixed-sex vials

casein

mixed-sex cage

Diet Restriction in D. melanogaster

AL ⫽ 16% Y, DR ⫽ 2% Y; cornmeal 5.2%, sugar 11% SY at 1, 5, 10, 15%; cornmeal 8% (larvae reared on Lewis medium) Champétières S ⫽ 0.75%, essential mix, casein at 0, 1, 2, 3, wild ⫻ vestigial 4, 5%; max. at 2% (F) and 1% (M) casein Champétières casein ⫽ 5%, essential media, S at 0, 1, 2, 3, wild ⫻ vestigial 4, 5%; max. at 3% Canton S 11% S, 1.1% agar and casein at 0.5, 1, 2 or 4%

119

0

1.0

Age (days) 40

60

80

0

0.8

Low yeast High yeast

⫺1

0.6

Mortality rate (In)

Survivorship

20

0.4 Low yeast High yeast

0.2 0

⫺2 ⫺3 ⫺4 ⫺5

0

40

20

a

60

80

b

Age (days)

⫺6

1.0

0

20

Age (days) 40

60

80

⫺1.5

⫺2.0 0.6

Mortality rate (In)

Survivorship

0.8

0.4 No yeast Fed yeast

0.2

⫺2.5

⫺3.0

⫺3.5

0 0

c

No yeast Fed yeast

20

40 Age (days)

60

80

d

⫺4.0

Fig. 1. Assessing the influence of DR on aging from survivorship and mortality. Evidence of retarded aging with DR [2]: reduced yeast increased survivorship (a) because it consistently decreased mortality rate (b). Evidence inconsistent with retarded aging with DR [10]: flies without dietary yeast increased survivorship (c) because many control flies (fed yeast) died in one census interval (d).

extends life span, Min and Tatar [16] assessed a range of diet that varied in yeast from 1 to 16% with a constant concentration of dietary sugar. Age-dependent mortality was minimized by a 2% yeast diet; this consistently reduced mortality relative to a 16% yeast diet and in turn increased the median life span by 16 days. Accordingly, the feeding rate was measured by dye intake and fecal deposition on medium with 2 and 16% inactive yeast (in agar-cornmeal-sucrose base) in mated females. In contrast to the compensatory feeding that occurs at low levels of dietary sugar, females on 16% yeast consumed more diet than those on 2% yeast. Since fecundity is elevated on yeast-rich diets, the high feeding rate of these females may correspond to the metabolic demands of egg production.

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Carvalho et al. [17] took a somewhat different approach. They varied the concentration of both sugar and yeast extract in agar-cornmeal base (SYE diet) and labeled diets with a soluble radioactive tracer in the form of the nucleotide [␣-32P]dCTP. Tracer uptake and survivorship were measured in virgin females. Survival was greatest upon a diet of 1% SYE. At diets between 2 and 15% SYE there were no clear differences in age-dependent mortality despite some variation in mean life span. Across the full range of diets, tracer uptake increased as nutrient concentration declined. These data show compensatory feeding and its consequences: tracer intake on the 1% diet was only 40% of its consumption on the 15-fold richer diet. Flies on diluted diet acquired fewer nutrients as expected, but the quantity of consumed marker was not proportional to the nutrient concentration of the diet. It is also notable that although Carvalho et al. [17] and Min and Tatar [16] both found less total consumption on diluted diets, these studies reported that feeding was stimulated by opposite conditions. The reproductive status of females may explain this difference. Carvalho et al. [17] studied virgin females, which have little metabolic demands from egg production. Compensatory feeding may be the default behavior in the absence of reproduction. Mair et al. [18] provide our final perspective. They independently varied the concentration of both sugar and yeast in agar base. Newly hatched females were mated and subsequently studied without males. Survivorship was markedly increased on diets that restricted yeast while holding sugar constant. On the other hand, when holding the amount of yeast constant, survivorship was only modestly increased by diets with less sugar. These data confirm the importance of dietary yeast in DR as reported in Chippindale et al. [2] and provide a potential way to assess the relative importance of specific nutrients and calories. Mair et al. [18] noted that solitary females extended their proboscides for the same amount of time on each type of diet and suggest from this behavior that females will acquire the same amount of calories if the diets have similar energetic contents. Since the low-yeast/high-sugar and the high-yeast/low-sugar diets were energetically equivalent, Mair et al. argued that DR mediates life span because it limits specific nutrients rather than calories. This inference, however, requires that nutrient acquisition is proportional to nutrient concentration. The actual relationship between proboscis extension and food consumption is unknown. Furthermore, different experimental conditions were used to measure proboscis extension (undisturbed, solitary females) than was used to measure longevity because females in the demographic trials had complex patterns of feeding behavior. If females in fact increase their food intake on high yeast diet under the demographic conditions, as they may to support egg production, their net caloric intake will be greater on high-yeast/low-sugar than on low-yeast/high-sugar media. On the other hand, if these females feed more when yeast or sugar is

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reduced, differences in net caloric intake will be less than anticipated from the energetic value of the diet. In either case, caloric intake and nutrient quality will be correlated and we cannot determine which factor modulates aging. To move forward we must directly control food intake or explicitly measure assimilated nutrients. Markers placed in the medium are useful but indirect; dyes measure gut content and radio-tagged nucleotides will correlate with gut content and assimilation. An additional complexity is that not all components of a diet are equally soluble, and this applies to markers as well as to sugar and yeast. Adult Drosophila feed by sponging material from the media surface; solutes of sugar (and of marker) will be readily acquired but insoluble metabolites from yeast that are embedded below the agar surface will be relatively inaccessible. One strategy to assess whether calories mediate Drosophila DR is to measure energy flux (caloric value of eggs, soma, excreta and heat). A specific role of calories can be ruled out if a restricted diet increases survival but these flies process the same amount of energy as ad libitum controls. An alternative approach would be to experimentally regulate consumption. This was feasible with the Mediterranean fruit fly [19] and the housefly [20] where adults were provided a defined volume of liquid diet that was fully consumed during a normal feeding cycle. In both cases and contrary to precedence, reduced intake of nutrients had no positive effect on life span. A fixed food intake study of DR should be a high priority with Drosophila, although the small scale of this fly makes for many technical challenges. A third option would be to label carbon and nitrogen of dietary sugar and yeast with stable isotopes and then track their acquisition and metabolic flux by elemental specific mass spectrometry [21]. Through such methods O’Brien et al. [22] showed how the butterfly Heliconius charitonius acquired essential amino acids from dietary pollen to produce eggs and perhaps to support its long-lived soma. Stable isotope labeling of fly nutrients might help identify specific metabolites that limit egg production but increase in somatic tissue when DR extends life span. Although we cannot yet resolve the relative importance of caloric intake and specific nutrients, it is clear that reduced dietary yeast is sufficient to extend life span. Yeast is a rich source of carbohydrates, sterols, fatty acids, vitamins, minerals and amino acids. Amino acids deserve attention because reduced methionine extends life span in rats and in mice [23, 24]. In mice this also retards the age-dependent decline in immunity, metabolism and stress resistance. Whether D. melanogaster survival can be improved by limiting dietary amino acids has proved difficult to address since defined diets optimized for larvae are not suitable for adults [25]. As an alternative we have measured the survival of flies maintained on sugar agar alone or when supplemented with casein at 0.5, 1, 2 and 4% [75]. In both males and females, survival was greatest

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at intermediate and low levels of casein, and this arose demographically because the age-dependent trajectory of mortality was consistently reduced. Importantly, females on 4% casein produced most eggs, although net reproduction was much less than when females feed on yeast. While preliminary, these observations meet the criteria for DR and suggest that amino acid restriction may specifically influence Drosophila aging. The low level of absolute fecundity indicates that other nutrients are essential for egg production and these may as well affect aging. Regardless of the metabolic currency, models to explain how restricted diet modulates life span largely focus on the use of nutrients for reproduction relative to somatic repair [27, 28]. Before discussing this allocation concept, we note that nutrients may mediate aging through a very different route – via sensory cues. Environmental cues that predict future conditions regulate major life history phenotypes of invertebrates. Dauer in Caenorhabditis elegans is induced by pheromones and the perception of low food concentration, while mutations within specific sensory neurons extend adult life span [29]. Among insects, each stage of the life cycle may be controlled by cues that predict environmental suitability for growth relative to quiescence [30]. Adult reproductive diapause is no exception [31]. The black blowfly Phormia regina can detect potential food supplies through taste hairs on its tarsi [32], and adults of this fly will not initiate egg production until it feeds on protein [33]. In a similar way Drosophila may sense dietary yeast to modulate neuroendocrine signals that control commitment to reproductive physiology. It should be fascinating to determine whether the perception of different concentrations of dietary yeast is sufficient to modulate Drosophila aging.

Mechanisms of Diet Restriction in Drosophila

Models of resource acquisition and allocation provide the classic explanation for how DR modulates aging [34]. Available resources are allocated from reproduction to somatic maintenance when nutrient acquisition is limited (fig. 2a). However, while many data are consistent with the correlations predicted by the allocation model, there are few data to test its assumptions. It will be important to discuss the type of evidence needed to evaluate resource allocation and to consider a mechanistic alternative – direct constraints of reproduction [7, 35]. Manipulative studies of life history traits often find negative correlations between reproduction and survival. For instance, the female Drosophila life span is increased when their reproduction is experimentally repressed [10]. Likewise, females on a restricted diet have increased survival but produce

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Soma

Resource acquisition

a

Reproduction Soma

Inhibitory

acquisition

Negative

Resource

b

Reproduction

Fig. 2. Conceptual models for how nutrients mediate life span. a The ‘Y model’ of resource acquisition and allocation. When nutrients become limited, acquired resources are preferentially allocated to somatic maintenance at the expense of current reproduction. b The ‘direct constraints’ model. The activity of reproduction directly constrains somatic maintenance and persistence by imposing direct damage or by inhibiting the process of somatic repair. When nutrients become limited, reproduction is reduced and constraints on somatic persistence are attenuated.

fewer eggs [3]. Each of these outcomes is anticipated if there is a limiting resource essential for both somatic and reproductive function but the data are equally consistent with an alternative explanation (fig. 2b). Nutrients mediate the level of reproduction, and reproduction imposes direct constraints on somatic functions that affect survival [7, 35]. Reproduction for instance may activate metabolism and thereby incur damage through exposure to reactive oxygen species. Reproduction may also inhibit the expression of somatic protection systems. For instance, misexpression of hsp-70 reduces age-specific mortality in young females but this simultaneously diminishes egg hatchability without reducing egg production [36, 37]. Selection will favor systems to negatively regulate hsp-70 because the fitness benefit of early reproduction is greater than the value of survival to old age. By such direct constraints from reproduction, if DR reduces fecundity it can increase survival without resource allocation.

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How can we experimentally distinguish between these alternative mechanisms since allocation and direct constraints both predict negative correlations between reproduction and survival? One approach is to jointly manipulate nutrient acquisition and reproduction. If resources are competitively allocated to somatic function at the expense of reproduction, experimentally reduced reproduction should have a larger effect on survival when resources are limited than when they are abundant [38, 39]. When testing this idea with the bean beetle Callosobruchus maculatus we found that resource allocation could explain only a portion of the mortality differential associated with egg production [35]. From this observation we proposed that direct reproductive constraints account for the remaining effects of reproduction on mortality. As with C. maculatus, resource allocation alone may not be sufficient to explain DR in D. melanogaster. Recent studies have physiologically decoupled age-specific fecundity and mortality. For instance, third instar larvae that complete development without dietary yeast produce females with low fecundity despite feeding on yeast as adults [40]. Yet, these females have the same age-dependent mortality trajectory as highly fecund controls. Diet manipulation during adulthood appears to have the same capacity to decouple reproduction and mortality [41]. Females maintained on a sugar-only diet survive for many weeks, produce no eggs and have relatively high age-specific mortality (fig. 3a). When these females are switched to a diet with yeast at late ages they rapidly acquire the mortality trajectory of control females heretofore maintained on full-yeast media (fig. 3b). Control females at this age lay few eggs but the switched females now produce many eggs; at these ages differences in fecundity are not accompanied by differences in mortality. An important extension of this design confirmed that age-specific mortality trajectories are similarly plastic when diet is varied in conditions where DR extends life span [42]. Adults were switched between DR and ad libitum conditions, and mortality rapidly adjusted to the level of the continuous diet treatment (fig. 3c). Although reproduction was not measured across these diets, Chippindale et al. [2] have previously documented that such a switch rapidly adjusts age-specific fecundity. It would be useful to simultaneously measure age-specific fecundity and mortality in a trial of ad-libitum-to-DR switch to determine if reproduction is independent of current mortality. Together these studies provide important insights into the nature of DR. Current mortality has no mechanistic connection to past nutrition and reproduction, contrary to expectation based on resource allocation. Rather, nutrition modulates the current level of susceptibility to fail in response to underlying systems that degenerate with age. Genetic analyses provide further challenges to the traditional view of resource allocation. We now recognize many genes that extend adult survival

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Age (days) 10

20

30

Age (days) 40

50

60

0

0

0

⫺1

⫺1

10

20

30

40

50

⫺2

⫺2 Mortality rate (In)

Mortality rate (In)

0

⫺3 ⫺4 ⫺5 ⫺6

⫺3 ⫺4 ⫺5 ⫺6 ⫺7

Full diet Switch week 4 No yeast

⫺7

a

⫺8

c

⫺9

Full diet Switch week 2 Restricted diet

Eggs

80 60 40 20 0

0

10

20

b

30 Age (days)

40

50

60

Fig. 3. Age-specific mortality and reproduction decoupled by diet switch. a, b Females maintained on sugar-only diet have a relatively high mortality and produce no eggs [41]. When switched to an SY diet (day 28, arrow), females rapidly assume the mortality trajectory of control females that were continuously fed the SY diet. This illustrates the amnesia of mortality with respect to diet. At ages after 35 days, females of the switched group produce many more eggs than the fully fed females, yet the groups have similar mortality rates. c The amnesia of mortality with respect to diet illustrated when females are switched from restricted to full diet [42].

when mis-expressed in D. melanogaster [43]. Some concomitantly reduce fecundity (e.g. InR), but many (e.g. INDY, foxo, rpd-3) do not reduce reproduction within the benign conditions of the laboratory [44–46]. While a trade-off might occur under more natural conditions [47], these cases demonstrate that somatic survival can be increased without diminishing the allocation of resources to reproduction. Sterility mutants demonstrate the same principle. The ovoD genotype blocks oogenesis at an early stage but DR remains an effective way to extend life span in these sterile females [48]. While these data challenge concepts about resource allocation, they also present a broader paradox: reproduction can be decoupled from mortality, either with or without nutrient interactions. In this case, is it still possible for DR to mediate aging through direct costs of reproduction? It may, providing

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that reproduction modulates somatic damage and maintenance through germline-correlated signals [49, 50]. Mortality was decoupled from reproduction in C. elegans in worms where the gonad was intact and or laser-ablated without affecting life span [51]. However, life span was extended when the germ line alone was eliminated [52]. To account for these observations, Kenyon et al. [51] proposed that there is a balance of longevity modulating signals between the germ line and the somatic gonad. These gonad signals could regulate direct constraints of reproduction on somatic function. Although we have yet to define roles for germ line and somatic gonad control of D. melanogaster aging, recent data make a strong case for nutrient regulation of germ line stem cells in the adult. Insulin is secreted by neurons within the adult brain in well-fed flies [40]. Germ line proliferation within the ovary is autonomously stimulated by these insulin-like ligands; reduced diet and reduced insulin-producing cell secretion is sufficient to slow germ line stem cell divisions [53, 54]. In parallel to these reproductive responses, aging is retarded by systemic reduction of the insulinlike receptor as well as by ablation of the neuronal insulin-producing cells [55–57]. It shall be important in the future with D. melanogaster to determine whether nutrients or insulin mediate demographic aging via their effects on the activity of germ line stem cells.

The Interaction of Genes and Diet Restriction

As we discover the nutrient conditions whereby reduced diet extends fly life span, we can apply Drosophila genetics to uncover the mechanistic basis for DR. It should be possible through mutant screens to identify genes that are essential for a restricted diet to extend life span. The focus of current work is on candidate genes, which asks if misexpression reduces the longevity gain induced by a restricted diet in matched wild-type controls. When they do, functions associated with the gene are considered to overlap with mechanisms by which diet modulates aging. To date this approach has been applied to genes that retard aging when misexpressed. Prominent among these is chico, which encodes the insulin-like receptor substrates 1–4 homolog. Ligand-activated insulin receptor phosphorylates insulin-like receptor substrate, which in turn signals through AKT to inactivate the dFOXO transcription factor [58] and to permit activity of the translation regulator target of rapamycin [59]. Here we discuss chico to understand the practice of gene-by-diet analysis and to assess whether DR modulates fly aging via insulin signaling. The mutant allele chico1 reduces age-dependent mortality in males and females [60, 61]. Homozygote chico1/chico1 are dwarf and infertile. Clancy et al. [62] investigated how survival of homozygote and wild-type females

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Mean life span (days)

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chico1/chico1

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Wild type 1.5

3.0

5.0

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Diet concentration (SY%)

65

Median life span (days)

Median life span (days)

a

Additional sir-2

55 Wild

45

type

Wild type

55

Mu

tan

45

tr pd -3

35

35 5

b

65

5

15

SY concentration (%)

c

10

SY concentration (%)

Fig. 4. Gene-by-diet interaction plots. a Median life span of wild-type and chico dwarf adults on SY diet of 7 concentrations [62]. b Median life span of adults with wild type or sir-2 overexpression on SY diet at 2 concentrations [64]. c Median life span of adults with wild type or mutant rdp-3 on SY diet at 2 concentrations [46].

changed as diet ranged from 1.25 to 15% of both sugar and yeast in agar base (SY diet). In general, the phenotypic response within a defined genotype across environmental conditions is called a reaction norm or a gene-by-environment interaction [63]. The reaction norm for control females, as expected, was concave; median survival was greatest at an intermediate diet concentration, 6.5% SY (fig. 4a). Females of chico1/chico1 produced a similar pattern except that the optimal diet was 8.0% SY. The reaction norm of mutant homozygote flies was shifted to the right such that DR produced the same maximum median survival as observed for wild-type females. In the analysis of reaction norms, we have evidence that some function of a gene interacts with the manipulated environmental parameter (diet in our case)

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Case 1 No : gene⫻ DR Yes : DR plasticity in mutant

Median life span

Case 2 Yes : gene⫻ DR No : DR plasticity in mutant

Case 3 Yes : gene⫻ DR Yes : DR plasticity in mutant

Case 4 Yes : gene⫻ DR Yes : DR plasticity in mutant DR

ad libitum

Diet continuum

Fig. 5. How reaction norms are interpreted to determine when a gene participates in the process whereby DR extends longevity. Schematic reaction norms are presented as continuous linear functions and are limited to the range of diet concentrations corresponding to DR physiology: with reduced diet, survival increases and fecundity declines. The diet concentration axis is continuous from most restricted to full ad libitum feeding. This scheme can represent many permutations. Our cases provide examples to understand when there is overlap of gene function and mechanisms of DR. The median life span of the wild genotype (‘wild type’; dashed line) strongly responds to DR; it is represented with the same reaction norm in each panel. The variant allele (‘mutant’; solid line) can be any well-defined genetic variation of the candidate gene, for instance a loss of function mutant, hypomorphic mutant, overexpression transgene or isolated natural polymorphism. For these examples, the variant allele increases life span in standard laboratory conditions. Significant gene-by-DR interaction (gene ⫻ DR) is evidence that the function of the candidate gene is involved in the mechanism by which DR extends life span (cases 2, 3, 4). Reaction norms with nonzero slope have ‘phenotypic plasticity’ – these show that the phenotype (median life span) varies across environments (diet). The allelic variant may abolish phenotypic plasticity (case 2) or change its extent (cases 3 and 4) – these equally imply DR functionality of the candidate gene. Lack of gene ⫻ DR evidence occurs only in case 1, where DR induces proportional reaction norms; the genotypes have equivalent phenotypic plasticity.

when the gene-by-environment interaction plots are not parallel (fig. 5). Data sufficient to reject the null hypothesis of parallel reaction norms indicate that there is some overlap between the function of the gene and how the environment affects the phenotype. Because Clancy et al. [62] measured survivorship across many levels of nutrition they were able to define the diet of maximum

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longevity for both chico1/chico1 and wild-type flies, and their data recorded survivorship on either side of this maximum, that is under conditions of malnutrition and when DR affects aging. As a consequence, the wild-type and chico1/chico1 reaction norms intersect. From this evidence and based on the apparent similarity among genotypes of their longevity maxima, Clancy et al. [62] concluded that ‘chico1 induces a stage equivalent to submaximal, DRinduced slowing of aging. These two pervasive interventions that slow aging therefore act through overlapping mechanisms.’ To assess this conclusion we must consider the different ways nutrition can affect survival and then how to test inferences from reaction norms of survival data. The chico1 mutation appears to affect the interaction with nutrition in two ways. First, chico1 is more prone to starvation due to malnutrition; in the range of diets where restriction has negative effects on life span, chico1 has a shorter life span than the wild type. Second, chico1 retards aging when nutrients are physiologically sufficient; in the range of diets where restriction increases life span, chico1 has superior survival. As originally noted by Chapman and Partridge [3], the mechanisms that affect life span during starvation level diets will differ from those that affect life span when DR slows aging [11]. When malnourished, both fecundity and survival decline as diet is progressively restricted, and physiology activated to ensure survival must be directed at coping with the effects of starvation. In contrast, in the range of nutrients that maintain sustenance, restricted diet increases survival as it decreases fecundity. While we cannot yet describe mechanisms of DR that regulate survival and fecundity, most likely they are distinct from processes that kill flies when starved [65]. Do the data of Clancy et al. [62], when viewed in this way, indicate that chico1 and DR slow aging through overlapping mechanisms? If there are distinct biological processes that modulate survival under conditions of malnutrition versus DR, we should separately evaluate how the candidate gene interacts with each process. In particular, to determine if functions of chico overlap with the mechanisms of slow aging induced by restricted diet, we must evaluate the reaction norms specifically in the diet range where nutrients were sufficient, not in the range when flies are starving. The relevant range of diets occurs at or above 8% SY for chico1 and 6.5% SY for wild-type flies, and here the reaction norms appear remarkably parallel. If so, these data provide no evidence that functions of chico overlap with the mechanism of DR. What is the likelihood that the reaction norms in the DR diet range of figure 4a are in fact not parallel, that the data implicate chico to function within the mechanisms of DR? Presently we cannot evaluate this question because point estimates of median survivorship do not describe the error variance of the mortality differences, and the log rank test does not estimate mortality differentials.

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Inferences on gene-by-environment (diet) interaction require survival regression analysis where the difference in mortality is statistically evaluated with a model where genotype (G) and diet level (D) are main effects and the interaction parameter (G ⫻ D) describes how genotype modifies the effect of diet on mortality. Survival analysis can be conducted with proportional hazard or accelerated failure (parametric) methods; both use every death observation to simultaneously estimate the main and interaction parameters and their associated standard errors. These statistics describe the extent mortality is modified by genotype and by diet, and whether these effects are significant. Central to the problem at hand, when the interaction parameter (G ⫻ D) is statistically significant, we can infer that the reaction norms are not parallel. Only then can we say that the way diet modifies aging depends on the function of the tested gene (fig. 5). In practice we test data to reject the null hypothesis of parallel reaction norms. However, failure to detect a significant interaction parameter must be interpreted with care. There may be a meaningful deviation from parallel reaction norms but the data may be too few or too variable to make a statistically powerful test. Furthermore, DR reaction norms may be nonlinear, or the interaction may be apparent only at diets beyond the studied range. These cautions apply to work beyond Drosophila. Notably, eat mutants are used to simulate restricted versus full diet in C. elegans. The apparent absence of interaction of median life span between these proxies for nutrient intake and the longevity gene daf-16 [66] is frequently interpreted to imply that DR is affected through functions independent of insulin/IGF signaling [67]. To support this conclusion we need data from multiple diet levels and survival analysis with enough power to detect meaningful differences in mortality. The framework of reaction norm analysis recommends that we apply two operations to assess when a gene participates in mechanisms of DR. First, use data within the range of nutrients where survival is modified by mechanisms of DR and not by starvation. We can identify this range from survival measured across a broad and fine-grained series of diets, as with chico [62, 68]. When a study uses only two diets there is a chance that the diet concentration used for the restricted condition is in the range of malnutrition. Reproduction provides an additional criterion to define the region of a physiologically sufficient diet since fecundity should vary inversely with survival in the nutrient range of DR. Second, evaluate whether the reaction norms in the DR range are not parallel. Evidence to reject the null hypothesis (parallel norms) suggests that there is a function associated with the candidate gene that contributes to the mechanism by which DR retards senescence. Short of such formal analysis for chico we can be relatively confident that there is no support to reject the hypothesis of parallel reaction norms within the nutrient regions of DR reported in Clancy et al. [62]. Yet, overall we see the

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gene-by-environment plots intersect. How then might we interpret these results? Since the molecular functions associated with chico1 affect both the response to malnutrition and to aging, the mutation must influence a common and upstream process, for instance by modulating nutrient perception or by changing the rate of food intake. In these interpretations, chico1 does not elucidate a specific pathway through which DR extends life span. Rather, chico functions at a broad level of nutrient interactions to affect many aspects of physiology and life history. Besides chico, data to assess gene-by-DR interaction are available for the histone deacetylases encoded by rpd-3 and sir-2. Misexpression of histone deacetylases retards aging in yeast, C. elegans and Drosophila [69]. In C. elegans, longevity extension induced by SIR-2 requires functional daf-16 [70]. In mammalian cells, SIRT deacetylates regulatory proteins such as p53, FOXO and Ku70 and thereby mediates the balance between stress resistance and apoptosis [71]. To understand whether such aging-associated outcomes work through pathways of DR, Rogina et al. [46] and Rogina and Helfand [64] assessed how D. melanogaster rpd-3 and sir-2 affect longevity on media of low and high SY diets. Figure 4b and c compares the median life span of rpd-3 mutants and of sir-2 overexpression relative to their respective wild types. In both cases, the reaction norms converge, and although the data were not tested by survival regression analysis it is clear from pairwise tests that the lines cannot be parallel. Unfortunately, with only two diet levels we cannot rule out that the diet used at the lower concentration is within the region of malnutrition. The risk here is for the apparent interaction of the reaction norms to involve both the consequences of starvation and of aging. It would be useful to make life tables for additional levels of diet and to measure fecundity. Some clues are presently provided by available data on mRNA abundance. The message of sir-2 is reduced 2-fold in long-lived rpd-3 mutants as well as in studies where diet is restricted [46, 72]. Since rpd-3 and restricted diet affect the sir-2 message in similar ways, the restricted diet used by Rogina and Helfand may indeed affect survival through processes of DR rather than through starvation. Taken together, these data provisionally demonstrate gene-by-DR interaction and present the first genetic evidence on a mechanism of Drosophila DR. Reduced diet retards aging through a pathway that involves processes of deacetylation.

What We Know of Diet Restriction in Drosophila

This paper focused on how we study DR in Drosophila and said little about the way DR might work to slow aging in this animal. The literature is replete with many attractive mechanistic hypotheses but these remain speculations

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until we establish meaningful experimental designs and data interpretation. Still, strong progress has been made with the fly [73, 74]. The first experimental treatment to successfully implement DR with the fly was published little more than a decade ago and the first genes to modulate fly aging have only recently been reported. In this short time we have collected a small number of important insights. (1) Yeast restriction is sufficient to increase survival. Whether this works because flies acquire less of some yeast-specific nutrient or because they eat fewer calories remains unknown. (2) Adult survival is influenced by two distinct processes: starvation due to malnutrition and longevity assurance physiology due to DR. (3) Mortality has no memory of its past with respect to nutrition. Nutrition modulates how susceptible individuals are to failure caused by systems that degenerate with age. (4) Age-specific mortality and reproduction can be decoupled; DR does not retard aging simply by reducing the allocation of resources to eggs. (5) The molecular operation of DR may involve processes of deacetylation; whether the mechanisms of DR function through insulin-related signaling remains unknown. Finally, we can also see the potential that lies ahead: with new experimental, demographic and genetic tools we expect to make rapid progress to understand the mechanistic basis for how DR retards Drosophila aging.

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Marc Tatar Associate Professor Division of Biology and Medicine Box G–W, Brown University Providence, RI 02912 (USA) Tel. ⫹1 401 386 3455, Fax ⫹1 401 386 2160, E-Mail [email protected]

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Mobbs CV, Yen K, Hof PR (eds): Mechanisms of Dietary Restriction in Aging and Disease. Interdiscipl Top Gerontol. Basel, Karger, 2007, vol 35, pp 137–158

Dietary Restriction in Aging Nonhuman Primates Julie A. Mattison, George S. Roth, Mark A. Lane, Donald K. Ingram Intramural Research Program, Laboratory of Experimental Gerontology, National Institute on Aging, Baltimore, Md., USA

Abstract Dietary restriction (DR) has been shown to benefit health and longevity in a wide variety of species, although most have maximal life spans of only a few years. In 1987, the National Institute on Aging began the first well-controlled long-term study in a species with a considerably longer life span and a closer physiology to humans. Using rhesus monkeys (Macaca mulatta), an extensive array of physiological measures have been conducted in both males and females to evaluate the effects of DR. Monkeys benefit from DR with a lower body weight, body fat, blood glucose and thus are at lower risk for developing diabetes. Changes in several endocrine measures indicate an altered hormonal axis; however, circadian patterns and timing relative to the onset of DR can obscure the differences. Despite the caloric deficit, female monkeys are not reproductively compromised, and both males and females may benefit immunologically. There remains much to be learned from this model of DR including whether long-term DR will increase life span in a primate species. Copyright © 2007 S. Karger AG, Basel

Although dietary restriction (DR) as a prolongevity intervention has been investigated since 1935, its application to a primate model is relatively new and still unproven. Moreover, despite the considerable existing literature on the topic, it will be several more years before any degree of certainty will emerge for evaluating whether nonhuman primates (NHPs) on DR will exhibit increased life span and health span. A considerable amount of data has been generated to suggest many positive effects of DR in both humans and NHPs, but it is uncertain whether or not this will translate to a longer life expectancy. The first experimental study of DR in an NHP model was initiated in 1987 by intramural investigators at the National Institute on Aging (NIA) [1]. Subsequently, a similar study was undertaken at the University of Wisconsin

(UW) [2]. Male and female rhesus monkeys (Macaca mulatta) in both of these longitudinal studies were grouped randomly into approximately equal numbers of control and DR subjects. Monkeys at the NIA ranged in age from 2 to 23 years when they were initiated into the study; thus, some monkeys were prepubertal while others were considered old. Monkeys in the UW study were all adults (8–14 years of age) at initiation. All information gleaned about DR in rhesus monkeys is derived from these two studies but with a median life span of 25 years and a maximum of 40 years, it will be several more years before longevity data are conclusive. A related investigation at the University of Maryland developed from studies of obesity and diabetes in rhesus monkeys in which weight titration implemented to prevent obesity has produced many physiological effects similar to DR [3]. However, the small sample of DR monkeys (n ⫽ 8), the lack of concurrent controls and random diet group assignment complicate the interpretation of data emerging from this study. While it would be tempting to conclude from their early evidence that DR increases survival in rhesus monkeys as Bodkin et al. [4] made claim, the interesting relationships of nutrition, aging and disease do not provide conclusive results at this time [5]. With such a long life span, studies of longevity in rhesus monkeys are challenging and costly to conduct. Additionally, although a demonstration of increased mean and maximum life span will be required for drawing definitive conclusions, evidence for preserved health and function is also required. Since conclusive survival data are still years away, the evidence for DR as an intervention for decreasing the incidence and delaying the onset of age-related diseases and pathology will be presented in the current review. The NIA study has been designed to examine the effects of DR on cellular, organ, physiological and behavioral function.

Body Composition and Dietary Intake

It would be expected that DR would produce a decline in body weight and fat mass. Data from both the NIA and UW confirmed this expected result [2, 6]. Maturing monkeys, both control and DR, continued to gain weight into adulthood, but over the course of these studies, both male and female DR monkeys weigh less than age-matched controls [6]. However, male monkeys have responded to DR to a greater extent than females. NIA female monkeys have maintained higher body weights compared to their respective controls at similar levels of DR relative to males (fig. 1) [7]. A moderate sex difference was also apparent in UW monkeys [8]. Factors contributing to this apparent sex difference including body composition and hormones are being explored, but UW

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attributed some of this sex discrepancy to the low body fat of the females at the start of the study [9]. In addition to lower body weight, examination by dual energy X-ray absorptiometry indicated that DR monkeys have relatively less fat and lean mass than controls [10]. DR monkeys also have less trunk fat and a reduced trunk-to-leg fat ratio (fig. 2) [6]. DR monkeys in the UW study also had less abdominal fat than controls [11]. Considering the health risks associated with abdominal adiposity, this reduction indicates a favorable shift for cardiovascular risk factors. After 15 years of study, DR monkeys in the NIA study continue to have lower body weights compared to controls [7]. Additionally, consistent with studies in humans, rhesus monkeys show a clear age-related decline in caloric intake (fig. 3). Aging is commonly associated with decreased food intake in humans [12]. This decline may be related to health issues, decreased energy expenditure, decreased motivation (detailed in behavior section), changes in body composition or hormonal status. There are several blood parameters that may correlate with changes in feeding behavior and the NIA identified a negative relationship between globulin and caloric intake independent of age [12]. The significance of this relationship is being explored further. Although consistent with humans, this observation of decreased intake complicates the logistics of a DR study and maintaining a 30% reduction in caloric intake compared to age-matched controls. The anorexia of aging and associated weight loss predisposes older humans to malnutrition and disease [13]. Thus, the NIA study has held food allotments constant for both the control

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and DR monkeys at their adult level for the duration of the study. Although the intake for the control monkeys has been decreasing as they age (they have been leaving behind more food), the DR monkeys have not been subject to a further reduction.

Glucose, Insulin and Metabolic Rate

Considering the lower body weight and fat, it would be expected that DR monkeys would be better able to regulate glucose than controls. A reduction in fasting glucose was not evident immediately in the NIA monkeys [14], but levels

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were reduced significantly after 3 years on DR [15]. This delayed glucose response was also evident in the UW monkeys [16]. The NIA DR monkeys also had a lower acute insulin response to a glucose challenge compared to controls [15]. The UW study assessed glucose kinetics during an intravenous glucose tolerance test. Compared to controls, DR monkeys had lower basal ␤-cell sensitivity to glucose, greater insulin sensitivity and a lower first-phase plasma insulin response [17]. Body fatness was highly predictive of glucose response in these monkeys, and these factors may contribute to an overall reduced disease risk. Both NHP studies have reported a reduction in metabolic rate in DR monkeys. In the NIA study, the reduction occurred early but equilibrated to levels similar to the controls as body composition changed [18]. Blanc et al. [19] reported that the reduction in total energy expenditure of DR monkeys in the UW study was attributable to resting energy expenditure and was significant even when adjusted for fat-free mass.

Endocrine Function

The endocrine system is important for regulating and maintaining complex systems of metabolism, thermoregulation, reproduction, stress response, behavior, homeostasis and immunity. Most of these processes are disturbed with aging, and these changes may be attributed to a decline in endocrine activation or signaling. Although the basal activity of some endocrine functions may not be altered with age, the response to stimuli may be drastically compromized.

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Thus, it was of interest to investigate how DR could affect various endocrine parameters. Melatonin Melatonin is a hormone secreted by the pineal gland in a diurnal pattern peaking during the night. It has been reported to improve sleep, lower blood pressure, strengthen the immune system, act as an antioxidant and even increase longevity [reviewed in 20]. Peak nocturnal concentrations are reached at the age of 1–3 years in humans with a gradual decline thereafter [21]. The pineal gland shrinks, melatonin secretion decreases and is often accompanied by a phase shift of the pulsatile release. A similar trend with age is seen in rodents [22] and monkeys [23]. It was of interest to determine if long-term DR would alter the age-related changes in melatonin secretion in the NIA monkeys. Early studies in Fischer 344 rats showed that 40% DR attenuated the age-related decline in pineal function and melatonin secretion [24]. However, the immune response in rats following melatonin supplementation was not improved as it was with DR [25]. Cross-sectional data from 52 control-fed male and female monkeys in the NIA study confirmed an age-related decline in peak melatonin levels [26]. However, unlike findings in a cross-sectional study in humans in which females accounted for the age-related difference [27], there was no gender difference in this cohort of monkeys. Additionally, monkeys that had been maintained on DR for 12 years did not show an age-related decline; in fact, there was an ageassociated increase (fig. 4) [26]. Moreover, the old DR monkeys had significantly higher serum melatonin levels than age-matched controls. This diet difference was not apparent in the younger adult monkeys but they may not have experienced a significant age-related decline yet. Similarly, urinary excretion of melatonin in the adult monkeys at the UW was not different between diet groups [8]. Additional diet differences may emerge as both the NIA and UW adult monkeys reach older ages. Dehydroepiandrosterone The androgenic steroids, dehydroepiandrosterone (DHEA) and its sulfated form DHEAS, are among the most abundant steroids in the body. In both humans and NHPs, circulating levels of DHEAS are very high during early adult life and then decrease markedly with aging. Elevated serum levels of DHEAS have been related to a protective function against age-associated diseases, such as diabetes [28], heart disease [29] and cancer [30]. As a result, this hormone has received considerable attention as a possible intervention against the decrements of aging. A large-scale survey of 792 laboratory-housed male and female rhesus monkeys at the UW reported an approximately 90% reduction in DHEAS from Mattison/Roth/Lane/Ingram

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Fig. 4. The effect of aging and DR on plasma melatonin in rhesus monkeys. a Relationship between age and melatonin levels (log transformed) for control (CON) and DR rhesus monkeys. b Mean (⫾ SEM) melatonin levels in adult and old monkeys. The age ⫻ diet interaction is significant (p ⬍ 0.04) by two-way analysis of variance. ap ⬍ 0.04: analysis of the simple main effect of age indicated a significant decrease in the control group but not in the DR group; bp ⬍ 0.01: analysis of the simple main effect of diet indicated a significant effect in the old group but not in the adult group. Reprinted from Roth et al. [26].

infancy through 36 years of age [31]. The magnitude of the decrease was greatest during the first few years of life followed by a more gradual decline, averaging 4.2% per year [31]. In humans, DHEAS peaks at around 20 years of age and then decreases continuously thereafter in both men and women [32, 33]. Although the slope of the decline clearly differs in these two species, the rate is about 2–2.5 times higher in rhesus monkeys compared to humans, a rate that is consistent with the humans’ approximately 3-fold greater life span [35]. Blood samples from NIA monkeys have been tested on several occasions to identify changes in DHEAS with age and DR. Roth et al. [34] reported an age-related decrease in DHEA in a cross-sectional sample of males that was not affected by 2–3 years of DR. Subsequently, Lane et al. [35] showed that the postmaturational decline in DHEAS was attenuated by DR in a 3-year longitudinal study. However, this report focused on the young adults and it remained

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unclear if the effect of DR would be maintained into older age. Additionally, measurements were based on blood samples collected at one time point in the day, although at a consistent time point, they could not detect circadian fluctuations. In the UW DR study, an effect of age on DHEAS levels was not detected in a cross-sectional analysis, although it was reported in the larger cohort cited above. However, this finding may be explained by the narrow age range of the monkeys, as all were considered middle-aged and thus subtle changes would not be evident. The effect of DR in the UW cohorts was somewhat inconsistent. One group of DR males had lower DHEAS levels than controls, but the DR females had slightly higher levels. Overall, the results suggested that DR did not alter DHEAS concentrations [8]. Using an advanced methodology to collect hormonal data, a cohort of NIA rhesus monkeys housed at the Oregon National Primate Research Center (ONPRC) was fitted with an indwelling catheter system. Serial blood samples were collected at hourly intervals for a 24-hour period in young (approx. 10 years) and old (approx. 26 years) male monkeys following 4 years of 30% restriction [36]. A clear diurnal pattern in DHEAS was evident in the young monkeys with a peak occurring in the morning when the lights came on. There was a dramatic drop in DHEAS concentration from young levels in both control and DR old males with little evidence of a diurnal pattern. In this crosssectional cohort of male monkeys, DR did not attenuate the age-related decline seen in DHEAS; in fact, DHEAS levels were slightly lower in the DR monkeys of both age groups [36]. It is possible that DR was initiated too late in the old monkeys, they were already 22 years of age, and a significant age-related decline may have already occurred that could not be reversed. Cortisol Glucocorticoids are of interest in the context of aging since most studies report that levels increase in rodents during late middle age and in humans during old age, a change that could indicate hippocampal dysfunction [37]. In the context of DR studies, glucocorticoids are of interest because elevated cortisol (the primary glucocorticoid in primates; corticosterone in rodents) may be the result of this low-intensity nutritional stress and actually mediating some of the positive effects of DR. In a cross-sectional study of rhesus monkeys, the UW group did not detect an age-related increase in cortisol during a 30-month assessment. Additionally, there was no consistent difference between DR and control animals [8]. In the monkeys housed at the ONPRC, both young (10 years) and old (26 years) males showed a diurnal pattern of release using an indwelling vascular catheter collecting 1-hour interval samples. There was no significant age-related change. Although mean and maximal levels were similar, an additional peak was

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apparent in the young DR monkeys that was not evident in the age-matched control or older monkeys. The peak was consistent with their increased locomotor activity. The old DR monkeys subjected to the same 4-year period of DR had slightly lower 24-hour cortisol levels possibly indicating that their stress response pathway was not activated comparably to the young monkeys [36]. Thyroid Hormones Thyroid hormones regulate metabolism and thereby may play a role in the mechanism for the metabolic adaptations that occur with DR. Additionally, normal aging has been associated with a slight decrease in thyroid-stimulating hormone (TSH) release [38] and decreased peripheral degradation of thyroxine (T4), resulting in lower serum triiodothyronine (T3) [39]. DR has been shown to have immediate effects on thyroid hormones in Sprague-Dawley rats. Decreases in T3 and T4 were sustained for 1 year but returned to normal levels within 7 days of refeeding [40]. In a cross-sectional analysis of the NIA monkeys ranging in age from 8 to 32 years, T3 did not change with age, while T4 and TSH decreased [41]. T3 was lower in monkeys within 1 month of initiating a DR diet when phased in gradually. However, the effect may be transient as the difference disappeared after 6 months on the diet. A diet effect was also evident in the old cohort of monkeys in which TSH levels were increased by long-term DR [41]. UW monkeys, which had been on DR for a briefer time, did not demonstrate changes in T3 [8]. This inconsistency between studies may relate to differences in body composition or macronutrients in the diet. Overall, results suggest that DR may contribute to an altered thyroid hormone axis.

Reproductive Function

Mechanisms that control energy balance are linked to those that control maturation and reproduction; therefore, it is plausible that when calories, thus energy, are decreased, growth and reproduction would also be affected. It was of interest to determine if it would also retard sexual maturation in rhesus monkeys as has been shown in rodents [40] and undernourished humans [42]. Male rhesus monkeys subjected to 30% DR at 1–2 years of age experienced an approximately 1-year delay in sexual maturation as evidenced by lower circulating testosterone levels compared to control monkeys [43]. Reproductive development was not monitored in the NIA female monkeys; however, currently there is an active program investigating ovarian aging and the transition to menopause. The pattern of reproductive senescence in rhesus females is similar to that in humans; however, relative to life span, menopausal

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Fig. 5. Total number (a) and percent normal (24–31 days; b) menstrual cycles in control (CON; n ⫽ 21) and 30% DR (n ⫽ 19) female rhesus monkeys over a 2-year period. Each point represents data for individual monkeys. Linear regression analysis revealed that both total number and percent normal menstrual cycles declined with age. DR did not affect menstrual cycling (p ⬎ 0.05). Reprinted from Lane et al. [44].

changes in monkeys occur slightly later in life [44, 45]. The similar hormonal changes leading to menopause offer rhesus monkeys as a valuable model for this area of study. Previous NIA data from a single time point blood sample of 40 monkeys aged 7–27 years indicated a significant age-related decrease in serum estradiol, increase in follicle-stimulating hormone and decrease in both number and length of menstrual cycles (fig. 5) [44]. Progesterone and luteinizing hormone did not change with age (fig. 6). None of these parameters were altered by 6 years of a 30% DR diet. More recently, the NIA has collected blood samples from these same female monkeys during 3 consecutive menstrual cycles, daily during the follicular phase and every third day during the luteal

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phase. Data from this more rigorous sampling strategy are consistent with previous findings in that all young monkeys cycled regularly, regardless of diet group. Additionally, elevated follicle-stimulating hormone levels were apparent in the older monkeys, and estradiol was not different between age groups [46]. Studies are under way to determine if DR delays the onset of menopause. Long-term DR data contrast with those from a recent report in young rhesus females (6–10 years) restricted to lose 20% of their body weight. The 4 initially lean monkeys became anovulatory in about 2 months while it took up to 10 months and a 46% reduction in body weight for the obese monkey [47]. Ovulation returned with refeeding but at a 28% greater caloric intake. This apparent discrepancy was likely caused by the immediate stress of caloric deficit which compensatory mechanisms can reverse over time.

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Immune Function

The role of the immune response in contributing to the prolongevity effects of DR has not been explored extensively in NHPs. DR retards several disease processes which have an immune component as has been shown by a decreased incidence of lymphomas, hepatomas and lung tumors in mice and rats [48]. Jolly [49] also reported that DR was effective in modulating the severity of symptoms in several autoimmune disease rodent models. Many studies of immune function have focused on T lymphocytes, which regulate both the type and the magnitude of the immune response. In rodents, the age-dependent loss in T lymphocyte interleukin 2 (IL-2) production is slowed by DR, while the population of naïve lymphocytes is maintained [50]. The mechanisms for these effects in rodents are still under investigation and have limited applicability to NHPs because many species differences exist in immune system biology. Information on aging of the NHP immune system is limited, and data on the effect of DR in NHPs are more scarce. Due to the nature of the studies, invasive procedures and immune challenges in a long-term study are problematic. Thus, much of the data are obtained from in vitro studies of lymphocytes. Old control monkeys from the NIA study were reported to have a reduced intracellular free Ca2⫹ response from CD4⫹ lymphocytes following stimulation compared to their young counterparts [51]. In this early study, 4 years of DR in the males and only 4 months in the female did not alter the response. In the same monkeys, lymphopenia was reported following 7 years on a DR diet, a finding similar to data in rodents. Additionally, those monkeys initiated to DR at 1 year of age tended to have a reduced proliferative capacity of mitogen-induced peripheral blood mononuclear cells (PBMCs), which was not apparent in monkeys initiated at a slightly older age [52]. Following 4 years on DR, monkeys at the UW showed no reduction in peripheral blood lymphocytes, but mitogen responses were reduced in DR monkeys compared to controls [53]. However, when NIA monkeys were examined after 14 years on DR, there was no indication of lymphopenia and only a slight trend toward decreased white blood cell count in those monkeys that had been initiated on the DR diet at 1 year of age [54]. Additionally, the difference in PBMC proliferative response to several mitogens in DR animals was not different when the animals had been maintained on the diet for 9 years or longer [54]. Although age-related changes in immune function are often contradictory, cytokine responses from male NIA monkeys are in general agreement with several mouse and human studies. Despite variability, the monkeys demonstrated a likely switch from a Th1 (␥-interferon) to a Th2-associated (IL-10, IL-6) cytokine profile during aging as evidenced by an increase in IL-10 and IL-6 response and a decreased ␥-interferon response following stimulation of the PBMCs [55].

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These same measures were conducted in a group of young (6–7 years) and old (22–25 years) monkeys following 2 years on a DR diet and showed that DR might be able to prevent this immune response switch. The old DR monkeys had significantly higher levels of the Th1-like cytokine ␥-interferon compared with old controls [56]. This improved responsiveness could be a key factor in decreasing the incidence of cancer and other age-associated diseases. To date, the only data available on the effect of DR on the composition of the peripheral T cell compartment comes from NIA monkeys housed at the ONPRC. Nikolich-Z¤ ugich and Messaoudi [54] reported that both males (3–5 years old) and females (5–7 years old) on long-term DR initiated during early adulthood have elevated percentages of naïve CD4 and CD8 cells and a decline in effector memory and senescent T cells compared to controls. This same preservation of naïve T cells was also evident in the PBMCs of adult animals on DR for only 2 years and the lymph nodes of older animals started on DR at a minimum of 17 years old [54]. Harper et al. [57] reported that the percentage of naïve T cells was a strong biomarker of aging that correlated with longevity in mice.

Gene Array/Oxidative Stress

UW investigators have used gene array technology to analyze age-related changes in the pattern of gene expression from skeletal muscle and the effects of DR in rhesus monkeys. In a comparison of young (mean age 8 years) and old monkeys (mean age 26 years), an upregulation of some transcripts involved in inflammation and oxidative stress and a downregulation in those associated with mitochondrial electron transport and oxidative phosphorylation were observed [58]. A downregulation of genes involved in energy metabolism and an upregulation of some structural genes involved in cellular growth were observed after 10 years of DR in middle-aged monkeys (mean age 20 years). DR did not affect the progression of age-related changes in gene expression which contrasts findings in mice [58]. The UW study has also focused on molecular markers of oxidative stress using electron microscopy. In a cohort of 2- to 34-year-old rhesus monkeys, there was an age-associated 2-fold increase in carbonyl levels from the vastus lateralis [59]. However, in the adult monkeys (17–23 years old) following 10 years on a 30% DR diet, carbonyls and nitrotyrosine levels were significantly lower than age-matched control-fed monkeys. The NIA has measured serum isoprostane as an indicator of oxidative stress. However, due to changes in assay techniques and sensitivity, results have been inconsistent. Additional analyses are under way.

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Fig. 7. Retrieval time (square root transformation) in a food motivation task (means ⫾ SEM) for female (a) and male (b) rhesus monkeys in different age groups on a control diet (CON) or DR for over 8 years. Reprinted from Mattison et al. [7].

Behavioral Assessments

Both humans and NHPs experience declines in several cognitive domains including the ability to recall recent events, acquire new information and shift from one problem-solving strategy to another, as well as reduced motor abilities. Tests of delayed responses taxing short-term memory are the best characterized in aging monkeys. The NIA is actively developing and implementing a behavioral program to evaluate age-related changes in memory and function to include tasks such as: object discrimination as an association memory task; delayed match-tosample as a recognition memory task; object reversal for set shifting; motor movement assessment panel to detect coarse and fine motor movement, and automated locomotor activity detection. Many of these tasks are currently under study.

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Fig. 8. Regression of age onto retrieval time (seconds) as a measure of motivation for food in rhesus monkeys fed both a long- and short-term control diet. Reprinted from Mattison et al. [7].

Behavior tasks used in nutritional studies can be confounded by the common practice of using a food reward; thus, it was necessary to establish that DR monkeys did not perform better in food-motivated tasks merely because they were more hungry than controls. Monkeys were observed in a novel task that measured the time they would spend working to get to a food reward [7]. Performance in this task was considered a measure of the activational aspect of motivation as the monkey initiated and maintained behavior required to retrieve the food [60]. Retrieval times between control and DR groups for the three age cohorts juvenile, adult, and old for males and females were not different (fig. 7). This phenomenon was observed in monkeys that had been on DR for 7–11 years and also in a cohort following only 3–5 years of DR [7]. Because motivational aspects appeared similar between diet groups, the NIA has developed additional behavioral experiments which use food rewards. Additionally, from this initial task, a clear age-related decline in motivation was observed when data from control monkeys only were analyzed (fig. 8) [7]. This finding paralleled the age-related decline in caloric intake. Changes in energy expenditure, body composition and hormonal influences may all contribute to behavior and feeding changes. Considering the metabolic changes associated with DR, it was of interest to determine age-associated changes in locomotor activity. Following 6 years on the study, NIA monkeys were monitored using ultrasonic motion detectors and videotape. Compared to their respective control group, adult DR males were the only cohort to display more pacing, gross movement, stereotypies and were less passive [61]. A subsequent study in the female cohort showed no generalized

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diet effect on activity although DR juveniles (6–8 years) were slightly less active than age-matched controls [62]. DR monkeys at the UW initially declined in activity while the controls increased [2], but these differences were not sustained [63, 64]. Duffy et al. [65] reported increased motor activity in DR rats which was associated with feeding time. The NIA is again monitoring the locomotor activity level to evaluate age-related changes and the influence of long-term restriction. Preliminary data indicate an age-related decline in home cage activity for control-fed males and females, and that old DR monkeys appear slightly less active than their control counterparts [unpubl. observation].

Brain

Analyses are ongoing to correlate behavior function with in vivo brain imaging. Thus far, age-related declines in the volume of the striatum and reduced binding potential of striatal dopamine D2 receptors have been identified [66]. DR did not attenuate this age-related decline in the striatum; in fact, volumes of the putamen were larger in the middle-aged and old control monkeys compared to DR animals. The difference remained even when corrected for body size [67]. The functional significance of this anatomical measure is uncertain, and follow-up studies are planned. A recent ancillary study conducted at the NIA has shown that a short-term 30% DR can lessen the severity of disease in a model of Parkinson’s disease [68]. Adult male rhesus monkeys were injected unilaterally in the right carotid artery with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) following 6 months on a DR or control diet. Following MPTP treatment, locomotor activity was markedly decreased in all monkeys; however, DR monkeys showed higher levels of activity compared to controls. Additionally, the MPTPinduced reduction in striatal levels of dopamine and dopamine metabolites was attenuated in DR monkeys. Another important finding was that levels of glialderived neutrophic factor, which promotes survival of dopamine neurons, were also higher in the caudate nucleus of DR monkeys. These findings suggest that DR or other nutritional interventions may beneficially alter the course of Parkinson’s disease.

Sensory Function

Several components of sensory function are affected with advanced age in primates. The gradual losses to both the auditory and the visual systems compromise late-life abilities and independence. Rhesus monkeys are an excellent

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model of human auditory capacity [69] and experience an age-related decline in cochlear and neural function [70]. Similarly, the pattern and relative timing of the age-dependent loss in accommodative function parallels that of humans [71, 72]. It was of interest to determine whether or not DR would influence agerelated changes in these sensory abilities. Both the NIA and the UW studies have reported a clear age-related decline in some measures of auditory function, but only the UW monkeys showed an indication of diet differences. The NIA tested only the male monkeys and showed evidence of an age-related decline in cochlear function as measured by a decrease in distortion product otoacoustic emissions [73]. These are associated with outer hair cell function within the cochlea [74], and a decline is indicative of cochlear degeneration. Additionally, the NIA monkeys had some age-associated decline in neural function as measured by a decline in some auditory brainstem responses [73]. Their waveforms are recorded by electroencephalography, and measures of wave amplitude, response latency and threshold indicate neural function. There were no significant effects of DR on any auditory parameters. UW monkeys also showed an auditory decline related to aging [75]. Additionally, sex and diet differences were apparent. Females were generally younger than the males tested; thus, sex differences were likely confounded by the age difference. There were poorer auditory brainstem response thresholds with age; however, older DR males were better than the control counterparts suggesting that DR may be beneficial in delaying presbycusis (high-frequency progressive hearing loss). Actual sex differences in the measures will become apparent in future studies when the females reach a similar old age. NIA monkeys were measured on several parameters of visual function. Consistent with previous studies in rhesus monkeys, lens thickness increased with age and accommodative amplitude decreased; however, neither were affected by diet (fig. 9) [76]. Lens thickening may combine with other lenticular factors to decrease the lens’ ability to change shape during accommodation. This study was the first to suggest that the effect of DR may not extend to the ocular accommodative mechanism or lens clarity. However, it was possible that the older animals may have already experienced age-related decline before initiation of the DR diet that could not be reversed. Future studies when the younger monkeys have reached an advanced age will clarify potential diet differences.

Conclusion

Although DR continues to show great promise for its health benefits and potential to extend life span, conclusive results from a long-living primate model are still years away. Rhesus monkeys exhibit many beneficial effects from DR

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Fig. 9. Carbachol-induced accommodative amplitude as a function of age in rhesus monkeys. Accommodative ability declined in both control (CON) and DR monkeys by 1.03 ⫾ 0.12 (p ⫽ 0.001) and 1.18 ⫾ 0.12 dpt/year (p ⫽ 0.001), respectively. There was no difference between diet groups (p ⫽ 0.374). Reprinted from Mattison et al. [76].

that have been similarly documented in DR rodents, such as decreased fat, improved glucoregulatory function, decreased risk factors for cardiovascular disease and diabetes. Additionally, factors that might be compromised by nutritional stress, such as reproduction and immune function, have not been affected detrimentally and, in fact, appear predictive of enhanced survival. Both the NIA and UW studies will continue to provide opportunities to assess aging parameters and the effectiveness of DR in maintaining better health and function into old age. Acknowledgements The authors wish to acknowledge the valuable work of the staff at Poolesville, Md., including Edward Tilmont, Jennifer Young, April Hobbs, Sue Pazzi, and the excellent veterinary care provided by Drs. Doug Powell and Rick Herbert. Additionally, we thank our many collaborators at other research institutions for their contributions. The work is supported by funds from the NIA provided to the Veterinary Research Program through an Inter-Agency Agreement.

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Roth GS, Lesnikov V, Lesnikov M, Ingram DK, Lane MA: Dietary caloric restriction prevents the age-related decline in plasma melatonin levels of rhesus monkeys. J Clin Endocrinol Metab 2001;86:3292–3295. Ng Ying Kin NMK, Nair NPV, Schwartz G, Thavundayil JX, Annable L: Secretion of melatonin in healthy elderly subjects: a longitudinal study. Ann NY Acad Sci 2004;1019:326–329. Small M, Gray CE, Beastall GH, MacCuish AC: Adrenal androgens and insulin-dependent diabetes mellitus. Diabetes Res 1989;11:93–95. Barrett-Connor E, Khaw K, Yen S: A prospective study of dehydroepiandrosterone sulfate, mortality, and cardiovascular disease. N Engl J Med 1986;315:1519–1524. Zumoff B, Levin J, Rosenfeld RS, Markham M, Strain GW, Fukushima DK: Abnormal 24-hour mean plasma concentrations of dehydroepiandrosterone and dehydroepiandrosterone sulfate in women with primary operable breast cancer. Cancer Res 1981;41:3360–3363. Kemnitz JW, Roecker EB, Haffa AL, Pinheiro J, Kurzman I, Ramsey JJ, MacEwen EG: Serum dehydroepiandrosterone sulfate concentrations across the life span of laboratory-housed rhesus monkeys. J Med Primatol 2000;29:330–337. Orentreich N, Brind J, Rizer R, Vogelman J: Age and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulthood. J Clin Endocrinol Metab 1984;59:551–555. Carlström K, Brody S, Lunell NO, Largrelius A, Mollerstrom G, Pousette A, Rannevik G, Stege R, von Schoultz B: Dehydroepiandrosterone sulfate and dehydroepiandrosterone in serum: differences related to age and sex. Maturitas 1988;10:297–306. Roth GS, Blackman MR, Ingram DK, Lane MA, Ball SS, Cutler RG: Age-related changes in androgen levels of rhesus monkeys subjected to diet restriction. Endocrine J 1993;1:227–234. Lane MA, Ingram DK, Ball SS, Roth GS: Dehydroepiandrosterone sulfate: a biomarker of primate aging slowed by caloric restriction. J Clin Endocrinol Metab 1997;82:2093–2096. Urbanski HF, Downs JL, Garyfallou VT, Mattison JA, Lane MA, Roth GS, Ingram DK: Effect of caloric restriction on the 24-hour plasma DHEAS and cortisol profiles of young and old male rhesus macaques. Ann NY Acad Sci 2004;1019:443–447. Sapolsky RM: Glucocorticoids, stress, and their adverse neurological effects: relevance to aging. Exp Gerontol 1999;34:721–732. Marriotti S, Franceschi C, Cossarizza A, Pinchera A: The aging thyroid. Endocrinol Rev 1995;16: 686–715. Rubenstein HA, Butler VP Jr, Werner SC: Progressive decrease in serum triiodothyronine concentrations with human aging: radioimmunoassay following extraction of serum. J Clin Endocrinol Metab 1973;37:247–253. Merry BJ, Holehan AM: The endocrine response to dietary restriction in the rat; in Woodhead AD, Blackett AD, Hollaender A (eds): Molecular Biology of Aging. New York, Plenum Press, 1985, pp 117–141. Roth GS, Handy AM, Mattison JA, Tilmont EM, Ingram DK, Lane MA: Effects of dietary caloric restriction and aging on thyroid hormones of rhesus monkeys. Horm Metab Res 2002;34:378–382. Bongaarts J: Does malnutrition affect fecundity? A summary of evidence. Science 1980;208: 564–569. Roth GS, Blackman MR, Ingram DK, Lane MA, Ball SS, Cutler RG: Age-related changes in androgen levels of rhesus monkeys subjected to diet restriction. Endocrine J 1993;1: 227–234. Lane MA, Black A, Handy AM, Shapses SA, Tilmont EM, Kiefer TL, Ingram DK, Roth GS: Energy restriction does not significantly alter bone mineral metabolism or reproductive cycling and hormones in female rhesus monkeys. J Nutr 2001;131:820–827. Black A, Lane MA: Nonhuman primate models of skeletal and reproductive aging. Gerontology 2002;48:72–80. Wu J, Mattison J, Ottinger M, Zelinski-Wooten M, Handy A, Roth G, Ingram D, Tilmont E, Lane M: Ovarian function and incidence of endometriosis in long-term calorie restriction. Gerontologist 2004;44:10. Lujan ME, Krzemien AA, Reid RL, Van Vugt DA: Developing a model of nutritional amenorrhea in rhesus monkeys. Endocrinol Online 2005;10:1210. http://endo.endojournals.org/cgi/rapidpdf/ en.2005–0821v1.

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Weindruch R, Walford RL: Dietary restriction in mice beginning at 1 year of age: effect on lifespan and spontaneous cancer incidence. Science 1982;215:1415. Jolly CA: Dietary restriction and immune function. J Nutr 2004;134:1853–1856. Pahlavani M: Caloric restriction and immunosenescence: a current perspective. Front Biosci 2000;5:580–587. Grossmann A, Rabinovitch PS, Lane MA, Jinneman JC, Ingram DK, Wolf NS, Cutler RG, Roth GS: Influence of age, sex, and dietary restriction on intracellular free calcium responses of CD4⫹ lymphocytes in rhesus monkeys (Macaca mulatta). J Cell Physiol 1995;162:298–303. Weindruch R, Lane MA, Ingram DK, Ershler WB, Roth GS: Dietary restriction in rhesus monkeys: lymphopenia and reduced mitogen-induced proliferation in peripheral blood mononuclear cells. Aging Clin Exp Res 1997;9:304–308. Roecker EB, Kemnitz JW, Ershler WB, Weindruch R: Reduced immune responses in rhesus monkeys subjected to dietary restriction. J Gerontol 1996;51A:276–279. Nikolich-Zugich J, Messaoudi I: Mice and flies and monkeys too: caloric restriction rejuvenates the aging immune system of non-human primates. Exp Gerontol 2005;40:884–893. Mascarucci P, Taub D, Saccani S, Paloma MA, Dawson H, Roth GS, Ingram DK, Lane MA: Agerelated changes in cytokine production by leukocytes in rhesus monkeys. Aging Clin Exp Res 2001;13:85–94. Mascarucci P, Taub D, Saccani S, Paloma MA, Dawson H, Roth GS, Lane MA, Ingram DK: Cytokine responses in young and old rhesus monkeys: effect of caloric restriction. J Interferon Cytokine Res 2002;22:565–571. Harper JM, Galecki AT, Burke DT, Miller RA: Body weight, hormones, and T cell subsets as predictors of life span in genetically heterogeneous mice. Mech Ageing Dev 2004;125:381–390. Kayo T, Allison DB, Weindruch R, Prolla TA: Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys. Proc Natl Acad Sci 2001;98:5093–5098. Zainal TA, Oberley TD, Allison DB, Szweda LI, Weindruch R: Caloric restriction of rhesus monkeys lowers oxidative damage in skeletal muscle. FASEB J 2000;14:1825–1836. Salamone JD, Correa M: Motivational views of reinforcement: implications for understanding the behavioral functions of nucleus accumbens dopamine. Behav Brain Res 2002;137:3–25. Weed JL, Lane MA, Roth GS, Speer DL, Ingram DK: Activity measures in rhesus monkeys on long-term calorie restriction. Phys Behav 1997;62:97–103. Moscrip TD, Ingram DK, Lane MA, Roth GS, Weed JL: Locomotor activity in female rhesus monkeys: assessment of age and calorie restriction effects. J Gerontol 2000;55:B373–B380. Ramsey JJ, Roecker EB, Weindruch R, Baum ST, Kemnitz JW: Thermogenesis of adult male rhesus monkeys: results through 66 months of dietary restriction. FASEB J 1996;10:A726. Ramsey JJ, Roecker EB, Weindruch R, Kemnitz JW: Energy expenditure of adult male rhesus monkeys during the first 30 months of dietary restriction. Am J Physiol 1997;272:E901–E907. Duffy PH, Feuers RJ, Hart RW: Effect of chronic caloric restriction on the circadian regulation of physiological and behavioral variables in old male B6C3F1 mice. Chronobiol Int 1990;7:291–303. Ingram DK, Chefer S, Matochick J, Moscrip TD, Weed J, Roth GS, London ED, Lane MA: Aging and caloric restriction in nonhuman primates: behavioral and in vivo brain imaging studies. Ann NY Acad Sci 2001;928:316–326. Matochick J, Chefer SI, Lane MA, Roth GS, Mattison JA, London ED, Ingram DK: Age-related decline in striatal volume in rhesus monkeys: assessment of long-term calorie restriction. Neurobiol Aging 2004;25:193–200. Maswood N, Young J, Tilmont E, Zhang Z, Gash DM, Gerhardt GA, Grondin R, Roth GS, Mattison J, Lane MA, Carson RE, Cohen RM, Mouton PR, Quigley C, Mattson MP, Ingram DK: Caloric restriction increases neurotrophic factor levels and attenuates neurochemical and behavioral deficits in a primate model of Parkinson’s disease. Proc Natl Acad Sci 2004;101: 18171–18176. Allen AR, Starr A: Auditory brain stem potentials in monkey (M. mulatta) and man. Electroencephalogr Clin Neurophysiol 1978;45:53–63. Torre P III, Fowler CG: Age-related changes in auditory function of rhesus monkeys (Macaca mulatta). Hear Res 2000;142:131–140.

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Bito LZ, DeRousseau CJ, Kaufman PL, Bito JW: Age-dependent loss of accommodative amplitude in rhesus monkeys: an animal model for presbyopia. Invest Ophthalmol Vis Sci 1982;23: 23–31. Kaufman PL, Bito LZ, DeRousseau CJ: The development of presbyopia in primates. Trans Ophthalmol Soc UK 1982;102:323–326. Torre P III, Mattison JA, Fowler CG, Lane MA, Roth GS, Ingram DK: Assessment of auditory function in rhesus monkeys (Macaca mulatta): effects of age and calorie restriction. Neurobiol Aging 2004;25:945–954. Brownell CL: Outer hair cell electromotility and otoacoustic emissions. Ear Hear 1990;11:89–92. Fowler CG, Torre P III, Kemnitz JW: Effects of caloric restriction and aging on the auditory function of rhesus monkeys (Macaca mulatta): the University of Wisconsin study. Hear Res 2002;169:24–35. Mattison JA, Croft MA, Dahl DB, Roth GS, Lane MA, Ingram DK, Kaufman PL: Accommodative function in rhesus monkeys: effects of aging and caloric restriction. Age 2005;27: 59–67.

Donald K. Ingram, PhD Laboratory of Experimental Gerontology National Institute on Aging, NIH, 5600 Nathan Shock Drive Baltimore, MD 21224 (USA) Tel. ⫹1 410 558 8180, Fax ⫹1 410 558 8302, E-Mail [email protected]

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Mobbs CV, Yen K, Hof PR (eds): Mechanisms of Dietary Restriction in Aging and Disease. Interdiscipl Top Gerontol. Basel, Karger, 2007, vol 35, pp 159–175

Caloric Intake and Alzheimer’s Disease Experimental Approaches and Therapeutic Implications

Giulio Maria Pasinetti, Zhong Zhao, Weiping Qin, Lap Ho, Yemul Shrishailam, Donal MacGrogan, Wendy Ressmann, Nelson Humala, Xunxian Liu, Carmen Romero, Breton Stetka, Linghong Chen, Hanna Ksiezak-Reding, Jun Wang Neuroinflammation Research Laboratories, Department of Psychiatry, Mount Sinai School of Medicine, New York, N.Y., and Bronx Veterans Affairs Medical Center, Mount Sinai School of Medicine, Bronx, N.Y., USA

Abstract Alzheimer’s disease (AD) is a rapidly growing public health concern with potentially devastating effects. Presently, there are no known cures or effective preventive strategies. While genetic factors are relevant in early-onset cases, they appear to play less of a role in late-onset sporadic AD cases, the most common form of AD. Due to the fact that the disease typically strikes very late in life, delaying symptoms could be as good as a cure for many people. For example, it is now widely accepted that if the onset of the disease could be delayed by even 5 years, the incidence could be cut in half. Both clinical and epidemiological evidence suggests that modification of lifestyle factors such as nutrition may prove crucial to AD management given the mounting experimental evidence suggesting that brain cells are remarkably responsive to ‘what somebody is doing’. Among other nongenetic factors influencing AD, recent studies strongly support the evidence that caloric intake may play a role in the relative risk for AD clinical dementia. Indeed, the effect of diet in AD has been an area of research that has produced promising results, at least experimentally. Most importantly, as mechanistic pathways are defined and their biochemical functions scrutinized, the evidence supporting a direct link between nutrition and AD neuropathology continues to grow. Our work, as well as that of others, has recently resulted in the development of experimental dietary regimens that might promote, attenuate or even reverse features of AD. Most remarkably, while we found that high caloric intake based on saturated fat promotes AD type ␤-amyloidosis, conversely we found that dietary restriction based on reduced carbohydrate intake is able to prevent it. This evidence is very exciting and is, in part, consistent with current epidemiological studies suggesting that obesity and diabetes are associated with a ⬎4-fold increased risk of developing AD. The clarification of the mechanisms through which

dietary restriction may beneficially influence AD neuropathology and the eventual discovery of future ‘mimetics’ capable of anti-␤-amyloidogenic activity will help in the development of ‘lifestyle therapeutic strategies’ in AD and possibly other neurodegenerative disorders. Copyright © 2007 S. Karger AG, Basel

The health-related risks associated with obesity are of great public health concern in view of the fact that obesity may causally promote the incidence of a number of chronic degenerative conditions. Some of these conditions, such as Alzheimer’s disease (AD) and Parkinson’s disease, involve the nervous system and are being increasingly linked to issues relating to nutrition. For example, accumulating evidence indicates that certain nutrition-related issues, such as diabetes [1–7], increasing caloric intake [8] or obesity [9, 10], may promote neurodegeneration. In contrast, other nutritional factors, such as dietary restriction [11–14] or consumption of fish oil [15, 16], may beneficially modulate neurodegenerative disorders. From an economic point of view, neurodegenerative disorders have the potential to become major public health burdens as life expectancy increases. An estimated 4.5 million people have AD in the USA, a number that has doubled since 1980 and is expected to reach as much as 16 million by 2050 [17]. Most importantly, it has been calculated that even delaying the onset of AD for a few years would decrease its prevalence and burden on public health systems [18]. Obesity is also an important health issue when it comes to forecasting future cash flows in the Social Security and Medicare systems. Not only does the likelihood of having comorbidities rise with the degree of obesity, ‘but the prevalence of having 2 or more health conditions’ has been found to increase with weight status [19]. Moreover, in addition to the rather easily quantifiable impact of obesity on morbidity and mortality, being overweight or obese frequently compromises individuals’ quality of life [20]. Thus, it is of concern that at the beginning of the twenty-first century, the fraction of Americans considered to be obese had reached ‘epidemic’ levels, according to a study published in the Journal of the American Medical Association [21]. This study, which was carried out between 1991 and 1998, observed a steady increase in weight in all states of the union, in both sexes, across age groups, races and educational levels, and occurred regardless of smoking status. It found that obesity had increased from 12.0% in 1991 to 17.9% in 1998. Likewise, ‘national survey data show that between 1976–80 and 1988–94 the age-adjusted prevalence of obesity increased by 8 percentage points, from 14.5 to 22.5%, in the US adult population ages 20–74’ [22]. This translates into increases in mean body mass index (BMI) and in the prevalence of overweight and obesity for US adults and children.

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In an effort to establish a basis to define what a ‘healthy weight’ is, the Dietary Guidelines for Americans [6th ed., 2005; http://www.healthierus.gov/ dietaryguidelines/] addresses issues of weight maintenance and weight loss by making long-term changes in physical activity and eating behavior. The health consequences of overweight and obesity are related to adverse health conditions such as diabetes, coronary heart disease and hypertension. One glaring fact about the recommended range of healthy weights in the Dietary Guidelines is that close to more than half of the adult population, in particular half of adult males, have been above that range at least since 1960. A discussion of the pros and cons of this latest edition shows how complex and elusive the subject of optimal weight can be [23]. Of interest is that leptin, a hormone important in energy homeostasis and food intake regulation, has been singled out in the guidelines as a metabolic indicator influencing food intake. Because levels of leptin rise when fat stores are high, leptin may play a role in public health monitoring of adiposity in the future [24]. Of particular interest to this review article is the accumulating evidence pointing to a relationship between obesity and dementia later in life. For example, Whitmer et al. [25] have recently reported an analysis of prospective data from a multiethnic population-based cohort obtained with the objective of evaluating a possible association between obesity in middle age, as measured by BMI and skinfold thickness, and risk of dementia later in life. Dementia was diagnosed in 713 (6.9%; in a cohort of 10,276 people) of the participants. Obese people (BMI ⱖ30) had a 35% greater risk of dementia compared with those of normal weight (BMI 18.6–24.9). The authors concluded that obesity in middle age increases the risk of future dementia independently of comorbid conditions. This evidence is very interesting especially in view of the recent studies suggesting that certain cardiovascular risk factors (e.g. diet) may be significant contributors to an increased risk of vascular-related dementia [1]. A large body of evidence indicates that cardiovascular risk factors, e.g. certain dietary ones, may also increase the relative risk of AD and clinical dementia even when vascular dementia cases are excluded from the analysis [26, 27]. Thus, it may be the case that additional ‘nonvascular’ events associated with certain cardiovascular risk factors may be involved in the increased risk for AD. Most interestingly, we also note that recent evidence suggests that type 2 diabetes may also be associated with an increased risk of developing AD and may affect cognitive systems differentially [2]. Thus, it is possible that potential risk factors associated with certain dietary regimens accepted as cardiovascular risk factors for vascular dementia may also independently contribute to the development and progression of AD. Further exploration of this phenomenon in AD and AD model systems will provide critical direction for future studies investigating mechanisms involved in the potentiation of AD neuropathology and

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possibly future therapeutic applications based on dietary modifications. However, as discussed below, dietary regimens including dietary restriction (DR) and weight reduction programs in neurodegenerative disorders such as AD can be a complex endeavor because they should be made on the basis of combined evidence from different sources such as (1) epidemiological studies, (2) experimental models and ultimately (and most importantly) (3) from controlled clinical studies. Based on these considerations, this review article will first discuss recent evidence indicating (1) the beneficial role of dietary regimens in health and disease and (2) recent experimental evidence suggesting that diet and possibly the control of caloric intake may beneficially influence AD and possibly other neurodegenerative disorders. Finally, based on this evidence, this review will identify potential therapeutic scenarios for eventual future interventions.

Dietary Implications in the Onset and Progression of Clinical Alzheimer’s Disease

The possibility that reactive oxygen species are a factor in the neuronal damage seen in AD has led to examine how antioxidants in foods, or as vitamin supplements in the form of tocopherol (vitamin E), ascorbic acid (vitamin C) and carotenes, can affect AD. Although the results appear promising in some cases, the data on the value of antioxidant supplementation remain inconclusive. Likewise, deficiencies in folate, vitamin B12 and vitamin B6 lead to high concentrations of homocysteine in the brain through different pathways. This has led to study the possible link between homocysteine and the development of AD and Parkinson’s disease [28]. Although epidemiological studies have implicated high concentrations of homocysteine in the brain in neuronal degeneration [11, 28], the data relating folate and vitamin B12 and B6 supplementation as palliatives to the cognitive decline seen in AD are inconsistent [28]. However, Lim et al. [15] have recently used a transgenic mouse model of AD-type neuropathology to evaluate the impact of n–3 fatty acid docosahexaenoic acid (DHA) in AD-type amyloid neuropathology. They found that DHA-enriched diets significantly reduced total AD-type amyloid neuropathology by ⬎70% when compared with low-DHA or control chow diets. Dietary DHA also decreased ␤-amyloid (A␤) 1–42 levels below those seen with control chow. The results suggest that DHA could be protective against AD-type amyloid deposition in the brain and eventually prevent downstream neurodegenerative conditions. Studies relating fat and fish intake to the risk for AD and cognitive decline have failed to establish a firm causal relationship. Despite the fact that there are

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no controlled clinical studies to support dietary recommendations, in an indirect way, a diet low in saturated and trans-fatty acids and high in monounsaturated, polyunsaturated and fish-related fats can be assumed to be beneficial in the prevention of cognitive decline and AD by means of promoting a viable vascular system [29]. Furthermore, dietary fats may also influence AD through lipid metabolism, insulin resistance [30], high concentrations of circulating insulin [3], oxidation [31] and the amyloid cascade hypothesis [32, 33]. In addition, APOE, the gene associated with sporadic AD, is related to lipid metabolism and modulates cholesterol concentrations in response to intake from fats [34, 35]. People with the APOE ␧4 allele, who are at high risk of AD, normally have high levels of cholesterol in the blood. In addition to nutrients, alcohol intake has also been suggested as a risk factor for AD. This is supported by evidence that ethanol consumption might lead to oxidative brain damage in rat models [36]. Paradoxically, accumulating epidemiological evidence indicates that moderate consumption of alcohol in the form of red wine may actually lower the risk of cerebrovascular disease in older adults [37]. A study by Goldberg et al. [38] sponsored by the American Heart Association has found that consumption of alcohol-containing beverages (e.g. wine) actually has cardiovascular and cognitive benefits. However, numerous other studies relating alcohol to AD have yielded mixed results. A case has been made for the elderly to consume red wine in moderate quantities because of its high contents of antioxidants, such as flavonoids, which may not be found in other alcoholic beverages [4, 39, 40]. But, in and of itself, alcohol consumption carries the potential for abuse and addiction. Observational studies on diet and disease may incur errors in the measurement of nutrients, a caveat that has been clarified by Luchsinger and Mayeux [4]. If the measurement error is not related to outcome, it will underestimate true associations. Also, the long latency period of AD may be the consequence of lifelong exposure to a number of factors that are difficult to isolate and analyze in their true context. Moreover, the validity of basing clinical decisions about individual patients on data from randomized trials remains to be settled [41]. So far, it appears unlikely that trials can address all questions regarding diet and AD, given the nature of AD as a chronic disease with a latency period. It would be difficult to conduct trials long and large enough to observe results. Luchsinger and Mayeux [4] also touched on the concept that nutritional supplements alone (e.g. carotenoids) may not be as effective as the whole foods in which they may be found (fruits and vegetables) such that the interactions of nutrients within foods, or patterns of diet, is what may actually be of benefit. Recent findings by Gardner et al. [41] appear to corroborate their viewpoint. According to their findings, plant-based diets may be superior to low-fat diets even if the two diets are identical in total fat, protein, carbohydrate and cholesterol

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content. These authors noted that national dietary guidelines have probably underestimated the potential low-density lipoprotein cholesterol-lowering effect of certain diets. In a randomized clinical trial, they set out to contrast plasma lipid responses to two low-fat diet patterns. They found that a plantbased low-fat diet reduces levels of low-density lipoprotein twice as much as a low-fat diet based on prepackaged foods. Such plant-based diets may provide an effective alternative to cholesterol-lowering drugs like statins [41]. Although there is enough evidence suggesting that dietary modification, such as low calorie intake, may prevent AD and other age-related neurodegenerative disorders, malnutrition in the elderly remains a concern. Hence, dietary recommendations may need to be made on the basis of comorbidities such as type 2 diabetes, cardiovascular disease and osteoarthritis.

Alzheimer’s Disease and Nutrition

While genetic factors are highly relevant in early-onset AD cases, their significance diminishes in late-onset sporadic AD cases, the most common form of AD [17]. Nongenetic factors, including modifiable lifestyle dietary regimens, are receiving great attention in AD, especially because of recent epidemiological studies indicating that caloric intake may influence the relative risk for AD clinical dementia. Dietary factors have been an area of research that has produced promising results, at least experimentally. Most importantly, the evidence supporting a direct link between nutrition and AD amyloid neuropathology discussed below [12, 13] continues to grow, as the mechanistic pathways are defined and their biochemical functions scrutinized. AD is a progressive neurodegenerative disorder marked by loss of memory, cognition and behavioral stability [17]. AD is defined pathologically by extracellular neuritic plaques comprised of fibrillar deposits of ␤-amyloid (A␤) and neurofibrillary tangles comprised of paired helical filaments of hyperphosphorylated tau. Current therapies for AD, such as cholinesterase inhibitors, treat the symptoms but do not modify the progression of the disease. The etiology of AD is unclear, and data from familial AD mutations strongly support the ‘amyloid cascade hypothesis’ of AD, i.e. that neurodegeneration in AD is initiated by the formation of neurotoxic A␤ aggregates, and all familial AD mutations increase levels of A␤ peptide or the density of A␤ deposits [17]. Current therapeutic strategies to treat AD are aimed at preventing the formation of amyloidogenic A␤ peptides [17]. For this reason, the ‘amyloidogenic’ ␤- and ␥-secretase activities necessary for the generation of amyloidogenic A␤ peptides have become central targets for development of therapeutic reagents in AD [17]. However, it has been difficult to find safe, selective ␤- and ␥-secretase

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inhibitors, mainly because of the influence of these inhibitors on other cellular substrates whose processing is vital [17]. Ongoing studies, discussed below, in our laboratory suggest that DR regimens based on low-carbohydrate content may beneficially influence AD-type neuropathology through the promotion of ‘nonamyloidogenic’ processing of amyloid precursor protein (APP) via the promotion of ␣-secretase activities. In addition, since the ␣-secretase cleavage of APP is known to involve the release of a soluble neuroprotective form of APP (sAPP-␣; also found in our studies), it is possible that DR, while promoting the nonamyloidogenic pathway in the brain, may also promote brain repair activities as a consequence of sAPP-␣ neurotrophic function [42]. As discussed below, there is increasing consensus that the production and accumulation of A␤ peptides is central to the pathogenesis of AD. The continual search for ways to manage if not reverse AD neuropathology has led to ongoing efforts to elucidate its underlying causes and possible treatments. The likely link between A␤ peptide aggregation and AD pathology emphasizes the need for a better understanding of the mechanisms through which dietary regimens may influence A␤ production.

Diabetogenic Dietary Regimens Resulting in Insulin Resistance Coincide with Promotion of Alzheimer’s Disease Amyloid Neuropathology

Abnormalities in insulin metabolism associated with type 2 diabetes resistance are among the central factors thought to influence the onset of AD by promoting the synthesis and/or interference of A␤ degradation [5, 6, 43–45]. For example, there is in vitro evidence indicating that insulin itself may significantly promote the generation of extracellular amyloidogenic A␤ peptides through mechanisms that involve the acceleration of APP/A␤ trafficking from the trans-Golgi network, a major cellular site for A␤ generation, to the plasma membrane [43]. While this evidence tentatively suggests that abnormal carbohydrate metabolism might play an important role in AD through mechanisms that involve A␤ peptide generation, experimental studies also suggest that insulin resistance may promote AD amyloid neuropathology in the Tg25876 mouse model of AD amyloid neuropathology, possibly by limiting A␤ degradation via competition with insulin for degradation by insulin-degrading enzyme (IDE) [46], a zinc-metallopeptidase that preferentially cleaves proteins with a propensity to form ␤-pleated sheet-rich amyloid fibrils [47], such as monomeric A␤ peptides [46]. While the role of insulin in AD has received major attention with respect to its potential role in amyloid neuropathology, recent evidence also suggests a role

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for insulin in normal memory function, supporting the hypothesis that insulin affects many mechanisms related to neuronal activity and cognitive function by itself. Of interest to us is the fact that chronic hyperinsulinemia and insulin resistance, or reduced insulin effectiveness, may exert a negative influence on memory [5]. For example, Hoyer [44] proposed that low concentrations in circulating insulin in the central nervous system, together with reduced expression of IR and subsequent altered downstream signaling AD, would ultimately lead to reduced levels of acetylcholine with a corresponding decrease in cerebral blood flow. Based on this evidence and the fact that type 2 diabetes appears to be associated with an increased relative risk for AD [5, 6, 44, 45], we have recently explored the role of experimental type 2 diabetes in the Tg2576 AD mouse model [6]. We found that a diabetogenic diet resulting in elevated circulating levels of insulin coincided with promoted amyloidogenic A␤1–40 and A␤1–42 peptide generation and amyloid plaque burden in the brain of Tg2576 mice that corresponded with increased ␥-secretase activities and decreased IDE activities. Moreover, the increased AD-type amyloid neuropathology also coincided with increased and impaired spatial memory functions assessed by performance in a spatial water maze task [6]. Further exploration of the apparent interrelationship of insulin resistance to brain amyloidosis revealed a functional decrease in IR-mediated signal transduction in the brain, as suggested by decreased IR ␤-subunit (IR-␤) Y1162/1163 autophosphorylation and reduced phosphatidylinositol 3-kinase/pS473-AKT/protein kinase B in these same brain regions [6]. Results from this study strongly suggested that one mechanism through which diet-induced insulin resistance in Tg2576 mice can significantly promote AD-type amyloidosis in the brain is by reducing IR signaling, resulting in elevation of ␥-secretase activities. The studies also suggested that type 2 diabetes may further contribute to AD amyloid neuropathology attenuating degradation of A␤ peptides through pathways associated with IDEs (fig. 1). Collectively, these findings indicate that clinical AD is a result of early life as well as later life risk factors, and that genetic predisposition to the disease may modify the constellation of predictors.

Dietary Restriction Based on a Low-Carbohydrate Diet

As discussed above, a fundamental problem of AD neuropathology is the aberrant generation of amyloidogenic A␤ amyloid peptides in the brain that lead to an abnormal deposition of the neuritic plaques that are a landmark in AD. Although evidence supports a potential neuroprotective role for DR in neurodegeneration, until recently there was no information as to whether reduced caloric intake could attenuate AD neuropathology. Findings of recent

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High-fat diet APP

Insulin resistance

␥-Secretase A␤ IDE

Degradation Amyloid plaque deposition

Fig. 1. Role of insulin resistance in AD-type neuropathology.

prospective studies indicate that increasing caloric intake may be a risk factor for AD [45, 48, 49]. Because of this evidence and the epidemiological evidence indicating that DR may influence the risk for AD [8, 45], we have continued to explore if a clinically applicable weight reduction/DR regimen based on an approximately 30% reduced carbohydrate content could (1) attenuate AD neuropathology and (2) decrease preexisting amyloid neuritic neuropathology (e.g. a reduction in plaque size), eventually resulting in recovery of amyloidassociated neuritic dystrophy as a function of time in the same strain of Tg2576 mice fed a low-carbohydrate/DR diet. Based on these considerations, we tested the hypothesis that low-carbohydrate/DR may be a beneficial intervention in AD through mechanisms that prevent A␤ generation and neuritic plaque deposition in the brain using a mouse model of AD type amyloidosis [50]. The aim of these studies was to test the hypothesis that DR may beneficially influence AD through mechanisms that prevent the development of amyloidosis associated with AD. To test this hypothesis, 3-month-old Tg2576 mice, which develop AD type amyloid neuropathology by 8–10 months of age [50], were fed for 9 months with a daily low-carbohydrate diet resulting in a 30% lower caloric intake compared to that consumed by age- and gender-matched control Tg2576 mice fed ad libitum (AL) with a standard laboratory rodent diet. Nutrient composition in the DR diet was achieved by selectively reducing the carbohydrate content of the diet while consumption of protein, fat, cholesterol, vitamins and minerals was identical to that of AL fed Tg2576 mice [12]. This dietary regimen resulted in body weight stabilization over the 9-month study period among DR Tg2576

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mice relative to the AL fed group, coinciding with an approximately 3-fold lower ependymal fat pad weight and improved glucose tolerance response as determined by an intraperitoneal glucose tolerance test. These physiological adaptations in the DR Tg2576 mice relative to AL fed controls are consistent with clinical evidence that low-carbohydrate DR considerably improves abnormal glucose control and obesity [9, 51–53], which are risk factors not only for diabetes but also for AD [7, 54].

Weight Reduction Dietary Restriction Results in Decreased Alzheimer’s Disease-Type Amyloid Neuropathology in Tg2576 Mice

When Tg2576 mice were examined for AD-type neuropathology at 12 months of age, we found that 9-month DR treatment almost completely prevented cortical and hippocampal AD-type amyloid plaque development [12] relative to animals in the AL fed group. Consistent with this evidence, we noted commensurately lower concentrations of amyloidogenic A␤1–40 and A␤1–42 peptides in the neocortex and hippocampus as evaluated by ELISA assay, relative to AL fed controls [12]. No detectable change in total full-length APP level was noted in either brain region of DR versus AL fed Tg2576 mice [12]. To further evaluate the antiamyloidogenic role of DR in the brain of Tg2576 mice, we explored APP processing and A␤ peptide generation using immunoprecipitation (IP) and mass spectrometry (MS). Consistent with the aforementioned ELISA evidence, using 4G8 antibody for A␤ IP, we confirmed decreased levels of A␤1–40 and A␤1–42 in the same neocortical samples we used for the A␤ ELISA assay [12]. In addition, a relative proportional reduction in A␤1–37, A␤1–38 and A␤1–39 peptide content was also observed in the neocortex of the DR group compared to the AL fed control group. This evidence, together with our observation that the concentration of the approximately 7-kDa carboxy-terminal fragment (CTF) ␥ cleavage product of APP, an index of ␥-secretase activity, was unchanged in the neocortex of the DR group relative to AL fed controls, suggested the possibility that ␥-secretase activity was not involved in the DR-associated antiamyloidogenic activity. To further identify A␤ carboxy-terminal peptide fragments that would have been otherwise undetected in the 4G8 IP-MS studies, we used 6E10 antibody in additional A␤ IP-MS studies. Consistent with the 4G8 IP-MS spectra, we noted decreased levels of A␤1–40 and A␤1–42 as well as A␤1–37, A␤1–38 and A␤1–39 peptide in the DR group relative to AL fed control animals. In addition, we found a major elevation in A␤1–16 peptide fragment concentration in the neocortex of the DR group that was not detected in the AL fed controls.

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Because ␣-secretase can cleave APP, eventually resulting in the generation of A␤ CTFs ending at the AA residue leucine 16 of A␤ [42], we continued to explore the role of DR in ␣-secretase activity in the brain. Cleavage of APP by ␣-secretase releases the amino-terminal extracellular domain known as sAPP-␣ domain coincidental with elevation in membranebound ␣-secretase-cleaved APP CTF-␣. We therefore explored the regulation of sAPP-␣ and CTF-␣ cleavage products of APP in the brain as indices of ␣-secretase activity in response to DR. Interestingly, we found that DR in Tg2576 mice resulted in a ⬎2-fold elevation in the concentration of neocortical sAPP-␣ and membrane-associated CTF-␣ relative to AL fed control Tg2576 mice. The increase in CTF-␣ was somewhat less, about 1.6-fold, presumably because of further cleavage of CTF-␣ by ␥-secretase. Compared with the CTF-␣ fragment, the abundance of CTF-␤ signal was at the limit of detection in the neocortex of both DR and AL fed Tg2576 mice, preventing reliable quantification [12].

Weight Reduction Dietary Restriction Diet May Influence ␣-Secretase Activity in the Brain in Part by Selectively Promoting the Generation of Mature and Catalytically Active ADAM10 Species

In light of recent evidence indicating that the proteinase ADAM10 (a disintegrin and metalloproteinase) may act as an ␣-secretase [42], we continued to explore the regulation of ADAM10 expression in the brains of Tg2576 mice in response to DR, relative to AL fed controls. Both mature (62-kDa) and proform (90-kDa) ADAM10 species were detected in the neocortex of the AL fed control animals, confirming previous evidence [12]. The 62-kDa mature ADAM10 protein species is known to act as an ␣-secretase in vitro and to cleave A␤-derived peptides at leucine 16 [12, 42]. Excitingly, we found that the DR diet regimen resulted in a 30% elevation of neocortical mature ADAM10 species concentration, coinciding with a commensurate elevation in neocortical ␣-secretase activity, compared to AL fed control mice. As shown in figure 2, this evidence supports the hypothesis that lowcarbohydrate DR may prevent AD-type amyloid neuropathology through mechanisms that influence ␣-secretase activity in the brain, possibly by promoting the generation of mature, catalytically active ADAM10 species. Since ␣-secretase proteolysis of the APP sequence within the A␤ peptide would preclude the generation of amyloidogenic A␤ peptides, our studies suggest that DR may provide an attractive antiamyloidogenic strategy by promoting ␣-secretase activity in the brain. In addition to promoting ␣-secretase activity, we found that DR led to a small, but significant elevation of IDE content in the brain of Tg2576 mice. The

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DR

APP Proform ADAM10

Mature ADAM10

␤-,␥-SeDRetase

␣-SeDRetase

A␤ ?

Degradation

Amyloidosis

Fig. 2. Role of DR in the prevention of AD-type amyloid neuropathology.

role of IDE in A␤ degradation was demonstrated by recent studies showing that mice deficient for IDE exhibit increased cerebral accumulation of endogenous A␤ peptides. Thus, it is possible that the attenuation of A␤ burden in the brain of DR Tg2576 mice might also derive from enhanced IDE-mediated clearance of A␤ peptides in addition to the promotion of the nonamyloidogenic ␣-secretase cleavage of APP [5, 12, 46]. In view of a recent study from Patel et al. [13] reporting the A␤-lowering efficacy of a DR diet in additional mouse models of AD amyloid neuropathology, it is likely that the beneficial effect of a ‘lowcarbohydrate/DR’ diet on A␤ neuropathology and cognitive function in the Tg2576 AD mouse model [12] may reflect the impact of DR, per se. However, it is possible that the low carbohydrate content in the ‘low-carbohydrate/DR’ dietary regimen may promote additional disease-modifying activities. As discussed above, current strategies to treat AD are aimed at preventing formation of amyloidogenic A␤ peptides. Therefore, ␤- and ␥-secretases that generate A␤ peptides by sequential cleavage of the APP or degrade released A␤ peptides are obvious and central targets for the development of therapeutic

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reagents. Our evidence showing that DR may positively influence ␣-secretase, possibly through mechanisms that may involve the generation of mature, catalytically active ADAM10 species in the brain, might prove in the future the basis of potential novel preventative measure aimed at delaying the onset of AD neuropathology. In addition, since ␣-secretase cleavage of APP releases sAPP-␣, which is well known for its neuroprotective properties, our study tentatively suggests that promoting a low-carbohydrate DR dietary regimen may also result in increased brain repair activities as a consequence of sAPP-␣ neurotrophic function. However, we cannot exclude the possibility that DR might also influence other mechanisms, eventually resulting in decreased amyloid deposition in the brain by promoting ␣-site cleavage of APP or degradation of released A␤ by other proteases such as plasmin and neprilysin, respectively. In addition to promoting A␤-lowering activity, DR may also benefit AD through mechanisms not directly related to generation and/or degradation of A␤ of peptides. In particular, DR is known to reduce inflammation [55] and oxidative stress [56], two of the major contributing factors in AD-type neurodegeneration [57, 58]. Therefore, it would not be unexpected that DR may beneficially modulate the onset and/or progression of neuropathology and neurodegeneration in AD through multiple mechanisms. Thus, the relationship between caloric intake and AD could have important implications in the prevention and/or therapy of AD [17]. DR is well known to improve insulin sensitivity responses, especially in insulin resistance conditions such as type 2 diabetes [59, 60]. Based on the observation that diet-induced insulin resistance promotes the generation of A␤ peptides, it would not be unexpected that A␤-lowering activity of DR may be related to promotion of insulin sensitivity responses. However, evidence indicates that insulin resistance and DR may have independent impacts on A␤ generation and that diabetogenic and DR diet appears to activate independent signal transduction pathways ultimately influencing APP processing and generation of A␤ peptides [6, 12]. While a diabetogenic diet induces A␤ generation by promotion of the AKT-GSK pathway [6], ongoing studies showed that DR may reduce A␤ generation by activating ␣-secretase activity (perhaps activation of ADAM10 activity), in part, via promotion of the MAPK-PKC signaling pathway. Further studies in our laboratory are presently aiming to better understand whether DR in obese-diabetic Tg2576 mice may reverse AD-type amyloidogenic activities via modulation of these specific signal transduction pathways.

Conclusion

Study findings support existing epidemiological evidence indicating that caloric intake is positively associated with the increased incidence of AD and

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raises the possibility that changes in dietary regimens may be used in future preventative measures aimed at delaying the onset of AD amyloid neuropathology. Investigations in experimental mouse models of AD neuropathology, such as ours, are of great potential benefit in terms of public health because they provide insights into possible interventions to prevent or ameliorate conditions associated with those over 65 years of age in the USA. This is the age group with the highest incidence of excess weight, obesity and diabetes, and it is the largest group associated with the highest risk to develop AD dementia. We want to point out, however, that decisions on diet recommendations in AD can be a complex endeavor because they should be made on the basis of combined evidence from different sources such as (1) experimental models, (2) prospective epidemiological studies and ultimately (3) controlled clinical studies. While we believe that the ultimate evidence to support such recommendations should come from controlled clinical trial studies, we are also aware of the potential limitation of this approach. For example, we point out that, in view of the chronic nature of AD dementia with a relatively long latency period, it may be difficult to execute appropriate clinical studies for enough time and in large enough samples to draw accurate and repeatable conclusions. However, despite these limitations, we believe the recent prospective studies showing that increased caloric intake is a risk for AD [8] and the recent observation that that DR [12, 13] in AD mouse models may beneficially influence AD neuropathology provide strong impetus to ascertain the validity of a DR diet in AD patients.

Acknowledgments This study is supported by the Dr. Robert C. Atkins Foundation, the Dana Foundation for Brain Research Initiative and the NIH AG14766, AG02219 and NCCAM AT002602 to G.M.P. We thank Ms. Isabela Diaconescu for editorial revision of the manuscript.

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Giulio Maria Pasinetti, MD, PhD Neuroinflammation Research Laboratories, Department of Psychiatry Mount Sinai School of Medicine, 1 Gustave L. Levy Place, Box 1230 New York, NY 10029–6574 (USA) Tel. ⫹1 212 659 8716, Fax ⫹1 212 876 9042, E-Mail [email protected]

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Mobbs CV, Yen K, Hof PR (eds): Mechanisms of Dietary Restriction in Aging and Disease. Interdiscipl Top Gerontol. Basel, Karger, 2007, vol 35, pp 176–192

Can Short-Term Dietary Restriction and Fasting Have a Long-Term Anticarcinogenic Effect? Simon Klebanov Obesity Research Center, St. Luke’s Roosevelt Hospital Center, New York, N.Y., USA

Abstract Long-term dietary restriction (DR) robustly inhibits various types of carcinogenesis in rodents. Because malignancies are a major cause of death in humans, reducing the incidence or, at least, delaying the time of onset of neoplasia may significantly increase longevity of a large proportion of the human population. Long-term DR may not however be practical in humans and, judging from religious practices, several days of fasting to several weeks of DR is what a large segment of the human population can adhere to. In contrast to long-term DR, a single episode of fasting or several fasting-refeeding cycles did not have any long-lasting beneficial and usually had even a deleterious effect on carcinogenesis in rodent models. On the other hand, DR of a relatively short (1–3 months) duration often significantly increased latency and reduced the incidence of cancer over the entire life span. These results suggest that the immediate anticarcinogenic action of DR is to slow down the expansion of initiated clones, but that several months of DR may be sufficient for the elimination of a significant portion of initiated precancerous clones through apoptosis. The development of optimized DR regimens for humans will be contingent on further advances in our understanding of the mechanisms of cancer suppression by DR. Copyright © 2007 S. Karger AG, Basel

Dietary restriction extends life span regardless of feeding pattern, diet composition and the age of onset. However, the level and duration of a dietary restriction regimen determine its effectiveness. Dietary restriction (DR) is a robust antiaging intervention; DR regimens almost uniformly result in the extension of life span. Life extension was achieved regardless of whether food was presented as a single meal a day with a reduced caloric content [1], as 2 [2] or even 6 [1] smaller meals a day, or ad libitum one day followed by a day of fasting [3]. Diet composition also had little

effect on the degree of life extension because DR worked even when diets were enriched with protein [4], or fat and mineral contents were independently manipulated [5]. Finally, DR did not have to be initiated at a very early age to be effective. A preweaning initiation of DR did not incur any additional life extension compared with a postweaning initiation [6], while restricting caloric intake from 6 months [7], 12–14 months [6, 8–10] and even 18 months [10] extended life span. While the antiaging effect can be achieved with a broad variety of DR regimens, two parameters strongly influenced the outcome. First, the degree of DR, in contrast to the pattern of feeding, significantly affected life span [11]. A suppressive effect of DR on induced carcinogenesis was also enhanced at more severe levels of restriction [12, 13]. Second, the duration of DR, rather than the timing of initiation, determined the degree of life extension [6–8]. The requirement of maintaining a rigorous DR regimen for an extended period of time severely limits direct applicability of DR to humans. At the same time, the deficiency in our understanding limits the development of mechanism-based DRmimetic interventions for life extension. Under these circumstances, human life extension still remains the ultimate, but distant goal. Life extension is the most striking, but not the only beneficial health effect of DR. A broad range of age-related diseases is ameliorated by DR [14]. If at least some of these ameliorating effects could be achieved with a milder and/or shorter DR regimen, presumably more easily tolerated by humans, it would be of a great benefit.

Suppression of Carcinogenesis Is Very Prominent and May Contribute Substantially to Life Extension

The ability of DR to inhibit transplanted tumor growth [15] had been shown well before DR was reported to extend longevity [16]. A substantial body of work has been accumulated since then that documents the inhibition of spontaneous carcinogenesis in normal [17, 18] and genetically modified [19, 20] animals, of chemically induced carcinogenesis [21, 22] and of transplanted tumors [23, 24]. Thus, the inhibition of various types of carcinogenesis in numerous organs seems to be as universal and robust a feature of DR regimen as life extension. Although specific cancers have different incidence rates in different rodent laboratory strains, overall, malignancies are a major cause of death in mice and rats (for reviews, see Crispens [25], pp 159–181, and Weindruch and Walford [14], pp 73–101). Inhibition of spontaneous carcinogenesis is believed to be a major contributor to the life-extending effect of DR. For example, two extensive studies on

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the effect of DR in two long-lived mouse populations, a standard C57BL6 mouse strain and a B6C3 F1 hybrid, reported that DR substantially increased life span and that that increase could mostly be accounted for by a 30–40% decrease in the incidence of lethal neoplasia and by a significant delay in the neoplasia onset [18, 26]. An extensive review of the effect of DR on spontaneous malignancies in rodents has been provided by Weindruch and Walford [14], pp 73–101. Malignancies are a major cause of death in humans as well. According to the National Center for Health Statistics, cancer mortality accounts for more than 20% of all deaths in the USA (http://www.cdc.gov/nchs/fastats/deaths. htm). Thus, reducing the incidence or, at least, delaying the time of onset of neoplasia will have a significant effect on longevity of a large proportion of the human population. While the mechanisms of DR anticarcinogenesis are not fully understood, this question is probably more tractable than the DR overall antiaging effect.

Carcinogenesis: Current Understanding. Promotion as the Most Promising Stage for Anticarcinogenic Interventions in Humans

The development of neoplasia in mammals involves at least three definable stages: initiation, promotion and progression [27, 28]. Tumor initiation involves DNA damage induced by endogenous or exogenous carcinogens. Such mutations may alter the behavior of initiated cells, providing them with growth advantage. While DR may inhibit certain aspects of initiation in models of chemical carcinogenesis [29], this is likely to have little direct applicability to humans. Spontaneous initiation events are ubiquitous and even if a DR regimen were effective in reducing initiation levels, only potential initiation events that would have occurred during the period of DR would be prevented, while the initiation events that had occurred before DR would not. Thus, to have a significant anti-initiation effect in humans, a DR regimen will have to be applied for a period of time comparable with human life span. The promotion stage of carcinogenesis is characterized by clonal expansion of initiated cells. Unlike initiation and progression, this stage does not involve alterations in the structure of the genome, but rather in its expression [27]. A very important and clinically promising feature of this stage is its reversibility, i.e. the loss or regression of preneoplastic lesions under certain circumstances. For example, a withdrawal of a promoting agent [30] or an application of DR [31] significantly suppresses proliferation, increases apoptosis and leads to a selective reduction in the volume and the number of preneoplastic lesions in the liver. Another clinically important consideration is that all cells that have been initiated and moved to the stage of promotion by the time that a

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DR regimen is applied, will be affected by DR. If promotion can indeed be reversed by DR, even relatively short-term DR interventions applied regularly may prove to be an effective anticancer intervention in humans. The progression stage involves additional clonal expansion and progressive genetic damage due to genomic instability [27]. During this stage, preneoplastic lesions develop into invasive tumors. While in some respect the effect of DR on the progression stage may be similar to that on the promotion stage, there is also a significant difference. Even if DR does not eliminate all, but only a significant portion of promoted cells, it can still significantly retard carcinogenesis. On the other hand, some cells in the stage of progression are already neoplastic and even a small portion of cells surviving DR will be able to develop into a tumor relatively quickly. Thus, a short-term DR will unlikely be able to postpone carcinogenesis significantly at this stage, necessitating longterm DR hardly sustainable in humans. In light of the arguments presented above, most attention in the further discussion will be given to the effect of DR on the promotion stage in several models of induced carcinogenesis.

Effect of Dietary Restriction on Cellular Proliferation and Apoptosis

An initiated cell, while not a tumor by itself, may give rise to a tumor. A selective growth advantage over normal tissue is one of the crucial properties of malignancy [30]. The rate of cellular proliferation is an important determinant of the growth of preneoplastic and neoplastic lesions during the promotion and progression stages of carcinogenesis. Fasting and various short- and long-term DR protocols almost uniformly result in reduced rates of cellular proliferation in the majority of tissues and organs, including the liver [31–35], bladder [36, 37], skin [32, 36], kidney, heart [32], mammary gland, esophagus, jejunum [36] and colorectum [36, 38] (table 1). This reduction in proliferation may in part explain the anticarcinogenic effect of DR. Proliferation, however, is just one determinant of the overall growth rate. The growth rate of preneoplastic and neoplastic lesions is determined by the difference between the proliferation rate and the rate of cell death [30]. Most studies focused on the effect of fasting and DR on the rate of an active cell death, apoptosis, in the liver. Apoptosis seems to be enhanced by DR or fasting under most circumstances [31, 33, 35, 39, 40], although a mild DR regimen applied to young, rapidly growing animals may not be sufficient to induce apoptosis [34] (table 1). Induction of apoptosis by DR has also been reported for the bladder [37], colon [38] and mammary gland [41] (table 1). Thus, in addition to reducing cellular proliferation, DR may

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Table 1. Effects of DR on cellular proliferation and apoptosis Tissue

Species

Dietary regimen

Effect

References

rat rat

reduced mostly early reduced

[32] [33]

rat mouse rat

50% of ad libitum, lifelong fasting, 5 days; refeeding, 2 days; fasting, 5 days 70% of ad libitum, 7 weeks 60% of ad libitum, 9 months 40% of ad libitum, 4 days

reduced reduced reduced

[34] [35] [31]

Bladder

mouse mouse

75% of ad libitum, 1 month 80% of ad libitum, 5 weeks

reduced reduced

[36] [37]

Skin

rat mouse

50% of ad libitum, lifelong 75% of ad libitum, 1 month

reduced mostly early reduced

[32] [36]

Kidney

rat

50% of ad libitum, lifelong

reduced mostly early

[32]

Heart

rat

50% of ad libitum, lifelong

reduced lifelong

[32]

Cellular proliferation Liver

Mammary

mouse

75% of ad libitum, 1 month

reduced

[36]

Esophagus

mouse

75% of ad libitum, 1 month

reduced

[36]

Jejunum

rat mouse

50% of ad libitum, lifelong 75% of ad libitum, 1 month

no effect reduced

[32] [36]

Colorectum

mouse rat

75% of ad libitum, 1 month fasting, 4 days

reduced reduced

[36] [38]

rat

increased

[33]

rat rat mouse rat rat

fasting, 5 days; refeeding, 2 days; fasting, 5 days fasting, 3 days 70% of ad libitum, 7 weeks 60% of ad libitum, 9 months 40% of ad libitum, 4 days 60% of ad libitum, 2 months

increased no effect increased increased increased

[39] [34] [35] [31] [40]

Bladder

mouse

80% of ad libitum, 5 weeks

increased

[37]

Mammary

rat

60% of ad libitum, 6 weeks

increased

[41]

Colon

rat

fasting, 4 days

increased

[38]

Apoptosis Liver

reduce initiated cell growth rate and inhibit carcinogenesis by increasing the rate of cell death. It is likely that DR effects on cell proliferation and apoptosis will vary through the course of DR treatment. In mature animals, during the initial stage of DR, body and most organ weights will rapidly decline, suggesting the prevalence of cell death over cell replication. At this stage, there is net cell loss. Eventually,

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a new steady state will be achieved. At this stage, there is no additional decrease in organ sizes, and cell proliferation and cell death ought to balance each other out. This seems in apparent conflict with the reports that long-term DR simultaneously decreases cell proliferation [31, 32, 35] and enhances apoptosis [35, 40]. However, apoptosis is only one kind of cell death, and it has been proposed that some necrotic cell death, characteristic of ad libitum fed animals, is completely replaced by apoptotic cell death in DR animals [35]. This transition from the necrotic to the apoptotic type of cell death, if confirmed, was proposed to be important for the anticarcinogenic effect of DR as apoptosis might be more selective than necrosis in eliminating preneoplastic cells. The importance of apoptosis in carcinogenesis is not, however, limited to its quantitative negative drag on the overall growth rate. Mathematical modeling predicts that, with a finite rate of cell death, there is a probability of extinction of an initiated clone. This probability depends on the initial clone size, observation (treatment) period and the ratio of cell death to cell proliferation [42, 43]. This notion is very important as it predicts that because DR increases the ratio of cell death to cell proliferation it may, if applied long enough and early enough (i.e. while initiated clones are small), completely eliminate some initiated clones.

Reversibility of Preneoplastic Lesions: Evidence for the Selective Elimination of Initiated Cells by Dietary Restriction

Initiation involves irreversible alterations in the cellular genome [27]. During the promotion stage, such initiated cells clonally expand. It has often been reported that the persistence of preneoplastic lesions is dependent on a continuous administration of a promoting agent. Upon promoter withdrawal, a majority of preneoplastic and even neoplastic lesions may spontaneously disappear [30, 44–46]. This behavior of preneoplastic lesions has led to the understanding that the promotion stage is reversible [27]. The eventual fate of the preneoplastic cells that disappeared has not been unequivocally determined but may include death through apoptosis [30, 46] and redifferentiation to morphologically normal cells [47, 48]. DR has been reported to impose a strong inhibitory effect on the promotion and progression stages of carcinogenesis in the liver [31], bladder [37], skin [49] and mammary gland [13]. When preneoplastic lesions can be traced, like in the liver, their growth is not only retarded, but a majority of such lesions disappear or significantly decrease in size, and the percentage of the liver volume occupied by such lesions is significantly reduced [31]. A selective increase in the apoptosis rates in the preneoplastic lesions, rather than in the surrounding tissue,

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may be one of the mechanisms for selective elimination of preneoplastic lesions in response to DR [31]. Thus, it is possible that DR may lead to the actual elimination of preneoplastic cells. It remains, however, to be determined whether apoptosis is responsible for a significant cell elimination or whether a substantial portion of such cells simply loses some of the characteristic markers, i.e. undergoes redifferentiation to the apparently normal cellular phenotype [47, 48].

Effectiveness of Short-Term Dietary Restriction Regimens in Inhibiting Carcinogenesis: Long-Term Outcomes of Short-Term Dietary Restriction Interventions

Religious practices may provide guidance for dietary interventions that can be adhered to by a large segment of the human population. They include complete fasting for 1 (Jewish Yom Kippur) to several days and milder DR regimens for up to several weeks (Christian Lent and Islamic Ramadan). A significant number of studies explored the effect of comparable dietary manipulation in diverse rodent models of induced carcinogenesis. Fasting and DR may quickly reduce the number and the size of preexisting preneoplastic lesions [31, 33, 50], however, the longerterm outcomes of fasting or short-term DR regimens may not be so positive. Fasting A number of studies explored the effect of fasting-refeeding during the promotion stage of carcinogenesis (table 2). Almost uniformly, a single episode of fasting or several fasting-refeeding cycles did not have any long-lasting beneficial effect and usually had even a negative effect. In the liver of rats subjected to partial hepatectomy and initiated with diethylnitrosamine intraperitoneally, 2 periods of 5 days of fasting separated by 2 days of ad libitum feeding led to an immediate reduction of the number and the volume of preneoplastic lesions by several fold [33]. However, just 2 weeks after refeeding, lesion number and volume were restored and 3 months later, the fasted-refed group had even a larger number of preneoplastic lesions than the group fed ad libitum throughout the entire study. In another study, rats were also initiated with diethylnitrosamine intraperitoneally and, a week later, subjected either to 1 or 3 periods of 3 days of fasting followed by 11 days of refeeding [39, 51]. Seven weeks after diethylnitrosamine, rats were given 2-acetylaminofluorene intragastrically, to block the proliferation of the majority of normal hepatocytes, followed by a necrogenic dose of CCl4, to stimulate surviving hepatocyte proliferation. Under these circumstances, only resistant, i.e. initiated hepatocytes would proliferate, giving rise to altered hepatocyte foci. When sacrificed 4 weeks later, fasted-refed rats had significantly larger preneoplastic lesions in the liver, and the

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Table 2. Effects of fasting-refeeding and short-term DR on carcinogenesis Tissue

Species Dietary regimen

Fasting-refeeding Liver rat

rat

rat

Mammary

rat

rat rat

fasting, 5 days; refeeding, 2 days; fasting, 5 days 1 or 3 cycles of fasting (3 days) and refeeding (11 days) 3 cycles of fasting (3 days) and refeeding (11 days) 3 cycles of fasting (3 days) and refeeding (10 days) fasting, 3 days 17 cycles of fasting (2 days) and refeeding (2 days)

Carcinogen

Effect

References

diethylnitrosamine

initially reduced; later (3 months) increased

[33]

diethylnitrosamine

increased; more by 3 cycles than by 1

[51]

diethylnitrosamine

increased 1 year later

[39]

methylnitrosourea dimethylbenz[a] anthracene

increased multiplicity of tumors (42 weeks later) increased later no effect

[53]

dimethylbenz[a] anthracene

[54] [55]

Colon

rat

5 cycles of fasting (4 days) and refeeding (7–10 days)

azoxymethane

no effect immediately; increased 4 weeks later

[52]

Short-term DR Liver

rat

60% of ad libitum, 3 months

spontaneous tumors promoted by nafenopin

reduced initially; still reduced 17 months later

[31]

Mammary

rat

50% of ad libitum, 5 weeks 60% of ad libitum, 5 weeks

dimethylbenz[a] anthracene 1-methyl-1nitrosourea

reduced 22 weeks later initially reduced; no effect 3 weeks later

[56]

variably restricted, for up to 10 weeks

spontaneous lymphomas in p53-null mice

reduced in the long term

Hematopoietic

mouse

[57] Klebanov, unpublished

negative effect of 3 cycles of fasting-refeeding was more pronounced than that of 1 cycle [51]. When rats were sacrificed 1 year after these dietary manipulations, the incidence of hepatocellular carcinoma was doubled, and tumor size and histological grade were increased in the fasted-refed group [39]. In the colon of rats initiated with azoxymethane subcutaneously, 5 periods of 4 days of fasting interspersed with periods of 7–10 days of refeeding did not

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increase the number of aberrant crypt foci, but increased crypt multiplicity, a good predictor of colon cancer outcome [52]. This effect was not present right after the last fasting period, but was detectable a week after that and was even more pronounced 4 weeks later. Mammary carcinogenesis was also usually enhanced or at least not inhibited by short-term fasting-refeeding protocols. In female rats, 3 cycles of 3 days of fasting and 10 days of ad libitum feeding, started a week after initiation with methylnitrosourea intraperitoneally, increased multiplicity of mammary tumors [53]. Even a single 3-day period of fasting, imposed a week after initiation with dimethylbenz[a]anthracene, enhanced mammary tumor growth and reduced tumor latency [54]. However, a more restrictive 10-week-long regimen of 2 days of fasting followed by 2 days of refeeding had no effect on mammary tumor incidence in rats initiated with a gavage of dimethylbenz[a]anthracene [55]. The failure of fasting to inhibit carcinogenesis probably indicates that several days are not sufficient to eliminate any significant number of initiated cells and that a longer period of a continuous negative energy balance is necessary to eliminate clones of initiated cells [42, 43]. An additional enhancement of carcinogenesis in fasted-refed animals may in part be due to a compensatory increase in proliferation and a decrease in apoptosis in response to refeeding [33, 39], which may even be more pronounced in preneoplastic lesions [52]. Suppression of the refeeding response is an obvious target in the search for an efficient anticarcinogenic intervention [58]. Short-Term Dietary Restriction Only few studies explored the effect of short-term DR on the long-term carcinogenesis outcomes (table 2). The results are not as uniform as with fasting, and additional studies will be required for optimizing DR anticancer interventions. One study explored the effect of 3 months of DR, at 60% of ad libitum intake, on the amount of liver tumors after additional 17 months of ad libitum feeding [31]. The tumor promoter nafenopin was fed for the entire period after DR to expose all preneoplastic lesions not eliminated by DR. Three months of DR reduced the number and the volume of spontaneous putative preneoplastic liver foci to just 15% of control values. Only 1 week of refeeding was sufficient to increase this number back to 65% of control values. However, the 35% difference persisted then for the remaining 17 months. The total tumor yield, including hepatocellular adenoma and carcinoma, was reduced by approximately 50% by DR administered for a period of 3 months, 17 months prior to sacrifice. When DR was administered 1 week prior to the initiation with dimethylbenz[a]anthracene intravenously, and during the first 4 weeks of promotion,

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mammary tumor size and number were significantly reduced 26 weeks later [56]. In another study, mammary carcinogenesis was induced by 1-methyl-1nitrosourea intraperitoneally [57]. DR, started 1 week after initiation, significantly suppressed tumor development over the 5-week period. However, just 3 weeks of ad libitum feeding almost completely abolished the effect of prior DR on tumor incidence and multiplicity. Thus, short-term DR failed to provide any long-term protection from carcinogenesis. One clear difference between the studies that can explain the difference in outcomes is the administration of DR during the initiation stage in the first study. However, the difference in the initiation agent and, in general, a much more aggressive carcinogenesis protocol used in the second study might have contributed to the difference in the outcome. Our own experience suggests that a relatively short-term DR can significantly suppress carcinogenesis and extend longevity of p53 knock out mice. During a routine screening at 5 weeks of age, all p53 knock out mice had normal body weight, but at 15 weeks, we found out that 3 out of 47 mice were underweight. Upon further examination, the mice were diagnosed as having malocclusion that led to self-restriction of their food intake. Judging from their body weight, mice were DR by approximately 50%. After mice had had this diagnosis of malocclusion, their teeth were regularly clipped and their body weight stayed in the normal range. The life span of self-restricted mice was extended by 14 weeks, from 26 to 40 weeks (p ⬍ 0.001). Our observation is noteworthy for two reasons. First, no more than 10 (and probably fewer) weeks of DR extended life span by 14 weeks! Second, DR administered over the entire duration of life has been reported to extend the life span of p53 knock out mice by only 9 weeks [20]. Thus, it seems that a relatively short-term DR may yield the same anticarcinogenic benefits as life-long DR.

The Endocrine System as a Mediator of the Anticarcinogenic Effect of Dietary Restriction and as a Convenient Target for Anticarcinogenic Pharmacological Interventions

While some optimism is warranted because of the effectiveness of shortterm DR against some forms of cancer, further progress in devising DR regimens that will be both effective in inducing the ‘therapeutic’ response and tolerable for a majority of humans will depend on improving the understanding of molecular and cellular mechanisms of cancer suppression by DR. Several endocrine systems affected by DR seem to be both likely mediators of the DRinduced cancer suppression and convenient targets for future pharmacological interventions.

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The somatotropic axis is involved in the regulation of life span (for reviews, see Barbieri et al. [59] and Bartke [60]), and insulin-like growth factor-1 (IGF-1) reduction by DR was proposed to be an important factor in ameliorating many age-related diseases, including various malignancies (for reviews, see Kari et al. [61] and Sell [62]). The link between IGF-1 levels and various aspects of carcinogenesis has been well established (for reviews, see Yu and Rohan [63], Ibrahim and Yee [64] and Pollak et al. [65]). IGF-1 is known to stimulate cellular proliferation and inhibit apoptosis in a wide array of tissues (for reviews, see Jones and Clemmons [66], Butt et al. [67] and Gallaher et al. [68]). In humans, higher levels of IGF-1 have been linked to an increased incidence of several kinds of cancer, including that of the breast and prostate [69–72], and, in animals, carcinogenesis is suppressed at very low and is enhanced at high IGF-1 levels [73–80] (for a review, see Yakar et al. [81]). While all these lines of evidence support the role of IGF-1 in modulating carcinogenesis, replenishing IGF-1 in DR animals abrogates the protective effect of DR only in some [37], but not in other [82] models. Thus, it still remains to be determined whether a modest reduction in IGF-1 levels observed in DR animals is indeed a significant factor in the anticarcinogenic effect of DR. Leptin is another hormone whose suppression by DR [83–85] has been hypothesized to contribute to life extension [86] and which has a procarcinogenic effect (for reviews, see Garofalo and Surmacz [87] and Somasundar et al. [88]). In vitro, leptin enhances cellular proliferation in a number of tissues and cell lines [89–96]. In humans, several types of cancer appear to be linked to elevated leptin levels (for a review, see Garofalo and Surmacz [87]), while spontaneous and induced carcinogenesis is suppressed in many tissues of leptin-null ob/ob mice [97–100]. All this evidence is compatible with a role of leptin reduction in the suppression of carcinogenesis by DR. Glucocorticoids are yet another class of hormones that may contribute to the anticarcinogenic action of DR [101, 102]. Total and/or free glucocorticoid levels are increased by DR [103–105]. Glucocorticoids suppress cellular proliferation and enhance apoptosis in a number of cell types, including osteoblasts, lymphocytes and keratinocytes (for reviews, see Weinstein [106], Herold et al. [107] and Budunova et al. [108]). In humans, glucocorticoids are effectively used for treating lymphoid neoplasms [109]. Importantly, adrenalectomy abolishes the protective effect of DR on skin and pulmonary carcinogenesis, while glucocorticoid replacement restores this protection [110–112]. As with IGF-1, the DR-induced shifts in both glucocorticoids and leptin are compatible with their role in the DR-induced suppression of carcinogenesis. However, similarly to IGF-1, it still remains to be determined whether, quantitatively, the changes in glucocorticoid and leptin levels are significant contributors to the anticarcinogenic action of DR.

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Conclusion and Future Directions

Spontaneous and induced carcinogenesis is significantly postponed by long-term DR interventions. Long-term DR may not, however, be well tolerated by humans. Therefore, the major question addressed in this chapter was whether relatively short-term DR might have a significant long-lasting anticarcinogenic effect. The majority of the studies suggests that fasting does not have a longlasting anticarcinogenic effect and that the subsequent refeeding can even promote carcinogenesis. On the other hand, DR of a relatively short (1–3 months) duration may significantly affect cancer incidence and latency over the entire life span. These results suggest that the immediate anticarcinogenic action of DR is to slow down the expansion of initiated clones through shifting the balance from proliferation to apoptosis. The long-lasting effects of DR of 1–3 months duration indicate, however, that several months of DR may be sufficient for elimination of a significant portion of initiated precancerous clones through apoptosis. The anticarcinogenic mechanisms of DR, discussed in the current paper, may be relevant only to the dividing cell populations and may not therefore explain the entire spectrum of protective effects afforded by DR. While understanding the mechanisms of DR for human life extension remains the ultimate goal, garnering the anticarcinogenic effect of DR may be in and of itself of a significant value. This task is also likely to be more tractable than finding universal life-extending mechanisms and may be an important practical step towards extending human life span. The heterogeneity of humans in their susceptibility to cancer, their body composition and their endocrine response to DR may preclude the development of a single DR regimen that fits all. The development of optimized DR regimens will be contingent on further advances in our understanding of the mechanisms of cancer suppression by DR. References 1 2

3 4

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5

6

7 8 9 10

11

12 13

14 15 16 17 18 19

20 21 22

23 24

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28 29

30

31

32 33

34

35

36 37

38

39 40

41 42 43 44

45 46 47 48 49

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57 58 59

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73 74 75

76 77

78

79

80

81

82

83 84

85

86 87 88 89 90 91 92 93

94

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Simon Klebanov, PhD Research Associate Obesity Research Center St. Luke’s Roosevelt Hospital Center 1111 Amsterdam Avenue, WH1020 New York, NY 10025 (USA) Tel. ⫹1 212 5231632, Fax ⫹1 212 5231632, E-Mail [email protected]

Klebanov

192

Author Index

Al-Regaiey, K.A. 69 Anderson, R.M. 18 Bartke, A. 69 Bonkowski, M.S. 69 Chen, L. 159 Cheng, H. 39 Finch, C.E. 83 Gems, D. 98 Ho, L. 159 Hof, P.R. VII Houthoofd, K. 98 Humala, N. 159

Johnson, T.E. 98 Klebanov, S. 176 Ksiezak-Reding, H. 159 Lane, M.A. 137 Liu, X. 159

Ressmann, W. 159 Romero, C. 159 Roth, G.S. 137 Shrishailam, Y. 159 Stetka, B. 159 Tatar, M. 115

MacGrogan, D. 159 Masoro, E.J. 1 Mastaitis, J.W. 39 Masternak, M.M. 69 Mattison, J.A. 137 Mobbs, C.V. VII, 39 Morgan, T.E. 83

Vanfleteren, J.R. 98 Wang, J. 159 Weindruch, R. 18 Wong, A.M. 83 Yen, K. VII, 39

Pasinetti, G.M. 159 Ingram, D.K. 137 Isoda, F. 39

Qin, W. 159

Zhang, M. 39 Zhao, Z. 159

193

Subject Index

ADAM10, dietary restriction effects in Alzheimer’s disease model 169–171 Adipose tissue, dietary restriction and aging retardation 27–29 Advanced glycation end products (AGEs), dietary restriction reduction and anti-inflammatory effects 90, 91 Alcohol, Alzheimer’s disease risks 163 Alzheimer’s disease (AD) dietary restriction studies in transgenic mouse model amyloid precursor protein processing effects 168–171 clinical implications 171, 172 experimental design 167, 168 rationale 166, 167 economic impact 160 insulin metabolism defects 165, 166 nutritional factors 160, 162–164 obesity as risk factor 161, 162 oxidative stress in pathology 59, 60, 162 pathology 164 therapeutic targets 164, 165 Ames dwarf mice dietary restriction effects on insulin signaling genes IGF-1 findings 76–79 IRS-1 findings 75 IRS-2 findings 76 longevity response 73, 74 PPAR-␥ findings 75, 76 overview of model 70–73 Apoptosis, dietary restriction effects 179–181

Astrocyte, age-related activation and glial fibrillary acidic protein role 86, 87 Behavior, dietary restriction effects in rhesus monkey 150–152 Body composition, dietary restriction effects in rhesus monkey 138–140 Caenorhabditis elegans clk-1 mutant studies 43, 44, 103 dietary restriction study limitations 100, 101 DNA silencing alterations in dietary restriction 108 growth 100–102 insulin signaling in dietary restriction 104, 105, 107, 108 metabolic rate in dietary restriction 103, 104 overview of model 99, 100 RNA interference and longevity gene studies 22, 23, 102 stress resistance in dietary restriction 104 TOR signaling in dietary restriction 108–110 Cancer, dietary restriction effects cell proliferation and apoptosis 179–181 endocrine system mediation 185, 186 preneoplastic lesion reversal 181, 182 promotion stage interventions 178, 179 prospects for study 187 short-term intervention effects fasting 182–184

194

mouse studies of short-term dietary restriction effects 184, 185 suppression of carcinogenesis 177, 178 tumor burden 56, 57 chico1, dietary restriction effects 127–132 clk-1, longevity studies 43, 44, 103 Cold stress, dietary restriction effects 9 Corticosterone, dietary restriction effects in rat 3, 4, 8 Cortisol, dietary restriction effects in rhesus monkey 144, 145 C-reactive protein (CRP), dietary restriction effects 89 Cytokines, dietary restriction effects in rhesus monkey 148, 149 DAF, dietary restriction effects 22, 23, 28, 30, 105, 107, 108 Dehydroepiandrosterone (DHEA), dietary restriction effects in rhesus monkey 142–144 Diabetes Alzheimer’s disease risks 165, 166 hyperglycemia in aging 46 mitochondrial dysfunction 25 oxidative stress in pathology 60 Drosophila melanogaster dietary restriction experimental diets 117–123 longevity effects 115, 116 mechanisms chico1 mutant studies 127–132 gene interactions 127–132 prospects for study 133 resource allocation 123–126 mortality analysis 116, 117 longevity-regulating genes 23, 24, 126 Electron transfer chain complexes, longevity regulation clk-1 mutant studies in Caenorhabditis 43, 44, 103 complex I activity reduction and increased life span 43 complex II and reactive oxygen species production 42, 43 metabolic shifts 46, 47 overview 42

Subject Index

Electron transport system, dietary restriction effects on gene expression 20, 21 FADH2, glucose switch profile 51 Fatty acid oxidation, dietary restriction effects 20 FOXO dietary restriction effects 26 longevity regulation in Drosophila 23, 24 Ganciclovir, response in dietary-restricted mice 4 Glial fibrillary acidic protein (GFAP) aging and neuroinflammation 86, 87 dietary restriction effects 87 Glucocorticoids anti-inflammatory effects in dietary restriction 90 dietary restriction and carcinogenesis suppression 186 hormesis and dietary restriction 3–12 levels and longevity 10 Glucose dietary restriction effects on levels in rhesus monkey 140, 141 hyperglycemia in aging 46 hypothalamic neuron toxicity and metabolism regulation 47, 48 hysteresis hypothesis 54–59 induced genes and longevity effects 53, 54 metabolic fate regulation and glucose switch gene profile 49–53 Glucose-6-phosphate dehydrogenase, polymorphisms and longevity 50, 51 GLUT-1, hypoglycemia induction 49, 50 Glycolysis, dietary restriction effects 44, 45 Gompertz mortality rate, dietary restriction analysis 83 Growth hormone (GH), receptor/binding protein knockout mice and dietary restriction effects on insulin signaling genes IGF-1 findings 76–79 IRS-1 findings 75

195

Growth hormone (continued) IRS-2 findings 76 longevity response 73, 74 overview of model 71–73 PPAR-␥ findings 75, 76 GSK3␤, dietary restriction effects 26, 27 Heat stress, life span extension in lower organisms 5, 10, 11 HIF-1␣, longevity effects 54 Hormesis dietary restriction mechanisms 3–12 overview 2, 3 HSF-1, stress response in C. elegans 7 Huntington’s disease, oxidative stress in pathology 60 Hypoglycemia antioxidant enzyme induction 51, 52 gene induction 49, 50 senescence reversal 60, 61 Hypothalamic neuron, glucose toxicity and metabolism regulation 47, 48 Hysteresis, metabolic regulation of gene expression 41 IGF-1, dietary restriction effects carcinogenesis suppression 186 expression in long-lived mutant mice 76–79 Immune function, dietary restriction effects 9, 10, 148, 149 Inflammatory response, dietary restriction effects advanced glycation end product reduction 90, 91 C-reactive protein response 89 DNA microarray studies of antiinflammatory effects 88–90 glucocorticoid role 90 neuroinflammation attenuation in aging animal models 87, 88 astrocytic activation and glial fibrillary acidic protein role 86, 87 microglial activation and white matter degeneration 85, 86 overview 84, 85 PPAR-␥ response 90, 91

Subject Index

Insulin Alzheimer’s disease and metabolism defects 165, 166, 171 Caenorhabditis signaling in dietary restriction 104, 105, 107, 108 dietary restriction effects secretion 45 sensitivity in rhesus monkey 141 signaling genes in mutant mice Ames dwarf mice 70–74 growth hormone receptor/binding protein knockout mice 71–74 IGF-1 findings 76–79 IRS-1 findings 75 IRS-2 findings 76 PPAR-␥ findings 75, 76 resistance, see Diabetes IRS-1, dietary restriction effects on expression in long-lived mutant mice 75 IRS-2, dietary restriction effects on expression in long-lived mutant mice 76 JNK, dietary restriction effects 26, 27 lac operon, metabolic regulation glucose switch hypothesis 52, 53 hysteresis 41 overview 40 Leptin, dietary restriction effects carcinogenesis suppression 186 expression 27, 28 Lipid oxidation, dietary restriction effects 44, 45 Meal number, dietary restriction response of longevity 176, 177 Melatonin, dietary restriction effects in rhesus monkey 142 Metabolic rate, dietary restriction effects in rhesus monkey 141 Metabolic reprogramming, dietary restriction 18–31 Methionine, dietary restriction effects on longevity 55 Microglia, age-related activation and white matter degeneration 85, 86

196

Mitochondria complexes, see Electron transfer chain complexes diabetic dysfunction 25 dietary restriction effects on function 24, 25 oxidative stress and longevity 25, 26 Monkey, see Rhesus monkey NADH, glucose sensing 48, 49 NADPH, glucose switch profile 50 Nonhuman primates, see Rhesus monkey Obesity age-related dementia association 161, 162 epidemiology 160 Oxidative stress aging pathology 59, 60 dietary restriction effects markers in rhesus monkey 14 reactive oxygen species on production 11 electron transfer chain complexes clk-1 mutant studies in Caenorhabditis 43, 44, 103 complex I activity reduction and increased life span 43 complex II and reactive oxygen species production 42, 43 metabolic shifts 46, 47 p38 mitogen-activated protein kinase, dietary restriction effects 27, 78 Parkinson’s disease dietary restriction effects in rhesus monkey model 152 nutritional factors 160 PGC-1␣ dietary restriction effects on adipose tissue expression 28, 29 functions 29, 30 knockout mouse 30 PNC1, dietary restriction response role in yeast 6 PPAR-␥, dietary restriction effects on expression

Subject Index

anti-inflammatory effects 90, 91 long-lived mutant mice 75, 76 overview 20 skeletal muscle expression 20 Pyruvate dehydrogenase, longevity effects 54 Reactive oxygen species, see Oxidative stress Reproductive function, dietary restriction effects in rhesus monkey 145–147 Respiratory quotient (RQ) aging effects 54 dietary restriction effects 54, 55 Rhesus monkey dietary restriction response behavior 150–152 body composition 138–140 cortisol 144, 145 dehydroepiandrosterone 142–144 DNA microarray studies 149 glucose levels 140, 141 immune function 148, 149 insulin sensitivity 141 melatonin 142 metabolic rate 141 oxidative stress markers 149 reproductive function 145–147 sensory function 152, 153 thyroid hormone 145 history of dietary restriction studies 137, 138 life span and limitations of model 138 RNA interference, longevity gene studies in Caenorhabditis 22, 23, 102 Rpd3, longevity regulation in Drosophila 23, 132 Senescence, hypoglycemia and reversal 60, 61 Sensory function, dietary restriction effects in rhesus monkey 152, 153 Sin3, longevity regulation in Drosophila 23 Sir2, dietary restriction response role Drosophila 132 yeast 6, 7

197

SIRT1, dietary restriction effects 26, 28 Sirtuin, dietary restriction response role 6, 7, 53, 108 SOD, hypoglycemia induction 51, 52 Stearoyl coenzyme A desaturase 1, longevity effects 53, 54

TOR signaling, dietary restriction effects 22, 108–110 Tumor burden, dietary restriction effects 56, 57

Thyroid hormone, dietary restriction effects in rhesus monkey 145

Wound healing, dietary restriction effects 9

Subject Index

UCP3, dietary restriction effects 24, 25

198