Hunger Does Not Diminish Over Time in Mice Under Protracted ...

REJUVENATION RESEARCH Volume 10, Number 4, 2007 © Mary Ann Liebert, Inc. DOI: 10.1089/rej.2007.0555

Hunger Does Not Diminish Over Time in Mice Under Protracted Caloric Restriction Catherine Hambly,1 Julian G. Mercer,1 and John R. Speakman1,2

ABSTRACT Caloric restriction (CR) is the only nongenetic manipulation known to reliably prolong lifespan. Modeling suggests that humans would need to restrict their intake for many years to reap any lifespan benefits. The feasibility of such prolonged restriction may hinge on whether hunger diminishes with the time period spent restricted. We used the magnitude of hyperphagia on release from restriction as a bioassay of hunger in restricted mice. During restriction, mice develop a characteristic pattern of neuropeptide signals in the arcuate nucleus that reflect their hunger. This pattern is normalized after the postrestriction hyperphagia, validating hyperphagia as an indicator of the hunger during restriction. Mice were food restricted (80% of ad lib.) for 50 days. They initially lost weight, but then became weight stable and were maintained in CR at this lower level of energy balance for between 0 and 50 days and were then fed ad lib. for 50 days. When released onto ad lib. food, the magnitude of the hyperphagic response was independent of the prior length of CR. Hyperphagia ended when body mass was normalized. Hunger therefore did not diminish even when they were restricted for 100 days, equivalent to about 11 years in humans. The pattern of hyperphagic response suggested that signals coding body mass drive hunger during restriction, and because body mass under restriction remains depressed, this suggests that hunger would never disappear, making restriction to prolong lifespan in humans difficult to accomplish. INTRODUCTION

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ALORIC RESTRICTION (CR) is at present the only experimental nongenetic paradigm known to increase both mean and maximum lifespan. It was discovered more than 70 years ago in rats,1 and since that time its effects have been shown to extend lifespan in a wide variety of species including rats, mice, yeast, Drosophila melanogaster, rotifers, bowl and doily spiders (Frontinella pyramitela), and nematodes (Caenorhabdites elegans). However, the effects of CR are

not universal, because it does not appear to be effective in houseflies (Musca domestica2) and it is also ineffective in some strains of mice.3 In some studies, the impact on lifespan has been dramatic, with increases in mean and maximum longevity of 50%.4 The impact of chronically reduced food intake has been shown to depend solely on the reduction of caloric intake, rather than intake of specific dietary nutrients.5–7, but see 8 Several studies have indicated that the impact on lifespan occurs because CR attenuates the onset of many age-related dis-

1Aberdeen Centre for Energy Regulation and Obesity (ACERO), Rowett Research Institute, Bucksburn, Aberdeen, Scotland, United Kingdom. 2ACERO, School of Biological Sciences, University of Aberdeen, Aberdeen, Scotland, United Kingdom.

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eases, particularly cancer,9–16 but see 17 and generally reduces the expression of markers of agerelated decline in function. Hence, the effect on lifespan is a consequence of a true reduction in the rate of aging. At present, CR is the only experimental manipulation of the environmental circumstances known that extends maximum lifespan through a reduction in the rate of aging. The prospects of extending human lifespan by adopting a similar paradigm have recently generated considerable interest. This has been stimulated by the fact that studies of CR in nonhuman primates18–20 have also reported an attenuation of age-related disease and markers of age-related decline.20–25 Unsurprisingly in the light of this evidence, and doubtless encouraged by a series of popular books that have strongly advocated the CR paradigm as a route to enhanced longevity (e.g.,26–28), groups of individuals have already started to voluntarily restrict themselves in the hope that this will slow their aging rate and extend their lives (see http://www.cron-web.org).29 Moreover, appropriately randomized controlled trials of the impacts of CR have also started (e.g., the CALERIE trial in the United States; see http:// calerie.dcri.duke.edu/). We have recently used the data available across several studies of small mammals to model the likely impact of commencing on a program of CR at different times of life,30 assuming that the magnitude of the response in humans will mirror that observed in the rodents (which might be an optimistic assumption31–35). This modeling has revealed that to reap a significant longevity reward (exceeding 5 years), it would be necessary to engage in many decades of CR. The possibility that human subjects might be capable of voluntarily restricting their intake for such long periods remains open to question. One aspect that may make this more possible, however, would be if the hunger that accompanies periods of short-term calorie restriction ultimately dissipated. Anecdotally there are reports from people engaged in restriction that after time the hunger wanes. This effect might be possible because after an initial period when the body is in a dynamic state of change, energy demands appear to stabilize at a new lower level

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(the body weight plateau; animals36–38 and humans39,40) and thereafter the subject remains in energy balance. The protracted absence of energy imbalance may no longer provide a hunger signal, making continued restriction (once the dynamic phase is complete) a more likely possibility. Unfortunately, to date no controlled trials of CR protocols in nonobese subjects have been continued beyond the dynamic phase when body mass is still changing. To explore the impact of protracted CR on perceptions of hunger, we studied the responses of MF1 mice. We have previously shown in these mice that the dynamic phase during which animals on restriction reach a new energy balance takes approximately 30–50 days to develop.41 After release from restriction, the mice show a profound hyperphagic response, increasing their food intake to well above the levels of age-matched control animals maintained without restriction. We used the magnitude of this hyperphagic response on release from restriction as a bioassay of the levels of hunger in the mice, although this is not the only method of hunger assessment available (other alternatives include behavioral operant tasks that evaluate how much the animal is willing to work for food). We placed mice on a 20% restriction for 50 days and then fed them in energy balance for further periods between 0 and 50 days, recording the magnitude of their hyperphagic responses on release from restriction. METHODS Animals and housing MF1 mice aged 6–8 weeks were purchased from Harlan (UK) and housed individually in M3 cages (NKP Kent, UK) under a 12 light:12 dark photoperiod at 20 2°C. All mice had ad lib. access to water throughout the study and were provided with wood shavings and shredded paper bedding for enrichment. The diet used in this study was pelleted rat and mouse, breeder and grower diet, (Special Diets Services, BP Nutrition, UK), which has a gross energy content of 17.4 MJ/kg (9.2% fat by energy).

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HUNGER DURING CALORIC RESTRICTION

Effect of duration of restriction on postrestriction hyperphagia To determine the hunger of mice under prolonged restriction, 40 mice were placed on 20% CR for 50 days. Ten control mice continued feeding ad lib. for the remainder of the study. Restriction was calculated on an individual basis relative to the measured food intake at the start of the study (prerestriction). After 50 days, the restricted mice were randomly assigned to one of 4 groups (n 10 in each). These continued to be fed in restriction but had body mass “clamped” to maintain energy balance for either 0, 10, 30, or 50 days. To achieve this, we adjusted the amounts of food they ate every second day to keep body mass constant. Actual achieved levels of restriction differed slightly, therefore, between the treatment groups and are reported in the results. After the period of body mass clamp, each group was released from the imposed restriction and allowed free access to food for a further 50 days. Body mass and food intake were carefully monitored throughout, while body composition was determined using dual-energy x-ray absorptiometry (Lunar Piximus methodology as described previously41 and calibrated as described previously42) on three separate occasions: at the start of the study when all mice were feeding ad lib, at the end of the restriction phase, and at the end of the final ad lib. phase. Validation of the “hunger bioassay” We have previously observed that when mice under CR are permitted access to ad lib. food, they exhibit a period of hyperphagia. We used the magnitude of this hyperphagic response as a measure of the hunger of the mice under restriction. To evaluate whether hyperphagia reflects “hunger,” we performed a validation study to compare the feeding behavior of the mice, under restriction and after release from restriction, with patterns of neuropeptide gene expression in their brains. When mice are food deprived, they develop a characteristic neuropeptide gene expression profile in the arcuate nucleus (ARC) of the hypothalamus. This profile involves upregulation of so-called orexigenic neuropeptides (notably neuropeptide Y [NPY] and Agouti-regulated peptide [AgRP])

that stimulate food intake, and downregulation of anorexigenic neuropeptides that inhibit food intake (notably pro-opiomelanocortin [POMC] and cocaine and amphetamine regulated transcript [CART]). These neuropeptide patterns are believed to underpin the drive to eat and are a molecular marker of the phenomenon of “hunger.” If the mice under restriction are “hungry,” we would expect to observe upregulation of NPY and AgRP and downregulation of POMC and CART in their arcuate nuclei, relative to control mice. If the hyperphagia serves to abolish this “hunger,” then we would anticipate that the gene expression would be normalized relative to ad lib.-fed controls after the hyperphagic period. To test this idea, we placed 30 mice on a 50% CR for a period of 25 days, and kept 10 control mice on ad lib. food. We split the 30 mice on restriction into 3 groups. Group 1 was culled after 25 days of restriction before being given the daily food ration on day 25. Group 2 was culled after 25 days of restriction 2 hours after being given the daily food ration on day 25. Group 3 was released from restriction on day 25 and fed ad lib. for 4 days and then culled. Mice were dissected immediately postmortem and their brains were removed and frozen on dry ice. Hypothalamic gene expression was quantified using in situ hybridization techniques.43 We measured POMC, AgRP, CART, and NPY in the ARC. Brain sections were collected onto sets of 8 slides spanning the ARC from approximately –2.7 to –1.22 mm relative to bregma, according to the atlas of the mouse brain.44 After undergoing in situ hybridization, the slides were exposed to film (Kodak, Biomax MR film) to determine the intensity of the hybridization signal, which was then quantified using Image Pro Plus (Media Cybernetics) after calibration using a standard curve. RESULTS Effect of duration of restriction on postrestriction hyperphagia Throughout the study, control animals maintained a constant food intake averaging 5.17 0.24 g on day 1 and 5.20 0.22 g on the last

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day of study. Their body mass, however, increased from 35.6 1.10 to 44.7 2.18 g, involving increases in both lean and adipose tissue. Lean tissue increased from 32.9 to 36.5 g, while fat increased from 3.4 to 6.9 g. Restricted mice did not grow at the same rate as controls (GLM [General Linear Model] using day of restriction and group as factors F19,998 80.94, p 0.0012), and consequently on day 50 of restriction, they had a significantly lower body mass than the controls by an average of 2.6 g (analysis of variance [ANOVA] F1,50 4.35, p 0.04). Fat mass was not significantly different from that of the controls. Lean mass, however, was significantly lower in the restricted mice (ANOVA F1,49 6.72, p 0.013). By the end of the 50-day restriction period, the restricted mice were in a new state of energy balance achieved by reducing both activity and resting metabolic rate.41 Control animals continued to increase in body mass beyond day 50. In contrast, the body mass of the mice on restriction remained depressed. Body mass was lower than that of the controls by an average of 5.6 g for the mice restricted for 50 days, 2.5 g for those restricted for 60 days, 2.8 g for the 80-day restriction, and 3.8 g for those restricted for 100 days. Actual achieved levels of restriction relative to the initial food intakes were 18% for those exposed for 60 days, 21% for those restricted for 80 days, and 23% for those restricted for 100 days. When mice were released from restriction, they all showed a marked hyperphagia; however, this only lasted for 1 day before they reduced their food intake to levels that were not significantly different from the control animals measured at the same time point (Fig. 1a). For the mice restricted for 50 and 100 days, this food intake level was not significant different from the intake rate in the same animals before they were placed on restriction. However, in the mice restricted for 60 and 80 days, the food intake after the period of hyperphagia was slightly but significantly lower (Max of 14%) than their initial rate of food intake (paired t-tests p 0.05). The extent of the hyperphagia did not vary significantly with the length of time that the mice had been on restriction (ANOVA F3, 37 0.37, p 0.78; Fig. 1b). When food intake was summed over the first week of release from re-

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FIG. 1. The extent of hyperphagic response after release from caloric restriction. a: Daily variations after release from clamp. b: Extent of hyperphagia on day 1 after release from clamp. c: Combined food intake over the first week after release from clamp. In the latter two cases there were no significant differences with time held on caloric restriction (n 10 per group).

striction, there was also no effect of the time on restriction on intake (ANOVA F3, 37 0.93, p 0.14), although a trend suggested that, if anything, hyperphagia increased with prolonged restriction (Fig. 1c). The response in body mass to re-feeding was extremely rapid (Fig. 2a), showing a marked increase by day 2 of re-feeding, to a level that in all cases was not significantly different from the controls. By the final measurement period, none of the restricted groups differed significantly in body mass or body composition from the controls or each other (Fig. 2b) (ANOVA Body mass: F4, 49 1.18, p 0.33, Fat mass: F4,49 0.68, p 0.61, Fat free mass: F4,49 0.70, p 0.60).

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not normalize relative to the controls. However, if mice were removed from restriction and fed ad lib. for 4 days, until they had completed their postrestriction hyperphagia, their gene expression patterns completely normalized relative to the controls (Fig. 3).

DISCUSSION Validation of the hunger bioassay by comparison to neuropeptide gene expression levels

FIG. 2. Body mass response after release from caloric restriction. a: There were rapid body mass increases after release to ad lib. feeding. b: After 50 days of ad lib. feeding, there were no significant differences in any clamped group compared to controls or each other (analysis of variance F4, 49 1.18, p 0.33) (n 10 per group). BM, body mass; NS, not significant.

Validation of the hunger bioassay by comparison to neuropeptide gene expression levels When the mice were killed after 25 days of 50% food restriction but before receiving their daily food ration, they exhibited the expected pattern of hypothalamic gene expression, with expression levels of the orexigenic NPY (ANOVA with Tukey test F3,35 6.00, p 0.002) and AgRP (ANOVA with Tukey test F3,35 10.30, p 0.001) elevated in the ARC relative to control ad lib.-fed mice, and expression levels of the anorexigenic neuropeptides POMC (ANOVA with Tukey test F3,35 10.23, p 0.001) and CART (ANOVA with Tukey test F3,35 7.26, p 0.001) reduced (Fig. 3). When mice were placed under the same level of restriction for 25 days but were given their daily ration of food 2 h before they were sacrificed, the pattern of restriction gene expression did

The pattern of gene expression in the hypothalamic ARC, with the orexigenic neuropeptides upregulated and the anorexigenic neuropeptides downregulated, clearly indicated that prior to receiving their daily food ration, mice on restriction were hungry relative to ad lib.-fed controls. Under this level of restriction, the data also clearly suggested that even after they had been given their daily food ration, they were still hungry relative to the control animals, because the neuropeptide gene expression levels were not normalized. After release onto ad lib. food, however, the mice underwent a postrestriction hyperphagia, after which their hypothalamic gene expression levels were normalized relative to the ad lib.-fed controls. These patterns provide us with confidence that the postrestriction hyperphagia is driven by these neuropeptide patterns and is a reflection of the extent of the animals’ “hunger.” Effect of duration of restriction on postrestriction hyperphagia When the mice were placed on restriction, they rapidly modified their behavior and reduced their energy demands to compensate for the reduced energy intake. We have previously shown that they achieved this by a combination of both reduced activity and reduced resting metabolism,41 consistent with the responses observed in other small rodents when placed under restriction.45–48 Despite the fact that the mice under restriction were feeding in energy balance for a protracted period, the magnitude of the hyperphagic response they demonstrated showed no abatement with the time on restriction. That the hyperphagia observed on the day after release from restriction was not related to

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FIG. 3. Neuropeptide gene expression in mice that had received 50% food restriction for 25 days. During restriction, the orexigenic neuropeptides expression levels of the orexigenic neuropeptide Y (NPY) and Agouti-regulated peptide (AgRP) were elevated in the arcuate nucleus relative to control ad lib.-fed mice, and expression levels of the anorexigenic neuropeptides pro-opiomelanocortin (POMC) and cocaine and amphetamine regulated transcript (CART) were reduced. The levels were not normalized by feeding during caloric restriction but had normalized after 4 days of ad lib. access to food. Values with common letters are not significantly different (n 10 per group).

the duration of prior restriction may have been because there is a limit in the amount of energy that these mice can process over a 24-h period. Possibly there was no point in the animals eating more than the 7.3 g that they ate (Fig. 1) because they could not process any additional intake. Considerable work has been performed to elucidate the limits on sustained energy intake.49–52 These studies suggest that a processing limit may occur at around 7 basal metabolic rate. In our mice, the food intake on the day after restriction was equivalent to 126.3 kJ/day. We have previously measured RMRt (the resting metabolic rate [RMR] at thermoneutral but not postabsorptive: slightly higher than basal metabolic rate [BMR];53) in these mice at 17.5 kJ/day.54 Hence, the mice were feeding at around the supposed limit of 7 BMR. Despite the fact the mice appeared to be feeding at around their physiological capacity on the day after restriction ended, the hyperphagia, in all but the 50-day clamp group, was not

extended beyond the first day of derestricted feeding. Hence, while there may have been a ceiling on the capacity of the animals to ingest and process food on the first day after restriction, the signals driving food intake (presumed to be the physiological basis of hunger) were insufficient to sustain the hyperphagia beyond that first day, independent of the period the animals had been on restriction. This evidence suggests that for these mice under these durations and magnitude of restriction, hunger does not dissipate over the duration of restriction. The maximum period of 100-day restriction used in our study was about one seventh the lifespan of an average MF1 mouse.55 In human terms, therefore, this would be similar to being on CR for a period of about 11 years (using the mean human life expectancy of around 78 years;56). If human responses are similar, this suggests that even after more than a decade of restriction, there would still be a feeling of hunger in subjects under restriction. Whether humans would respond in the same manner,

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however, is questionable and data from rhesus monkeys (Macaca mulatta) suggested that the drive to perform an operant task for food did not differ between monkeys under prolonged CR compared to controls.57 The authors suggested that this was an indication of diminished hunger during prolonged CR. Our understanding of the physiological factors that drive food intake has expanded enormously in the last decade. In particular, the roles of hormones released from the alimentary tract, in combination with other peripheral signals (such as leptin and insulin) and their targets in the brain, have been elucidated.58–65 In the current animals, the hyperphagic response abated and neuropeptide levels in the ARC were normalized as soon as the mice had returned to the same body mass as the control animals. This suggests that signals from the periphery reflecting the body mass may have been a key component driving the neuropeptide gene expression profiles and the postrestriction hyperphagia. Once the body mass and these signals were “normalized,” the animals stopped overeating. If this interpretation that the drive to eat was signaled primarily by the low body mass is correct, it has some important implications. First, because body mass of subjects under restriction remains permanently reduced relative to control subjects not under restriction,38,66 then hunger on restriction would never dissipate. Second, if these peripheral signaling compounds were supplied exogenously to subjects under restriction, this might remove the sensation of hunger and make it more likely that subjects would remain compliant with the restriction protocol. However, we have also shown here that during restriction there are profound changes in the gene expression levels of neuropeptides that are involved in the regulation of food intake. It is possible that these neuroendocrine responses are also an integral part of the mechanism that triggers the beneficial physiological responses to restriction. Consequently, by normalizing these patterns to remove the “hunger” pain of restriction, one might also inadvertently remove the longevity gains. How tightly these pathways are linked, if at all, would merit additional study.

ACKNOWLEDGMENTS Sarah Johnston, Lynn Bell, and Sarah Murray provided invaluable assistance with animal care and measurements. Zoe Archer and Kim Moar assisted with the gene expression work. We are grateful to Sarah Johnston and Gerald Lobley for helpful discussions throughout this study. Work was conducted under Home Office Project Licence # 60/3073 and funded by SEERAD (Scottish Executive Environment and Rural Affairs Department).

REFERENCES 1. McCay CM, Crowell MF, Maynard LA. The effect of retarded growth upon the length of the life-span and ultimate body size. J Nutr 1935;10:63–79. 2. Cooper TM, Mockett RJ, Sohal BH, Sohal RS, Orr WC. Effect of caloric restriction on life span of the housefly, Musca domestica. FASEB J 2004;18:1591–1593. 3. Forster MJ, Morris P, Sohal RS. Genotype and age influence the effect of caloric intake on mortality in mice. FASEB J 2003;17:690–692. 4. Merry BJ. Molecular mechanisms linking calorie restriction and longevity. Int J Biochem Cell Biol 2002;34:1340–1354. 5. Weindruch R, Walford RL. The retardation of aging and disease by dietary restriction. Springfield, IL: Charles C. Thomas, 1988. 6. Masoro E. Caloric restriction: a key to understanding and modulating aging. Amsterdam: Elsevier, 2002. 7. Masoro EJ. Life span extension and food restriction. Compr Ther 1988;14:9–13. 8. Miller RA, Buehner G, Chang Y, Harper JM, Sigler R, Smith-Wheelock M. Methionine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-1 and insulin levels, and increases hepatocyte MIF levels and stress resistance. Aging Cell 2005;4:119–125. 9. Tannenbaum A. Genesis and growth of tumors. II. Effects of caloric restriction per se. Cancer Res 1942;2: 460–467. 10. Tannenbaum A. The dependence of tumor formation on the degree of caloric restriction. Cancer Res 1945;5:609–615. 11. Ross MH, Bras G. Tumor incidence patterns and nutrition in the rat. J Nutr 1965;87:245–260. 12. Cheney KE, Liu RK, Smith GS, Leung RE, Mickey MR, Walford RL. Survival and disease patterns in C57BI/ 6J mice subjected to undernutrition. Exp Gerontol 1980;15:237–258. 13. Grasl-Kraupp B, Bursch W, Ruttkay-Nedecky B, Wagner A, Lauer B, Schulte-Herman R. Food restriction eliminates preneoplastic cells through apoptosis and

540

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

antagonizes carcinogenesis in rat liver. Proc Natl Acad Sci U S A 1994;91:9995–9999. Volk MJ, Pugh TD, Kim M, Frith CH, Daynes RA, Ershler WB, Weindruch R. Dietary restriction from middle age attenuates age-associated lymphoma development and interleukin 6 dysregulation in C57Bl/6 mice. Cancer Res 1994;54:3054–3061. Lee JH, Jung KJ, Kim JW, Kim HJ, Yu BP, Chung HY. Suppression of apoptosis by calorie restriction in aged kidney. Exp Gerontol 2004;39:1361–1368. Spindler SR. Rapid and reversible induction of the longevity, anticancer and genomic effects of caloric restriction. Mech Ageing Dev 2005;126:960–966. Pugh TD, Oberley TD, Weindruch R. Dietary intervention at middle age: caloric restriction but not dehydroepiandrosterone sulfate increases lifespan and lifetime cancer incidence in mice. Cancer Res 1999;59: 1642–1648. Ingram DK, Cutler RG, Weindruch R, Renquist DM, Knapka JJ, April M, Belcher CT, Clark MA, Hatcherson CD, Marriott BM, et al. Dietary restriction and aging: the initiation of a primate study. J Gerontol 1990;45:B148–B163. Kemnitz JW, Weindruch R, Roecker EB, Crawford K, Kaufman PL, Ershler WB. Dietary restriction of adult male rhesus monkeys: design, methodology, and preliminary findings from the first year of study. J Gerontol 1993;48:B17–B26. Hansen BC, Bodkin NL. Primary prevention of diabetes mellitus by prevention of obesity in monkeys. Diabetes 1993;42:1809–1814. Cefalu WT, Wagner JD, Wang ZQ, Bell-Farrow AD, Collins J, Haskell D, Bechtold R, Morgan T. A study of caloric restriction and cardiovascular aging in cynomolgus monkeys (Macaca fascicularis): a potential model for aging research. J Gerontol A Biol Sci Med Sci 1997;52:B10–B19. 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 U S A 2001;98:5093–5098. Lane MA, Ball SS, Ingram DK, Cutler RG, Engel J, Read V, Roth GS. Diet restriction in rhesus monkeys lowers fasting and glucose-stimulated glucoregulatory end points. Am J Physiol 1995;268:E941–E948. Lane MA, Tilmont EM, De Angelis H, Handy A, Ingram DK, Kemnitz JW, Roth GS. Short-term calorie restriction improves disease-related markers in older male rhesus monkeys (Macaca mulatta). Mech Ageing Dev 2000;112:185–196. Roth GS, Ingram DK, Lane MA. Calorie restriction in primates: will it work and how will we know? J Am Geriatr Soc 1999;47:896–903. Walford RL, Walford L. The anti-aging plan: the nutrient-rich, low-calorie way of eating for a longer life—the only diet scientifically proven to extend your healthy years. New York: Marlowe & Company, 2005. Walford RL, Walford L. The anti-aging plan: strategies and recipes for extending your healthy years. New York: Marlowe & Company, 2005.

HAMBLY ET AL. 28. Delaney BM, Walford L. The longevity diet: discover calorie restriction—the only proven way to slow the aging process and maintain peak vitality. New York: Marlowe & Company, 2005. 29. Fontana L, Meyer TE, Klein S, Holloszy JO. Long-term calorie restriction is highly effective in reducing the risk for atherosclerosis in humans. Proc Natl Acad Sci USA 2004;101:6659–6663. 30. Speakman JR, Hambly C. Starving for life—what animal studies can, and cannot, tell us about the use of caloric restriction to prolong human lifespan. J Nutr. 2007;137:1078–1086. 31. Shanley DP, Kirkwood TBL. Caloric restriction does not enhance longevity in all species and is unlikely to do so in humans. Biogerontology 2006;7:165–168. 32. Braeckman BP, Demetrius L, Vanfleteren JR. The dietary restriction effect in C. elegans and humans: is the worm a one millimetre human? Biogerontology 2006;7:127–133. 33. Demetrius L. Aging in mouse and human systems: a comparative study. Ann N Y Acad Sci 2006;1067: 66–82. 34. Phelan JP, Rose MR. Why dietary restriction substantially increases longevity in animal models but won’t in humans. Ageing Res Rev 2005;4;339–350. 35. de Grey AD. The unfortunate influence of the weather on the rate of ageing: why human caloric restriction or its emulation may only extend life expectancy by 2–3 years. Gerontology 2005;51:73–82. 36. Sprott RL. Diet and calorie restriction. Exp Gerontol 1997;32:205–214. 37. Weindruch R. Caloric restriction, gene expression and aging. Internat Congress Ser 2004;1260:13–20. 38. Evans SA, Messina MM, Knight WD, Parsons AD, Overton JM. Long-Evans and Sprague-Dawley rats exhibit divergent responses to refeeding after caloric restriction. Am J Physiol Regul Integr Comp Physiol 2004;288:1468–1476. 39. Fanghänel G, Cortinas L, Sánchez-Reyes L, Berber A. Second phase of a double-blind study clinical trial on Sibutramine for the treatment of patients suffering essential obesity: 6 months after treatment cross-over. Inter J Obes 2001;25:741–747. 40. Goldstein DJ, Potvin JH. Long-term weight loss: the effect of pharmacologic agents. Am J Clin Nutr 1994; 60:647–657. 41. Hambly C, Speakman JR. Contribution of different mechanisms to compensation for energy restriction in the mouse. Obes Res 2005;13:1548–1557. 42. Johnston SL, Peacock WL, Bell LM, Lonchampt M, Speakman JR. PIXImus DXA with different software needs individual calibration to accurately predict fat mass. Obes Res 2005;13:1558–1565. 43. Simmons DM, Arriza JL, Swanson LW. A complete protocol for in situ hybridisation of messenger RNAs in brain and other tissues with radiolabeled single stranded RNA probes. J Histotechnol 1989;12: 169–181. 44. Franklin KBJ, Paxinos G. The mouse brain in stereotaxic coordinates. San Diego: Academic Press, 1997.

HUNGER DURING CALORIC RESTRICTION 45. Ballor DL. Effects of dietary restriction and/or exercise on 23-h metabolic rate and body composition in female rats. J Appl Physiol 1991;71:801–806. 46. Boakes RA, Dwyer DM. Weight loss in rats produced by running: effects of prior experience and individual housing. J Exp Psychol 1997;50B:129–148. 47. McCarter R, McGee J. Transient reduction of metabolic rate by food restriction. Am J Physiol 1989; 257:E175–E179. 48. Sherwin CM. Voluntary wheel running: a review and novel interpretation. Anim Behav 1998;56:11–27. 49. Drent RH, Daan S. The prudent parent: energetic adjustments in avian breeding. Ardea 1980;68:225–252. 50. Peterson CC, Nagy KA, Diamond J. Sustained metabolic scope. Proc Nat Acad Sci U S A 1990;87: 2324–2328. 51. Hammond KA, Diamond J. Maximum sustained energy budgets in humans and animals. Nature 1997; 386:457–462. 52. Johnson MS, Thomson SC, Speakman JR. Limits to sustained energy intake. I. Lactation in the laboratory mouse Mus musculus. J Exp Biol 2001;204:1925–1935. 53. Krol E, Speakman JR. Limits to sustained energy intake. VI. Energetics of lactation in laboratory mice at thermoneutrality. J Exp Biol 2003;206:4255–4266. 54. Speakman JR, Krol E, Johnston MS. The functional significance of individual variations in BMR. Phys Biochem Zool 2004;77:900–915. 55. Speakman JR, Talbot DA, Selman C, Snart S, McLaren JS, Redman P, Krol E, Jackson DM, Johnson MS, Brand MD. Uncoupled and surviving: individual mice with high metabolism have greater mitochondrial uncoupling and live longer. Aging Cell 2004;3:87–95. 56. Sardon JP. Recent demographic trends in developed countries. Population 2006;61:227–300. 57. Mattison J, Black A, Huck J, Moscrip T, Handy A, Tilmont E, Roth G, Lane M, Ingram D. Age-related decline in caloric intake and motivation for food in rhesus monkeys. Neurobiol Aging 2005;26:1117–1127. 58. Schwartz MW, Woods SC, Porte D Jr, Seeley RJ, Baskin DG. Central nervous system control of food intake. Nature 2000;404:661–671.

541 59. Stanley S, Wynne K, McGowan B, Bloom S. Hormonal regulation of food intake. Physiol Rev 2005;85: 1131–1158. 60. Berthoud HR. Neural control of appetite: cross-talk between homeostatic and non-homeostatic systems. Appetite 2004;43:315–317. 61. Mercer JG, Speakman JR. Hypothalamic neuropeptide mechanisms for regulating energy balance: from rodent models to human obesity. Neurosci Biobehav Rev 2001;25:101–116. 62. Chaudhri O, Small C, Bloom S. Gastrointestinal hormones regulating appetite. Philos Trans R Soc Lond B Biol Sci 2006;361:1187–1209. 63. Trayhurn P, Bing C. Appetite and energy balance signals from adipocytes. Philos Trans R Soc Lond B Biol Sci 2006;361:1237–1249. 64. Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW. Central nervous system control of food intake and body weight. Nature 2006;443: 289–295. 65. Baynes KC, Dhillo WS, Bloom SR. Regulation of food intake by gastrointestinal hormones. Curr Opin Gastroenterol 2006;22:626–631. 66. Thurman JD, Bucci TJ, Hart RW, Turturro A. Survival, body weight, and spontaneous neoplasms in ad libitum-fed and food-restricted Fischer-344 rats. Toxicol Pathol 1994;22:1–9.

Address reprint requests to: Catherine Hambly ACERO School of Biological Sciences University of Aberdeen Aberdeen, AB24 2TZ Scotland, United Kingdom E-mail: [email protected] Received: March 23, 2007 Accepted: July 10, 2007

This article has been cited by: 1. Aubrey D.N.J. de Grey . 2008. Rejuvenation Research in 2007. Rejuvenation Research 11:4, 837-839. [Citation] [PDF] [PDF Plus]