Sexual Dimorphism in the Response of Adipose Mass and Cellularity to Graded Caloric Restriction

Objective: To investigate the effects of mild to moderate caloric restriction on parameters of body growth, fat mass, and adipose tissue cellularity in female and male Wistar rats. Research Methods and Procedures: Three‐month‐old female and male Wistar rats were subjected to a chronic, mild to moderate caloric restriction paradigm (5%, 10%, or 20% reduction in caloric intake from ad libitum values) for 6 months. This was accomplished using a unique automated feeder system tailored to the food consumption levels of individual rats. Body weight and length, weight of lean organs, regional adipose mass, and adipose cellularity were measured before and after the diet restriction. Results: Caloric restriction produced proportional decelerations in body weight increases in both genders, without significant changes in body length or lean organ mass. Marked and disproportional reductions in regional adipose tissue mass were produced at all levels of food restriction (even at 5% restriction). An unexpected finding was that in response to graded caloric restriction, female rats preserved adipose fat cell number at the expense of fat cell volume, whereas the converse was seen for male rats. Discussion: These studies demonstrate a sexual dimorphism in the response to mild to moderate degrees of chronic caloric restriction. At low levels of caloric restriction, it is possible to affect regional adipose mass and cellularity while preserving lean organ mass.     

Cold acclimation in food-restricted rats

Food intake, body weight and brown adipose tissue (BAT) mass and composition of rats exposed at 6 degrees C either with food ad libitum or food-restricted were compared with those of rats in the thermoneutral zone, with food ad libitum. Cold acclimation with food ad libitum increases food intake and prevents body weight gains. IBAT (interscapular BAT) increases its mass and changes its composition after 3 weeks of cold exposure. Cold acclimation with food restriction produces a progressive decrease in body weight. IBAT mass increases after 3 weeks but changes in composition occur sooner. It is concluded that the overfeeding that accompanies cold acclimation is not necessary for non-shivering thermogenesis in BAT.     

Caloric restriction and the aging process: a critique

The main objective of this review is to provide an appraisal of the current status of the relationship between energy intake and the life span of animals. The concept that a reduction in food intake, or caloric restriction (CR), retards the aging process, delays the age-associated decline in physiological fitness, and extends the life span of organisms of diverse phylogenetic groups is one of the leading paradigms in gerontology. However, emerging evidence disputes some of the primary tenets of this conception. One disparity is that the CR-related increase in longevity is not universal and may not even be shared among different strains of the same species. A further misgiving is that the control animals, fed ad libitum (AL), become overweight and prone to early onset of diseases and death, and thus may not be the ideal control animals for studies concerned with comparisons of longevity. Reexamination of body weight and longevity data from a study involving over 60,000 mice and rats, conducted by a National Institute on Aging-sponsored project, suggests that CR-related increase in life span of specific genotypes is directly related to the gain in body weight under the AL feeding regimen. Additionally, CR in mammals and “dietary restriction” in organisms such as Drosophila are dissimilar phenomena, albeit they are often presented to be the very same. The latter involves a reduction in yeast rather than caloric intake, which is inconsistent with the notion of a common, conserved mechanism of CR action in different species. Although specific mechanisms by which CR affects longevity are not well understood, existing evidence supports the view that CR increases the life span of those particular genotypes that develop energy imbalance owing to AL feeding. In such groups, CR lowers body temperature, rate of metabolism, and oxidant production and retards the age-related pro-oxidizing shift in the redox state.     

Unusual increase of Scd1 and Elovl6 expression in rat inguinal adipose tissue

It is generally accepted that the location of body fat deposits may play an important role in the risk of developing some endocrine and metabolic diseases. We have studied the effect of food restriction and food restriction/refeeding, often practiced by individuals trying to lose body weight, on the expression of genes which are associated with obesity and certain metabolic disorders in inguinal, epididymal, and perirenal rat white adipose tissues. Gene expression was analyzed by real time semi-quantitative polymerase chain reaction and by Western blot. We found that prolonged food restriction caused a significant decrease of body and adipose tissue mass as well as the increase of Scd1 and Elovl6 gene expressions in all main rat adipose tissue deposits. Food restriction/refeeding caused increases of: a) Scd1 and Elovl6 mRNA levels in adipose tissue, b) Scd1 protein level and c) desaturation index in adipose tissue. The increased expression of both genes was unusually high in inguinal adipose tissue. The results suggest that the increase of Scd1 and Elovl6 gene expressions in white adipose tissue by prolonged food restriction and prolonged food restriction/refeeding may contribute to accelerated fat recovery that often occurs in individuals after food restriction/refeeding.     

Differential effect of long-term food restriction on fatty acid synthase and leptin gene expression in rat white adipose tissue.

Long-term food restriction (85%, 70% and 50% of ad libitum energy intake for one month) induced a substantial fall in serum leptin concentration and leptin mRNA levels in epididymal white adipose tissue in rats. Surprisingly, this suppression was not reversed by refeeding ad libitum for 48 h. The reduction in serum leptin concentration and leptin mRNA level did not strictly correlate with reduction in fat or body mass. Unlike serum leptin concentration and epididymal adipose tissue leptin mRNA levels, fatty acid synthase activity, fatty acid synthase protein abundance and fatty acid synthase mRNA levels increased significantly in white adipose tissue after refeeding rats subjected to food restriction. The increase in serum insulin concentration was observed in all groups on different degrees of food restriction and refed ad libitum for 48 h compared to controls. A decrease in serum insulin concentration was found in the rats not refed before sacrifice. Long-term food restriction did not significantly affect serum glucose concentrations in either refed or non-refed rats. The data reported in this paper indicate that there is no rapid rebound in serum leptin concentration or leptin gene expression in contrast to the increase in serum insulin concentration and fatty acid gene expression in white adipose tissue of rats refed ad libitum after one month's food restriction.     

食物限制对雄性大绒鼠能量代谢特征的影响

为探讨横断山区大绒鼠适应食物匮乏的适应对策,将成年雄性大绒鼠随机分为自由取食组和饲喂正常摄食量的80% 限食组。测定了自由取食组和限食组雄性大绒鼠的体重、静止代谢率、非颤抖性产热以及体脂含量、血清瘦素含量、肝脏鲜重、褐色脂肪组织重量和消化道形态。结果显示:限食使雄性大绒鼠的体重、体脂含量、静止代谢率、非颤抖性产热、褐色脂肪组织重量和大肠、小肠长度显著降低,使盲肠内容物重量显著增加。血清瘦素含量与体重、体脂含量呈极显著正相关。在限食条件下,大绒鼠主要通过降低体重、基础代谢和产热的能量支出以及动用体内脂肪以应对食物资源短缺的环境条件,瘦素可能参与了能量代谢和体重的适应性调节。     

Effect of Chronic Caloric Restriction on the Synchronization of Various Physiological Measures in Old Female Fischer 344 Rats

-A variety of physiological and behavioral parameters which relate to metabolism were continuously monitored in 18 month old female Fischer 344 rats which were maintained on either ad libitum or reduced calorie diets. Caloric restriction (CR) stimulated average motor activity per day, the duration of each feeding episode, food consumed per episode, and water consumed per gram lean body mass (LBM). However, CR limited total food consumption, feeding time, number of feeding episodes per day, total eating and drinking time, and the daily ratio of food consumed to water consumed, CR also decreased average body temperature per day, O₂ consumption, CO₂, production, and respiratory quotient. A variety of parameters concerning water consumption were not affected. CR rats ate their food immediately when food was presented during the light span, while ad libitum fed animals ate numerous small meals throughout the entire dark span. An anticipatory response to restricted feeding was also noted. Total motor activity, metabolism, and body temperature increased just prior to scheduled feeding and reached maximum values shortly after feeding, suggesting that these parameters were highly synchronized to feeding. Females and males were found to respond to caloric restriction in a similar fashion. Dramatic changes in respiratory quotient and body temperature suggest rapid shifts between metabolic pathways (glycolysis to giuconeogenesis) to obtain optimal efficiency. Lower body temperature and metabolism may provide protection against DNA damage, thereby increasing the survival potential of restricted rats. These responses may provide insight into the mechanisms by which caloric restriction acts to extend life span.     

Food restriction increases life span of hypertensive animals

Food restriction was used to increase the life span of normotensive (WKY) and Spontaneously Hypertensive Rats (SHR). When SHR's were maintained on 40% of an otherwise typical lab rat diet, their mean life spans increased from 18 months to over 30 months. The mean life times of normotensive rats which were similarly food restricted were expanded from 24 months to over 32 months. Histological examination of heart, adrenals, kidneys and brain showed that freely fed hypertensive rats died of end-organ damage associated with high blood pressure. In contrast, deaths of food restricted hypertensive rats appeared due to changes associated with old age, rather than specific lesions due to hypertension. Thus, food restriction allows a genetically hypertensive animal to reach a normal life span and to die of age-related rather than hypertension-related events.     

Aging and neural control of the GI tract: III. Senescent enteric nervous system: lessons from extraintestinal sites and nonmammalian species

Functional changes in GI motility associated with advanced age include slowing of gastric emptying, decreased peristalsis, and slowing of colonic transit. These changes appear to be associated with region-specific loss of neurons and impaired function. The mechanism(s) underlying physiological aging are likely to be multifactorial. Alterations in specific signal transduction pathways have been reported at the level of the receptor and postreceptor events including kinase expression and function, mitochondrial function, and activation of the apoptosis cascade. Advanced age is associated with increased oxidative stress and its concomitant effects on cellular function. Whereas no specific genes have been causally linked to life span in mammals, studies involving nonmammalian species suggest that specific genes are involved in determining life span and age-related changes in cellular function. Caloric restriction is the only intervention shown to slow aging in a variety of species. Recent studies implicate a possible role for an insulin/IGF-I cascade in the region- and tissue-specific changes associated with physiological aging.     

Brown Adipose Tissue Thermogenesis During Aging and Senescence

We have found that cold- and norepinephrine-induced brown adipose tissue (BAT) nonshiveringthermogenesis (NST) is significantly lower in old male Fischer 344 rats and is associatedwith the decreased ability of these animals to maintain homeothermy. This decline in BATthermogenesis is not as great in females. Although the mechanism(s) underlying this genderdifference in the age-related decrease in brown fat NST are not completely elucidated, theydo not appear to reflect decreased sympathetic neural activity of BAT in the older males vs.females. Rather, our investigations, strongly suggest that the blunted cold-induced heatproduction of BAT reflects less functional BAT. The fact that the older animals have less functionalBAT than do their younger counterparts may predispose them to the accumulation of excessbody fat. Our studies have also found that near the end of the natural life of these rats, theyenter a state of senescence that can be identified by spontaneous rapid body weight loss,resulting from decreased food intake. In this state, the rats are considerably more susceptibleto cold than are comparably aged presenescent (body weight stable) rats of the samechronological age. The greater hypothermia exhibited by the senescent vs. presenescent rats during coldexposure is associated with a significant reduction in the amount of functional brown fat andin the amount of heat each brown fat cell can generate. It is the intent of this review to discussthe findings of these investigations.     

An intervention resembling caloric restriction prolongs life span and retards aging in yeast.

The yeast Saccharomyces cerevisiae has a finite life span that is measured by the number of daughter cells an individual produces. The 20 genes known to determine yeast life span appear to function in more than one pathway, implicating a variety of physiological processes in yeast longevity. Less attention has been focused on environmental effects on yeast aging. We have examined the role that nutritional status plays in determining yeast life span. Reduction of the glucose concentration in the medium led to an increase in life span and to a delay in appearance of an aging phenotype. The increase in life span was the more extensive the lower the glucose levels. Life extension was also elicited by decreasing the amino acids content of the medium. This suggests that it is the decline in calories and not a particular nutrient that is responsible, in striking similarity to the effect on aging of caloric restriction in mammals. The caloric restriction effect did not require the induction of the retrograde response pathway, which signals the functional status of the mitochondrion and determines longevity. Furthermore, deletion of RTG3, a downstream mediator in this pathway, and caloric restriction had an additive effect, resulting in the largest increase (123%) in longevity described thus far in yeast. Thus, retrograde response and caloric restriction operate along distinct pathways in determining yeast longevity. These pathways may be exclusive, at least in part. This provides evidence for multiple mechanisms of metabolic control in yeast aging. Inasmuch as caloric restriction lowers blood glucose levels, this study raises the possibility that reduced glucose alters aging at the cellular level in mammals.     

Metformin slows down aging and extends life span of female SHR mice

Studies in mammals have led to the suggestion that hyperglycemia and hyperinsulinemia are important factors both in aging and in the development of cancer. It is possible that the life-prolonging effects of calorie restriction are due to decreasing IGF-1 levels. A search of pharmacological modulators of insulin/IGF-1 signaling pathway (which resemble effects of life span extending mutations or calorie restriction) could be a perspective direction in regulation of longevity. Antidiabetic biguanides are most promising among them. Here we show the the chronic treatment of female outbred SHR mice with metformin (100 mg/kg in drinking water) slightly modified the food consumption but decreased the body weight after the age of 20 months, slowed down the age-related switch-off of estrous function, increased mean life span by 37.8%, mean life span of last 10% survivors by 20.8%, and maximum life span by 2.8 months (+10.3%) in comparison with control mice. On the other side, treatment with metformin failed influence blood estradiol concentration and spontaneous tumor incidence in female SHR mice. Thus, antidiabetic biguanide metformin dramatically extends life span, even without cancer prevention in this model.     

Caloric restriction increases adiponectin expression by adipose tissue and prevents the inhibitory effect of insulin on circulating adiponectin in rats

Aging is associated with redistribution of body fat and the development of insulin resistance. White adipose tissue emerges as an important organ in controlling life span. Caloric restriction (CR) delays the rate of aging possibly modulated partly by altering the amount and function of adipose tissue. Adiponectin is a major adipose-derived adipokine that has anti-inflammatory and insulin-sensitizing properties. This study examined the effects of CR on adiposity and gene expression of adiponectin, its receptors (AdipoR1 and AdipoR2) in adipose tissue and in isolated adipocytes of Brown Norway rats that had undergone CR for 4 months or fed ad libitum. The study also determined plasma concentrations of adiponectin and insulin in these animals and whether insulin infusion for 7 days affects adiponectin expression and its circulating concentrations under CR conditions. CR markedly reduced body weight as anticipated, epididymal fat mass and adipocyte size. CR led to an increase in plasma free fatty acid and glycerol (both twofold), and adipose triglyceride lipase messenger RNA (mRNA) in adipose tissue and isolated adipocytes (both >2-fold). Adiponectin mRNA levels were elevated in adipose tissue and adipocytes (both >2-fold) as was plasma adiponectin concentration (2.8-fold) in CR rats. However, CR did not alter tissue or cellular AdipoR1 and AdipoR2 expression. Seven days of insulin infusion decreased adiponectin mRNA in adipose tissue but did not reverse the CR-induced up-regulation of circulating adiponectin levels. Our results suggest that the benefits of CR could be, at least in part, dependent on enhanced expression and secretion of adiponectin by adipocytes.     

Long-term intermittent feeding, but not caloric restriction, leads to redox imbalance, insulin receptor nitration, and glucose intolerance

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance.     

Maternal food restriction delays weaning in the guinea pig, Cavia porcellus

In mammals, the duration of lactation varies much more than other life history parameters in relation to body mass, both within and between species. The causes of this variation are poorly understood and seem to result from varying conditions of mothers and young. We studied the effect of long-term maternal food restriction on litter mass at birth, duration of lactation and offspring development in the precocial guinea pig. Mild experimental food restriction during reproduction resulted in prolonged nursing behaviour of mothers. Evidence for a threshold mass at weaning was, however, equivocal. In the guinea pig, benefits of prolonged lactation prove hard to understand, because nutritional benefits are minor. Independently of maternal food regime, pups terminated suckling attempts several days after mothers ceased nursing behaviour. The time between the last nursing behaviour and the last suckling attempts was not longer in litters with higher need, that is, in litters of food-restricted females, than in litters of females fed ad libitum. Under food restriction, mothers maintained their own body mass, leading in pups to lower mass at birth, reduced postnatal growth and lower body mass at maturity. Guinea pig mothers appear to be selected to value their own condition more than that of a litter. We suggest that lengthening of the nursing period under poor conditions is a life history response primarily of precocial mammals.     

Tissue-specific effect of refeeding after short- and long-term caloric restriction on malic enzyme gene expression in rat tissues

Restricting food intake to a level below that consumed voluntarily (85%, 70% and 50% of the ad libitum energy intake for 3 or 30 days) and re-feeding ad libitum for 48 h results in an increase of malic enzyme (ME) gene expression in rat white adipose tissue. The increase of ME gene expression was much more pronounced in rats maintained on restricted diet for 30 days than for 3 days. The changes in ME gene expression resembled the changes in the content of SREBP-1 in white adipose tissue. A similar increase of serum insulin concentration was observed in all groups at different degrees of caloric restriction and refed ad libitum for 48 h. Caloric restriction and refeeding caused on increase of ME activity also in brown adipose tissue (BAT) and liver. However, in liver a significant increase of ME activity was found only in rats maintained on the restricted diet for 30 days. No significant changes after caloric restriction and refeeding were found in heart, skeletal muscle, kidney cortex, and brain. These data indicate that the increase of ME gene expression after caloric restriction/refeeding occurs only in lipogenic tissues. Thus, one can conclude that caloric restriction/refeeding increases the enzymatic capacity for fatty acid biosynthesis.     

Lysosomes and Brain Aging in Mammals

Hypotheses about the factors controlling the rate of brain aging are usually derived from 1) correlates of maximum life span across mammals or 2) investigations into the causes of age-related neuropathologies in humans. With regard to the former, the strong correlation between metabolic rate and longevity prompted a variety of free radical hypotheses of aging. There is also evidence that brain size affects life span independently of body metabolism rates. The second approach has led to a diverse array of pathogenic mechanisms and, importantly for the development of general hypotheses, the discovery of animal analogues. The present paper discusses the possibility that age-associated lysosomal dysfunction constitutes a generalized mammalian phenomenon that accounts for specific features of the aged human brain. Immunocytochemical studies using rats and dogs have identified lysosomal changes that begin early in adulthood and are most pronounced in brain areas known to be particularly vulnerable to age-related pathogenesis in humans. Experimentally induced lysosomal dysfunction in cultured brain slices from rats and mutant mice triggers a wide array of changes associated with the aged human brain, including meganeurites and intraneuronal tangles. Finally, there is evidence that at least some forms of proteolysis decrease with increasing brain size across the mammals. The above observations lead to the suggestion that the expansion of neuronal arborizations that occurred in conjunction with increases in brain size secondarily slowed both neuronal metabolism and protein turnover. These events could have served to reduce the rate at which lysosomes (and other organelles) fail.     

Chaperone-mediated regulation of hepatic protein secretion by caloric restriction

Calorie restriction (CR) delays age-related physiological changes, reduces cancer incidence, and increases maximum life span in mammals. Here we show that CR decreased the expression of many hepatic molecular chaperones and concomitantly increased the rate and efficiency of serum protein secretion. Hepatocytes from calorie-restricted mice secreted twice as much albumin, 63% more alpha1-antitrypsin, and 250% more of the 31.5-kDa protein 2 h after their synthesis. A number of trivial explanations for these results, such as differential rates of protein synthesis and cell leakage during the assay, were eliminated. These novel results suggest that CR may promote the secretion of serum proteins, thereby promoting serum protein turnover. This may reduce the circulating level of damaging, glycoxidated serum proteins.     

Life-long calorie restriction in Fischer 344 rats attenuates age-related loss in skeletal muscle-specific force and reduces extracellular space.

The decline in muscle function is associated with an age-related decrease in muscle mass and an age-related decline in strength. However, decreased strength is not solely due to decreased muscle mass. The age-related decline in muscle-specific force (force/muscle cross-sectional area), a measure of intrinsic muscle function, also contributes to age-related strength decline, and the mechanisms by which this occurs are only partially known. Moreover, changes in the extracellular space could have a profound effect on skeletal muscle function. Life-long calorie restriction in rodents has shown to be a powerful anti-aging intervention. In this study, we examine whether calorie restriction is able to attenuate the loss of muscle function and elevations in extracellular space associated with aging. We hypothesize that calorie restriction attenuates the age-associated decline in specific force and increases in extracellular space. Measurements of in vitro contractile properties of the extensor digitorum longus (type II) and soleus (type I) muscles from 12-mo and 26- to 28-mo-old ad libitum-fed, as well as 27- to 28-mo-old life-long calorie-restricted male Fischer 344 rats, were performed. We found that calorie restriction attenuated the age-associated decline in muscle mass-to-body mass ratio (mg/g) and strength-to-body mass ratio (N/kg) in the extensor digitorum longus muscle (P < 0.05) but not in the soleus muscle (P > 0.05). Importantly, muscle-specific force (N/cm2) in the extensor digitorum longus, but not in the soleus muscle, of the old calorie-restricted rats was equal to that of the young 12-mo-old animals. Moreover, the age-associated increase in extracellular space was reduced in the fast-twitch extensor digitorum longus muscle (P < 0.05) but not in the soleus muscle with calorie restriction. We also found a significant correlation between the extracellular space and the muscle-specific force in the extensor digitorum longus (r = -0.58; P < 0.05) but not in the soleus muscle (r = -0.38; P > 0.05). Hence, this study shows a loss of muscle function with age and suggests that long-term calorie restriction is an effective intervention against the loss of muscle function with age.     

食物限制对黑线仓鼠能量代谢和产热的影响

为阐明动物应对食物短缺的能量学对策与其自身的代谢水平的关系,测定了不同限食程度下黑线仓鼠的体重、基础代谢率和非颤抖性产热。结果发现,限食使基础代谢率、非颤抖性产热、褐色脂肪组织细胞色素c氧化酶活性降低。90% 限食驯化4 周后,存活率为80% ,60% 限食驯化4 周后,存活率为30% 。低温驯化使黑线仓鼠基础代谢率和非颤抖性产热显著增加,使80% 限食动物的体重和存活率显著降低。高基础代谢率组的摄食量比低基础代谢率组多23.8% ,80% 限食后两组体重降低的幅度和存活率差异不显著。结果表明:高水平的代谢率使黑线仓鼠对食物资源短缺的敏感性增加;支持“代谢率转换假说”,符合“具有储食习性的动物对食物短缺的生理耐受性较低” 的预测。