Anti-aging effects of anti-lipolytic drugs

Genetic disruption of insulin and insulin-like signaling pathways may extend lifespan. Hyperinsulinemia and insulin resistance may accelerate aging. The hypothesis was tested that a once-a-week life-long inhibition of insulin secretion by the administration of anti-lipolytic drugs might have anti-aging effects. Groups of 3-month-old male Sprague-Dawley rats were (a) given standard laboratory food ad libitum (AL); (b) fed AL 6 days and fasted 1 day every week (FW); (c) fed AL every other day (EOD), (d) fed like FW and given Acipimox (50 mg/kg b.w.) on the day of fasting (FWA) by the gastric tube. The AL, FW and EOD groups received saline intragastrically. Treatment with ACIPIMOX transiently decreased plasma free fatty acids, glucose and insulin and increased valine plasma levels, and had no long-term effect on food consumption and body weight. By age 6, 12 and 24 months subgroups were taken and the age-related changes in liver dolichol and autophagic proteolysis--which are correlated with life-expectancy--were measured. Liver dolichol levels increased and autophagic proteolysis decreased in mature and older AL rats; EOD and FWA fully counteracted these changes; FW rats had significant but smaller beneficial effects. It is concluded that life-long weekly-repeated transient inhibition of insulin secretion by antilipolytic drugs may have an anti-aging effect, additive to the anti-aging effect of a milder caloric restriction. Speculation is that transiently lower plasma insulin levels might stimulate the anti-aging cell-repair mechanism autophagy, which has longer lasting effects on cell housekeeping.     

Food restriction in female Wistar rats. III. Thermotropic transition of membrane lipid and 5'-nucleotidase activity in hepatocytes

The effect of diet restriction was measured on the anisotropy parameter of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 5'-nucleotidase enzyme activity in liver plasma membrane preparates. Diet restriction was applied to rats 3.5 months old on an every-other-day schedule (EOD) and the rats were killed at the age of 28-29 months. Six months and 24 months rats, fed ad libitum (AL), were used as controls. The Arrhenius plots of anisotropy parameter of liver membranes from young, old AL and old EOD animals exhibited well defined breakpoints at 16.3 degrees C, 19.5 degrees C and 16.7 degrees C, respectively. The breakpoint temperature of 5'-nucleotidase activity was lower in samples from young rats as compared to those from old AL rats, whereas no difference was observed comparing young and EOD fed rats. Present results support the hypothesis that diet restriction modifies lipid composition of liver plasma membranes in such a way that the appearance of age-dependent alterations is delayed.     

Food restriction in female Wistar rats. VI. Effect of reduced glutathione on the proliferative response of splenic lymphocytes from ad libitum fed and food restricted animals

The effect of reduced glutathione (GSH) on the proliferative response of splenic lymphocytes from young, adult and old ad libitum (AL) fed as well as from old food-restricted rats was investigated. Food restriction was applied on an every-other-day schedule (EOD) starting from the age of 3.5 months. As was expected, the cells from EOD fed animals responded to concanavalin A (Con A) much better than those from age-matched ad libitum fed rats. The presence of the antioxidant GSH in the culture medium increased the response of lymphocytes in all the models taken into account; furthermore, it decreased the differences due to aging and application of food restriction. According to present knowledge, mitogenic stimulation induces free radical production, and GSH has, among others, a strong antioxidant activity. Thus, present data suggest that splenocytes from EOD animals tolerated the peroxidative stress resulting from mitogenic stimulation better than those from AL fed ones.     

Food restriction in female Wistar rats. I. Survival characteristics, membrane microviscosity and proliferative response in lymphocytes

The effect of food restriction on the survival characteristics, membrane microviscosity and proliferative response in lymphocytes of female Wistar undernourished rats has been evaluated. Diet restriction was applied starting from the age of 3.5 months by feeding the animals on an every-other-day schedule (EOD). Diet restricted animals showed an increase of both mean, median and maximal life span as compared to the rats fed ad libitum (AL). Analyzing the survival curves by a parametric model, it emerged that undernutrition increased the individual resistance to environmental insults. In particular, it could be speculated that the positive influence was more pronounced in individuals with the lowest physiological capacities. The membrane microviscosity of lymphocytes was lower in EOD animals as compared to the AL ones even if one assumes a decrease in body temperature of 1-2 degrees C in EOD groups. The improvement of membrane microviscosity due to diet restriction may in part explain the improvement of proliferative response of lymphocytes from EOD groups.     

Comparison of the effects of DHEA and food restriction on serum calcitonin

In the first of two studies, female Wistar rats were fed ad libitum or 60% of the ad libitum intake. In the second study, female Sprague Dawley rats were given subcutaneous injections of DHEA (2-4 mg/day) five times per week or received similar volumes of the solvent vehicle. Animals in both studies were maintained on their respective regimens for six months. At the termination of the study, the food restricted animals weighed significantly less than the animals fed ad libitum; in addition, their serum calcitonin concentration was markedly lower and was over 60% less than that of the ad libitum fed animals. In contrast, DHEA treatment had no significant effect on the body weight or on the plasma calcitonin of the Sprague Dawley rats. Since food restriction maintains calcitonin concentrations toward youthful levels, it is clear that at least one of the anti-aging effects of food restriction is not mediated by DHEA.     

Influence of age and long-term dietary restriction on plasma insulin-like growth factor-1 (IGF-1), IGF-1 gene expression, and IGF-1 binding proteins.

The purpose of these studies was to determine more accurately the relationship between IGF-1 and life span, and to determine whether moderate dietary caloric restriction alters the age-related changes in IGF-1. Studies included an assessment of plasma IGF-1, hepatic IGF-1 mRNA, and plasma IGF-1 binding proteins (IGF-1-BP). Rats (6, 12, 22, and 28 months of age) were fed ad libitum or maintained on a diet 60% of ad libitum. In ad libitum fed animals, plasma IGF-1 decreased by 20% between 6 and 28 months of age. Similar age-related declines were observed in dietary restricted animals but levels were generally 14-25% lower at each age group. IGF-1 mRNA levels demonstrated similar decreases with age in ad libitum fed animals, but in dietary restricted animals, levels plateaued at 22 and 28 months. IGF-1 binding protein analysis revealed 3 bands at approximate molecular weights of 40k, 30k, and 24k. All bands demonstrated a decrease in relative IGF-1-BP concentration with age, as well as a decrease in the 40k and 30k binding proteins after dietary restriction. These results indicate that (a) aging in ad libitum fed animals is associated with decreases in plasma IGF-1, IGF-1-BP, and IGF-1 mRNA levels; and (b) long-term dietary restriction decreases plasma IGF-1 and IGF-1-BP levels in each age group although the age-associated decreases in IGF-1 mRNA levels are prevented by dietary restriction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dietary restriction alters characteristics of glucose fuel use.

A longitudinal study of plasma glucose and insulin concentrations in ad libitum fed and dietary restricted male F344 rats was carried out. The life span diurnal pattern of plasma glucose concentration was such that through most of the day dietary restricted rats have significantly lower plasma glucose levels than ad libitum fed rats. Throughout the life span, dietary restricted rats maintain mean 24-hour plasma glucose concentrations about 15% below those of ad libitum fed rats. Plasma insulin levels are maintained in dietary restricted rats at about 50% of the levels in ad libitum fed rats. Although plasma glucose and insulin levels are lower, dietary restricted rats use glucose fuel at the same rate per unit of metabolic mass per day as rats fed ad libitum. While these findings are consistent with the glycation hypothesis of aging and with our hypothesis that dietary restriction retards the aging processes by altering the characteristics of fuel use, they do not establish the validity of either. It is possible that this effect of dietary restriction on carbohydrate metabolism plays no role in its antiaging action. Further studies are required to define the role of these altered characteristics of carbohydrate metabolism in the aging processes.     

Food restriction in female Wistar rats. VII. Mitochondrial parameters in resting and proliferating splenic lymphocytes

The effect of food restriction on the mitochondria of resting and proliferating rat splenocytes was examined, measuring the membrane potential and mass of these organelles, by means of the specific fluorescent probes Rhodamine-123 and Nonyl Acridine Orange, respectively. Food restriction was applied on an every-other-day schedule (EOD) starting at the age of 3.5 months. The ad libitum fed (AL) animals were killed when they were 4, 11 and 24 months old, whereas the EOD rats were killed at 11 and 26 months. Resting lymphocytes from AL rats showed an age-dependent increase of both membrane potential and mass of their mitochondria. However, the mitochondrial mass increased to a larger extent when compared with the membrane potential resulting in a decrease of the respiratory quotient (RQ), i.e. of the respiratory activity per unit of mitochondrial mass. In EOD animals, the mitochondrial membrane potential was lower and the mitochondrial mass was higher in the corresponding age-matched controls, resulting in a further decrease of RQ. Following mitogenic stimulation, most of the cells from young and adult AL rat showed an increase of membrane potential and mass of their mitochondria. In contrast about 50% of cells from old AL rats had depolarized organelles after 72 h from the stimulation. Food restriction was able to prevent these alterations allowing the majority of cells, including those from old animals, to maintain the hyperpolarization of their mitochondria during the 3-day culture. In light of the well known sensitivity of mitochondrial membrane potential to peroxidative stress, present data suggest that the increase of respiration occurring during mitogenesis may increase free radical production, which is better tolerated by cells from EOD animals than by those from AL animals.     

Effects of intermittent feeding upon growth,activity, and lifespan in rats allowed voluntary exercise

From weaning until death, male Wistar rats were housed in activity-wheel cages with one group maintained on an ad libitum (AL) diet and another provided the diet every-other-day (EOD). EOD-fed rats had a mean lifespan of 124 weeks compared to 103 weeks for AL-fed rats. While post-weaning body weight and growth rates were reduced among the EOD-fed animals compared to AL-fed animals, there was no significant difference in growth duration. Positive correlations were observed between lifespan and estimates of growth rate and duration in the AL group but not in the EOD group; thus, little evidence was produced to support the hypothesis that growth rate is inversely related to longevity. While the EOD feeding regimen resulted in higher activity levels later in life, wheel activity levels were actually lower in this group in early life compared to the AL group. The observation of reduced wheel activity among young rats fed EOD was replicated in a second experiment. Thus, little support was obtained for the hypothesis that increased activity mediates the beneficial effects of dietary restriction on longevity, unless this mechanism is active late in the lifespan.     

Aging and dietary modulation of rat skeleton and parathyroid hormone

Studies were carried out on SPF F344 male rats to evaluate the effects of aging and life-prolonging food restriction, without malnutrition, on rat skeleton and circulating PTH. Six-week-old F344 rats were divided into five groups. Group 1 rats were fed ad libitum a diet that contained 21% protein. Group 2 rats were fed 60% of the mean food intake of group 1 rats from 6 weeks of age for the rest of their lives. Group 3 rats were fed 60% of the ad libitum food intake until 6 months of age and then switched to ad libitum feeding. Group 4 rats were fed ad libitum until 6 months of age, and then switched to 60% of the ad libitum food intake. Group 5 rats were fed ad libitum a diet that contained only 12.6% protein so that these animals ingested the same amount of protein per day as the group 2 rats. In group 1 animals, bone length, weight, density, and calcium content increased rapidly with age and plateaued at about 12 months of age. There was no evidence of bone loss in these animals until about 24 months of age, but by 27 months, the animals had lost appreciable amounts of bone. The circulating immunoreactive PTH levels of the animals increased with advancing age, with a marked rise at 27 months. The age-related changes in bone and serum PTH levels of rats in groups 3 and 5 were similar to those of group 1 animals, except that a terminal increase in serum PTH did not occur in group 5 rats. In the groups 2 and 4 animals which were food restricted for the longest period, bone growth and maturation were slowed down, but the animals did not experience senile bone loss or marked terminal increase in circulating PTH. The salutary effects of food restriction were, therefore, not due specifically to the restriction of protein intake or to restricting food intake only during the period of rapid growth.     

Effect of a caloric restriction regimen on the angiogenic capacity of aorta and on the expression of endothelin-1 during ageing

Ageing is accompanied by impaired angiogenesis, as well as by a deficient expression of several angiogenic growth factors and the alteration of endothelial functions. Caloric restriction (CR) is the only intervention that can extend lifespan and retard age-related-decline functions in mammals by reducing the rate of ageing and the progression of the associated diseases. Herein, we have investigated the effects of ageing and of a caloric restriction regimen (mild or severe) on the angiogenic response and on the expression of endothelin-1 (ET-1) in the aorta of male 3-, 12- or 24-month-old Sprague-Dawley rats fed ad libitum (AL), fed ad libitum and fasted 1 day a week (mild CR) or fasted every other in alternate days (severe CR). Our findings, using the rat aorta ring assay, show that the angiogenic capacity of aorta decreases with ageing in the oldest rats only. Furthermore, caloric restriction counteracts the age-related changes caloric restrictions actually give raise to a similar recovery. Interestingly, the mRNA ET-1 levels as well as ET-1 expression in aorta sprouting decreases both in middle and in aged animals. Mild and severe caloric restriction regimens prevents ET-1 changes.     

短期能量限制对正常大鼠胰岛素敏感性的影响

目的探讨短期能量限制（CR）对正常大鼠胰岛素敏感性的影响及其机制。方法将24只F344/NSIc系雄性大鼠随机分为对照组（AL组）和能量限制组（CR组）各12只。AL组自由摄入食物及水,CR组限制食物摄入（不禁水）8周（饲料总摄入量为对照组的64%）。采用高胰岛素—正常血糖钳夹试验测定两组葡萄糖输注率（GIR）判断其胰岛素敏感性（GIR越高,胰岛素敏感性越高）;比较两组体质量增加及内脏脂肪重量;Western blot法检测骨骼肌葡萄糖转运体4（Glu T4）、蛋白激酶B底物蛋白160（AS160）及磷酸化AS160（p-AS160）蛋白表达。结果CR组GIR明显高于对照组,体质量及内脏脂肪重量明显低于对照组;两组Glu T4、AS160及p-AS160蛋白表达无显著差异。结论短期CR可提高大鼠正常状态下的胰岛素敏感性,其机制可能与AS160上游的胰岛素信号转导蛋白有关。     

Restricted diet rescues rat enteric motor neurones from age related cell death

BACKGROUND: Alone among autonomic neurones, enteric neurones are known to be vulnerable to age related cell death; over 50% may be lost in aging rodents. A previous study demonstrated unexpectedly that neurones of the myenteric plexus from rats fed a restricted diet appeared not to suffer from extensive cell death in contrast with previous studies of ad libitum fed animals. AIMS: To compare myenteric neurone numbers in the ileum of young and aging male Sprague-Dawley rats fed either ad libitum or a restricted diet. METHODS: Neurones were counted in whole mount preparations of rat ileum stained immunohistochemically for the pan-neuronal marker PGP9.5, for choline acetyltransferase, or for nitric oxide synthase, or with NADH or NADPH histochemistry. RESULTS: Neurone numbers in the rat myenteric plexus were substantially affected by the dietary regimen: ad libitum feeding (50-60 g per day of standard rat chow) resulted in the death of about 50% of myenteric neurones in 24 month Sprague-Dawley rats, while numbers were unchanged when the daily dietary intake was halved between the ages of six and 24 months. Animals fed a double restricted diet (15 g per day) showed no cell loss at 30 months, as well as the predicted increase in longevity. Neurone loss was largely complete by 16 months in ad libitum fed animals. Numbers of cholinergic (possibly motor) neurones, as demonstrated by choline acetyltransferase immunohistochemistry, were substantially reduced in ad libitum fed aging rats but not in animals fed a restricted diet. Loss of cholinergic neurones after ad libitum feeding was confirmed by reduced numbers of neurones of a size range matching that of cholinergic neurones. CONCLUSIONS: Ad libitum feeding of adult rats has adverse effects on the survival of myenteric neurones, neurone loss commencing before 16 months of age. Cholinergic neurones appear to be particularly vulnerable to the effects of diet. Restricting dietary intake from six months of age prevents neurone loss almost entirely up to 30 months of age in these rats.     

Lifelong dietary modulation of calcitonin levels in rats

Studies were carried out on specific pathogen-free rats to evaluate the effects of aging and dietary manipulation on serum and thyroid calcitonin (CT) levels. Male Fischer 344 rats were randomized at 6 weeks of age to six dietary groups and subsequently maintained on the following dietary regimens. Group 1 rats were fed ad libitum throughout life; group 2 rats were fed 60% of the ad libitum food uptake, but received the same amounts of calcium, phosphorus, and vitamin D; group 3 rats were fed as the group 2 animals until 6 months of age and from then on were fed ad libitum; group 4 rats were fed ad libitum until 6 months of age and then switched to 60% food restriction; group 5 rats were fed ad libitum on food isocaloric with that of group 1 rats, but containing only 60% of the protein. Group 6 rats were killed at 6 weeks of age to serve as baseline controls. Ten rats were killed in each of the remaining five groups 15 h postprandial at 6-month intervals. The following observations were made. Serum CT increased with age similarly in the ad libitum fed group 1 and 5 rats. Food restriction markedly inhibited the increase in serum CT, and the effect was more profound in animals whose food intake was restricted after 6 months of age (group 4) than in animals on lifelong food restriction (group 2). In rats switched from food restriction to ad libitum feeding (group 3) at 6 months of age, serum CT increased with age to levels identical with those of lifelong ad libitum fed group 1 animals. Thyroid CT showed a similar pattern of age-dependent and dietary modulated changes. In contrast, aging and dietary modulation had no appreciable effect on serum calcium levels, except at 27 months of age when the serum calcium level of group 1 animals increased dramatically from the level for 24-month-old animals. There was a weak positive correlation between serum calcium and serum CT (r = 0.627; P = 0.02) and a highly significant positive correlation between serum CT and thyroid CT (r = 0.917; P = 0.001). These findings indicate that elective and therapeutic restriction of food intake might also attenulate CT levels in humans, with potentially adverse implications for skeletal homeostasis.     

Effects of lifelong restricted feeding on complex maze performance in rats

Male Wistar rats reared on a regimen of every-other-day (EOD) feeding (24% protein) since weaning were compared in complex maze learning at 30 mo of age to young (6 mo) and old (22 mo) ad libitum (AL) fed groups. Maze learning performance for the aged, EOD-fed group was equivalent to that of young AL-fed rats, and superior to that of aged AL-fed rats.     

Dietary restriction diminishes early glycation adducts on murine hepatic cytosolic proteins

The effect of three dietary regimes on the amount of Schiff base adducts, as well as acid-stable (Amadori) glycation adducts in murine hepatic cytosolic fractions was studied. The dietary regimes consisted of an unrestricted diet or two different diets in which the caloric restriction was 25 and 50% of the caloric intake of unrestricted animals. The concentration of the Schiff bases in ad libitum and slightly restricted mice (25%) was higher than that observed in severely restricted mice (50%). The concentration of the Schiff bases in the severely restricted mice did not reach values observed in ad libitum or slightly restricted mice even in 45-month old animals. The concentration of the Amadori products was essentially the same for all three dietary groups. Our results show that a severe caloric restriction decreases the formation of early glycation adducts which might influence the rate of aging of laboratory animals under caloric restriction.     

The diet restriction paradigm: a brief review of the effects of every-other-day feeding

It has been known since the early 1900s that restriction of dietary intake relative to the ad libitum (AL) level increases stress resistance, cancer resistance, and longevity in many species. Studies investigating these phenomena have used three paradigms for dietary restriction. In the first, the AL intake of a control group is measured, and an experimental group is fed less than that amount in a specified proportion, e.g., 40%. In the second, food is provided AL to both the control and experimental groups: however, the experimental group is subjected to periods of fasting. Recent studies using this paradigm provide food every other day (EOD). Both of these paradigms have been in use since the early 1900s. A third paradigm that combines them was developed in the early 1970s: one or more days of fasting separate the provision of a limited amount of food. It was assumed for many years that the physiological responses to these paradigms were due exclusively to a net decrease in energy intake. Recently, however, it was found that some species and strains of laboratory animals, when fed AL every other day, are capable of gorging so that their net weekly intake is not greatly decreased. Despite having only a small deficit in energy intake relative to control levels, however, these animals experience enhanced longevity and stress resistance is enhanced in comparison to AL controls as much in animals enduring daily restriction of diet. These observations warrant renewed interest in this paradigm and suggest that comparisons of the paradigms and their effects can be used to determine which factors are critical to the beneficial effects of caloric restriction.*R.M. Anson and B. Jones contributed equally to this review.     

Caloric restriction protects mitochondrial function with aging in skeletal and cardiac muscles

In skeletal muscles and heart in vitro complex IV activity is lower in young adult caloric restricted (CR) animals despite normal aerobic function in situ and in vivo. On the other hand, whereas markers of oxidative capacity decline 25% to 46% between 8 and 10 months and 35 months in ad libitum fed (AL) animals, in most muscles there is no decline in CR across the same absolute age (35 mo old) or relative age (35% survival rate) span and PGC-1alpha gene expression in gastrocnemius muscle declines more slowly with aging. The present results show that CR largely prevents the age-associated decline in mitochondrial function in heart and skeletal muscles, and suggest that this is secondary to a better-maintained drive on mitochondrial biogenesis.     

Age-dependence of the lateral mobility of lipids in hepatocyte plasma membrane of male rats and the effect of life-long dietary restriction

The lateral diffusion constant of lipids (D(1)) in hepatocyte plasma membranes was measured in liver smears by means of the fluorescence recovery after photobleaching (FRAP) method, applying the label, N-4-nitrobenzo-2-oxa-1,3-diazolyl phosphatidylethanolamine (NBD-PE). Nineteen ad libitum fed, male Fischer-344 rats in four age groups (2.1-29.8 months of age) were studied. A highly significant negative linear age-correlation of D(1) (cc = 0.958) was found. D(1) values were 1.39 x 10(-9) cm2/s in the young rats, and only 6.77 x 10(-10) cm2/s in the oldest rats. Lipid lateral mobility is changing in parallel with that of proteins, having been measured previously also with the FRAP method by the authors. Fractional recovery values (FR%) of the lipids were lower than those of proteins even in the young ages, but also decreased linearly with age, therefore, the parameter, D, x FR decreased even steeper with age than D(1) itself. D(1) was also measured in a group of six male Fischer 344 rats having been kept on dietary restriction (DR) since their age of 1 month until 30 months of age (applying the every-other-day (EOD) feeding). DR caused an increase of D(1), compared with the age-matched ad libitum fed animals: the mean was 9.24 x 10(-10) cm(2)/s. FR% and D(I), x FR again increased considerably under DR. The results are interpreted in terms of the increased protein and lipid turnover under DR.     

Feeding Patterns of Rats Chronically Ingesting an Ethanol-Containing Liquid Diet

We compared the feeding patterns of rats ingesting a 36% ethanol-containing liquid diet for 30 days with those of rats pair-fed an isocaloric liquid control diet or provided control diet or ground rat chow ad libitum. Ethanol-fed rats consumed fewer calories per day and gained less body weight than rats fed control diets ad libitum. Daily caloric intakes were ∼50% lower during the first 10 days and 20% thereafter. Lower intakes in ethanol-fed rats occurred through a decrease in mean meal size rather than number of meals per day, although meals were more evenly distributed diurnally. Pair-fed rats ingested most of their food in one or two meals within a few hours of presentation. In a related experiment, a 4-hr duodenal infusion of ethanol at a rate comparable to that of ethanol ingestion resulted in plasma ethanol levels of 28 ± 4 mM and suppressed 5-hr intake by ∼40% by increasing the mean postmeal interval and satiety ratio. These results suggest that the suppressive effect of ethanol ingestion on food intake may be mediated in part by a post-gastric mechanism of ethanol action.