Energy-restricted Rats Research Articles

Effects of Prolonged Energy Restriction and Dietary Protein on Muscle Protein Synthesis and Proteome Dynamics in Obese Zucker Rats

Abstract Objectives High protein (HP) diets during short-term energy restriction (ER) attenuate energy-mediated reductions in muscle protein synthesis (MPS). MPS-adaptive responses to HP diets during prolonged ER are not well described. This study examined the effects of prolonged ER and HP on MPS and the synthesis rates of numerous individual muscle proteins. Methods Female 6-wk-old obese Zucker (leprfa+/fa+, n = 48) rats were randomized to one of four diet groups for 10 weeks: ad libitum-standard protein (AL-SP; 14% protein), AL-HP (35% protein), ER-SP, and ER-HP (both fed 60% of intake of AL-SP). At the start of week 10, D2O was administered by intraperitoneal injection and isotopic equilibrium was maintained daily by providing D2O in drinking water. Rats were euthanized after 1 week of labeling, and mixed-MPS (gastrocnemius), absolute mixed-MPS (mixed-MPS x muscle protein content), proteome dynamics, and protein half-lives [rate/d (k) = –ln(1-f)/d, where f is mixed-MPS and t is time in days; t1/2 (days) = ln(2)/k] were quantified. Results Mixed-MPS was not altered by energy status and protein intake. Gastrocnemius mass was lower (P < 0.001) in ER-fed rats than AL-fed rats and higher (P = 0.034) for AL-HP than AL-SP. As a result, absolute mixed-MPS was lower (P < 0.005) in ER than AL, regardless of dietary protein. Absolute synthesis in 24 of 26 myofibrillar, 32 of 61 mitochondrial, and 55 of 60 cytoplasmic measured proteins were lower in ER than AL (P < 0.05), regardless of dietary protein. The difference in absolute synthesis of myofibrillar, mitochondrial, and cytoplasmic proteins due to ER compared to AL was 28%, 16%, and 27%, respectively. Comparison of HP and SP within each energy state revealed lower turnover rates and prolonged half-lives for a majority of measured muscle proteins in HP than in SP in both ER and AL conditions (P < 0.001). Conclusions Prolonged ER in obese Zucker rats exerted a strong suppressive effect on myofibrillar, mitochondrial, and cytoplasmic MPS, suggesting reduced protein accretion contributed to lower gastrocnemius mass in ER-fed rats. Lower turnover rates of most muscle proteins in HP-fed rats without reductions in protein pool size (i.e., tissue mass) suggests prolonged HP intake, independent of energy, may prolong muscle protein lifespan of in obese Zucker rats. Funding Sources Supported by USAMRDC; authors’ views not official U.S. Army or DoD policy.

N-3 Fatty acids preserve muscle mass and insulin sensitivity in a rat model of energy restriction.

In obese subjects, the loss of fat mass during energy restriction is often accompanied by a loss of muscle mass. The hypothesis that n-3 PUFA, which modulate protein homoeostasis via effects on insulin sensitivity, could contribute to maintain muscle mass during energy restriction was tested in rats fed a high-fat diet (4 weeks) rich in 18 : 1 n-9 (oleic acid, OLE-R), 18 : 3 n-3 (α-linolenic acid, ALA-R) or n-3 long-chain (LC-R) fatty acid and then energy restricted (8 weeks). A control group (OLE-ad libitum (AL)) was maintained with AL diet throughout the study. Rats were killed 10 min after an i.v. insulin injection. All energy-restricted rats lost weight and fat mass, but only the OLE-R group showed a significant muscle loss. The Gastrocnemius muscle was enriched with ALA in the ALA-R group and with LC-PUFA in the ALA-R and LC-R groups. The proteolytic ubiquitin-proteasome system was differentially affected by energy restriction, with MAFbx and muscle ring finger-1 mRNA levels being decreased in the LC-R group (-30 and -20 %, respectively). RAC-α serine/threonine-protein kinase and insulin receptor substrate 1 phosphorylation levels increased in the LC-R group (+70 %), together with insulin receptor mRNA (+50 %). The ALA-R group showed the same overall activation pattern as the LC-R group, although to a lesser extent. In conclusion, dietary n-3 PUFA prevent the loss of muscle mass associated with energy restriction, probably by an improvement in the insulin-signalling pathway activation, in relation to enrichment of plasma membranes in n-3 LC-PUFA.

Metabolic programming of sirtuin 1 (SIRT1) expression by moderate energy restriction during gestation in rats may be related to obesity susceptibility in later life

In rats, 20% gestational energy restriction programmes offspring for higher food intake, which in adulthood results in higher body weight in males but not in females. Here, we aimed to assess whether the effects of moderate energy restriction during gestation and the sex-related outcomes on adult body weight may be related to the metabolic programming of sirtuin expression in different tissues. For this purpose, 25-d-old offspring of control and 20% energy-restricted (ER) rats (from days 1-12 of pregnancy) were studied. Body weight and the weight of white adipose tissue (WAT) depots and liver were recorded and mRNA expression of sirtuin 1 (SIRT1) and selected genes in the WAT, liver, muscle and hypothalamus were analysed. No differences were found in body weight or the weight of WAT and liver between the control and ER animals. A similar pattern of SIRT1 mRNA expression was found in the WAT, liver and skeletal muscle of ER animals, but in a sex-dependent manner: ER males showed lower SIRT1 mRNA levels than the controls, while no differences were found in females. A sex-different pattern was also observed in the hypothalamus. ER males, but not females, also showed lower mRNA levels of adipose TAG lipase (ATGL) and uncoupling protein 2 in WAT and of sterol response element binding protein 1c and stearoyl-CoA desaturase-1 in the liver. Both sexes of ER animals showed lower mRNA levels of 5' adenosine monophosphate-activated protein kinase and ATGL in the liver. In conclusion, moderate maternal energy restriction during gestation programmes a particular, sex-dependent gene expression profile of SIRT1 in different peripheral tissues, which may be related to obesity predisposition in adulthood; therefore SIRT1 expression emerges as a potential early biomarker of obesity susceptibility.

The Nature of the Ingested Protein Has No Effect on Lean Body Mass During Energy Restriction in Overweight Rats

Severe energy restriction in obesity not only leads to fat mass loss but also to lean mass loss. The aim of this study was to compare the capacity of casein, a slowly digested protein, and milk soluble proteins (MSP; rapidly digested) to limit the loss of lean mass induced by energy restriction. Obesity was first induced in male Wistar rats by a 5-week feeding with a high-fat high-sucrose diet. The impact of energy restriction was then studied with high-protein (32%) diets containing either casein, MSP, or a 50/50 mixture of both proteins for 3 weeks (n = 10 per group). Food intake, body weight, nitrogen balance, creatinine, and 3-methyl-histidine excretion were measured during energy restriction. Then, tissue weights, plasma metabolic parameters (amino acids, glucose, insulin, cholesterol, triglycerides), and in vivo liver and extensor digitorum longus (EDL) muscle protein synthesis rates were measured in postabsorptive state at the end of the experimental period. Although significant differences relevant to protein metabolism were observed between groups (protein intake, plasma amino acid concentrations, fecal nitrogen excretion, muscle protein synthesis rates), week per week, there were no significant differences in nitrogen balance whatever the protein used. In conclusion, our results show that in young overweight energy restricted rats, using a high-protein diet, the nature of protein intake has no influence on body protein retention.

Low dose parathyroid hormone maintains normal bone formation in adult male rats during rapid weight loss

A persistent negative energy balance results in bone loss. It is not clear whether the bone loss associated with chronic negative energy balance can be prevented. The objective of this study was to assess the efficacy of intermittent low dose parathyroid hormone (PTH) treatment in maintaining normal bone formation during severe energy restriction. Six-month-old male Fisher 344 rats were divided into 4 treatment groups: (1) baseline, (2) ad libitum (ad lib)-fed control, (3) energy-restricted (to consume 40% ad lib caloric intake), or (4) energy-restricted+low dose (1μg/kg/d) PTH. Severe energy restriction for 14d decreased body weight and serum leptin levels. Compared to ad lib-fed controls, energy-restricted rats had lower cancellous bone formation, higher osteoclast perimeter/bone perimeter and higher bone marrow adiposity in the proximal tibial metaphysis. Also, the energy-restricted rats had a lower periosteal bone formation rate at the tibia–fibula synostosis. Administration of PTH to energy-restricted rats had no effect on weight loss or osteoclast perimeter/bone perimeter. In contrast, energy-restricted rats treated with PTH had higher rates of cancellous and cortical bone formation compared to energy-restricted rats, and did not differ from the ad lib-fed control animals. Furthermore, PTH treatment maintained normal bone marrow adiposity. In conclusion, rapid weight loss in adult male rats was accompanied by decreased bone formation and increased bone marrow adiposity and these changes were prevented by low dose PTH treatment. Taken together, the results suggest that the energy cost of bone formation in adult rats is low and PTH therapy is effective in preventing the reduced bone formation associated with rapid weight loss.

Longitudinal Adaptations to Very Low–carbohydrate Weight‐reduction Diet in Obese Rats: Body Composition and Glucose Tolerance

Longitudinal effects of a very low-carbohydrate (VLC) and a calorie-matched high-carbohydrate (HC) weight reduction diet were compared in dietary obese Sprague-Dawley rats exhibiting impaired glucose tolerance and insulin resistance. Obese rats were divided into weight-matched groups: (i) VLC rats consumed an energy-restricted 5% carbohydrate, 60% fat diet for 8 weeks, (ii) HC rats consumed an isocaloric 60% carbohydrate, 15% fat diet, and (iii) HF rats consumed a high-fat diet ad libitum. HC and VLC rats showed similar reductions in body fat and hepatic lipid at the midpoint of the weight-reduction program, indicating effects due to energy deficit. At the end point, however, HC rats showed greater reductions in total and percent body fat, hepatic lipid and intramuscular lipid than did VLC rats, suggesting that diet composition induced changes in the relative efficiencies of the HC and VLC diets over time. HC rats showed marked improvement in glucose tolerance at the midpoint and end point, whereas VLC rats showed no improvement. Impaired glucose tolerance in VLC rats at the end point was due to insulin resistance and an attenuated insulin secretory response. Glucose tolerance in energy-restricted rats correlated negatively with hepatic and intramuscular lipid levels, but not visceral or total fat mass. These findings demonstrate that adaptations to diet composition eventually enabled HC rats to lose more body fat than VLC rats even though energy intakes were equal, and suggest that the elevated levels of hepatic and intramuscular lipid associated with VLC diets might predispose to insulin resistance and impaired glucose tolerance despite weight loss.

Energy restriction with high‐protein diets decreases visceral fat mass but not fasting and postprandial inflammation in overweight insulin‐resistant rats

Visceral obesity may worsen the low‐grade postprandial inflammatory response to a high‐fat meal. To test this hypothesis, we studied the effects of energy restriction in rats with diet‐induced overweight. Overweight rats were given free‐access to a high‐fat diet (HF), or restricted to 60% of the spontaneous intake of HF rats, with either the same diet (HFR) or protein‐rich diets (HPR) for 5 weeks. Before and at the end of this period, rats were administered a high‐fat meal and plasma insulin, triglycerides and cytokines were monitored after meal. Rats were killed at the end of the experiment and the epididymal fat pads were weighed.At the end of the experiment, the weights of the restricted rat were similar between HFR and HPR groups and 19%‐lower than in HF rats. However, the epididymal fat masses were slightly but significantly lower in HPR rats than in HFR and HF rats (14.5±0.6, 17.4±1.2 and 24.0±2.3 g, respectively). Fasting insulin did not change in restricted rats, while almost doubling in HF rats. At the end of the experiment, fasting and postprandial increases in IL‐1β, IL‐6, MCP‐1 and PAI‐1 did not differ between groups. Altogether, these results suggest that decreasing visceral adiposity in insulin‐resistant overweight rats does not impact fasting and postprandial low‐grade inflammation.

High protein and branched‐chain amino acid diet alleviates intestinal tract and kidney atrophy during energy restriction in rats

The effect of consuming a high protein or a high branched-chain amino acid (BCAA) diet during energy restriction on body composition was studied in rats. Rats were given a control diet (C, AIN-93G,...

Energy restriction in pregnant and lactating rats lowers bone mass of their progeny

Bone growth and metabolism are studied in 2 groups of 14-day-old rats, a control group and an energy-restricted group whose mothers are submitted to energy restriction (24-hour fasted versus 24-hour standard diet intake) from the 14th day of pregnancy until day 14 after delivery. We obtained a decrease in body weight (−46%) and in plasma FT 4 (−47%) and FT 3 (−15%) levels. Energy restriction in rats altered bone mineral composition in their pups, especially calcium and phosphorus levels of bone, which decreased by 33% and 19%, respectively. Calcium levels increased by 15% in plasma and decreased by 64% in urine. In contrast, phosphorus levels decreased by 23% in plasma and increased by 61% in urine. These results suggested that fasting accelerated bone resorption activity, where plasma acid phosphatase (ACP) and plasma alkaline phosphatase are increased by 41% and 21%, respectively. We found a negative correlation between FT 3 and ACP ( r = −0.750, P < .003) and between FT 4 and ACP ( r = −.905, P < .001). In conclusion, energy restriction in rats during late pregnancy and early postnatal periods seems to engender disorders in bone growth and metabolism of their offspring.

A protein-free diet alters small intestinal architecture and digestive enzyme activities in rats

The consequences of feeding a protein-free (PF) diet, as compared to casein, on gut characteristics were studied in slightly energy-restricted rats fed similar amounts of feed for 10 d. The weight and pH of fresh digesta in the stomach were lower (P = 0.045 and P = 0.016). However, the weight of fresh digesta in the other segments and gut tissue weight were not significantly affected by the diet (P > 0.05). Small intestinal crypt depth, width and area were reduced by 13, 23 and 37%, respectively (P = 0.011, P = 0.004 and P = 0.001), and villus width tended to be smaller (P = 0.057), with the PF diet. Villus height to crypt depth ratio was also lower with the PF diet in the duodenum and ileum, respectively (P < 0.05). Finally, the specific activities of alkaline phosphatase and aminopeptidase N were reduced by 36 to 38% at different sites of the small intestine in the rats fed the PF diet (P < 0.05). In conclusion, chronic consumption of a protein-free diet altered the intestinal villus-crypt architecture and enzyme activities in rats.

Energy restriction prevents the development of type 2 diabetes in Zucker diabetic fatty rats: coordinated patterns of gene expression for energy metabolism in insulin-sensitive tissues and pancreatic islets determined by oligonucleotide microarray analysis

Energy restriction (ER) causes metabolic improvement in the prediabetic and diabetic state. Little information exists on the mechanism of action of ER, for example, on the changes at the transcriptional gene level in insulin-sensitive tissues. To gain further insight, we have investigated changes in gene expressions in skeletal muscle, liver, fat, and pancreatic islets after ER in male Zucker diabetic fatty rats. Eighteen Zucker diabetic fatty rats were divided at the age of 7 weeks into a control group (ad libitum diet) and an ER group (30% ER compared with the control group). Blood glucose, weight, and food intake were measured weekly. After 5 weeks, blood samples, and skeletal muscle, liver, visceral fat (epididymal fat pads), and islets tissue were collected. Gene expression was quantified with high-density oligonucleotide, microarray GeneChip technology. ER ameliorated the development of hyperglycemia, increased the levels of plasma insulin, and reduced plasma total cholesterol and the glucagon-insulin ratio ( P < .05). In skeletal muscle, the expression of 55 genes increased and 245 decreased involving genes related to glucose metabolism (eg, phosphorylase kinase, pyruvate dehydrogenase kinase 4), lipid metabolism (eg, carnitine palmitoyltransferase 1, fatty acid transporter), and signaling pathways (eg, mitogen-activated protein kinases, protein kinase C). In the liver, the expression of 123 genes increased and 103 decreased involving genes related primarily to lipid metabolism. In pancreatic islets, the expression of 110 genes increased and that of 127 decreased, whereas in visceral fat, the expression of 279 genes increased and that of 528 decreased. ER counteracts the development of diabetes and causes changes in the expression of multiple genes involved in glucose and lipid metabolism in skeletal muscle, liver, and pancreatic islets, which may play an important role for the prevention of diabetes.

Exercise Restores Immune Cell Function in Energy-Restricted Rats

To evaluate the effect of chronic moderate-intensity exercise upon the alterations of immune system cell function induced by energy restriction. Forty male Wistar rats were randomly assigned to the following groups: sedentary animals fed ad libitum (SF, N = 10) or submitted to energy restriction (SER, N = 10, receiving 50% of the mean amount of chow consumed by SF); and trained animals fed ad libitum (TF, N = 10) or submitted to energy restriction (TER, N = 10), who exercised on a treadmill (at 60-65%VO(2max) 5 d.wk(-1) for 10 wk(-1), after 30 d under the restriction protocol. The incorporation of [2-(14)C]-thymidine by lymphocytes obtained from the spleen and mesenteric lymph nodes, plasma glucose and glutamine concentration, and cytokine production by cells cultivated in the presence of glutamine were measured in all groups, 24 h after the last exercise session. Two-way ANOVA and Tukey's posttest were employed for the statistical analysis. Training induced an increase in the proliferative response and in the production of gamma-interferon and interleukin-1 (P < 0.05) in cells from the spleen and lymph nodes of SER, in which these parameters were diminished when compared with SF (P < 0.05). SER spleen and lymph node cells produced more TNF (26 and 42%, respectively) and IL-2 (49 and 42%, respectively) than SF. The Th1-like diversion of the immune response observed in SER persisted after training. Partial recovery of the decreased SER plasma glutamine concentration and muscle glutamine synthase mRNA was observed. Training induced the recovery of the proliferative capacity of lymphocytes from SER, probably due to the partial restoration of plasma glutamine levels, but did not interfere with the diversion towards a Th1-type immune response induced by food restriction.

Increasing the Protein Content in a Carbohydrate-Free Diet Enhances Fat Loss during 35% but Not 75% Energy Restriction in Rats

The purpose of the present study was to test the influence of the amount of protein in a carbohydrate-free diet during a weight reducing program using severe (75%) or more moderate (35%) energy restriction in rats. In Expt. 1, 3 groups (n = 6) consumed ad libitum a high-carbohydrate, low-fat diet [P21C69L10 containing 21% of energy as protein (P21), 69% carbohydrate (C69) and 10% lipids (L10)], a high-carbohydrate, high-fat diet (P21C34L45), or a carbohydrate-free, high-fat, high-protein diet (P55L45). In Expt. 2, 7 groups (n = 7) were studied. For 20 d, groups 1-4 consumed ad libitum diets containing macronutrients at the proportions indicated in their designations [P14C56L30 (control diet), P30L70, P50L50, and P90L10]. Groups 5-7 were pair-fed the same diets at the level of the spontaneous intake of the P90L10 group on the previous day (35% energy restriction). In Expt. 3, 5 groups (n = 7) were fed 1 of the following diets for 20 d. Group 1 consumed the control diet (P14C56L30) ad libitum. Groups 2-5 were energy restricted to 25% of the daily energy intake of group 1 with diets varying in their protein and lipid concentrations (P14C56L30, P50L50, P70L30, and P90L10). A high-fat content in the diet devoid of carbohydrate did not increase energy intake and body adiposity and neither body weight nor body composition was significantly affected by the protein to lipid ratio when energy restriction was 75%; however, a protein content > 50% preserved lean body mass at the expense of fat mass when energy restriction was 35%. Our results show that the absence of carbohydrates from the diet induces a low energy intake and the preferential deposition of protein.

Effect of dietary energy restriction on vascular density during mammary carcinogenesis.

Inhibition of mammary carcinogenesis by dietary energy restriction is associated with a decrease in cell proliferation and the induction of apoptosis. Although changes in the metabolism of insulin-like growth factor I and glucocorticoids have been proposed to modulate these cellular processes, limitations in blood supply could induce similar effects. To investigate this possibility, female Sprague Dawley rats were given an injection of 1-methyl-1-nitrosourea and fed purified diets ad libitum or at 60% ad libitum intake, i.e., 40% dietary energy restriction. Premalignant mammary pathologies and mammary adenocarcinomas obtained from these rats were processed for vascular density analysis via CD-31 immunostaining. Vascular density, measured as vessels/unit area, of premalignant mammary pathologies and adenocarcinomas from dietary energy restriction rats was reduced 31 and 39%, respectively (P < 0.01). This effect, which was observed in a 50-microm wide band of tissue surrounding each pathology, was exerted on blood vessels > 25 microm2. Conversely, intratumoral vascular density was unaffected by dietary energy restriction. cDNA microarray and Western blot analyses of adenocarcinomas for evidence of dietary energy restriction-mediated effects on vascularization revealed that only the level of vascular endothelial growth factor receptor protein Flk-1 was significantly reduced (P < 0.001). It appears that dietary energy restriction imposes limitations in the supply of blood to developing pathologies, an effect that could directly inhibit the carcinogenic process. The vascular density data imply that dietary energy restriction inhibited the growth of endothelial cells but leave unresolved the question of whether dietary energy restriction had a specific effect on angiogenesis. The factors that account for the failure of dietary energy restriction to limit intratumoral vascularization are not obvious and merit additional investigation.

Energy restriction restores the impaired immune response in overweight (cafeteria) rats

Impaired immune function linked to obesity has been shown in both human and animal studies. The purpose of this work was to evaluate the effects of a 4-week energy restriction (50% of total energy intake) on immune function in previously diet-induced (cafeteria) overweight rats. Flow cytometric analysis revealed that the number of spleen T helper cells were significantly ( P < 0.05) elevated in control and overweight energy-restricted rats as compared with groups fed ad libitum groups. The proliferative response of splenocytes to phytohaemaglutinin and concanavalin A from overweight rats after energy restriction was significantly ( P < 0.05) higher compared to overweight nonrestricted rats. The cytotoxic activity of natural killer cells tended to be lower in overweight rats compared to controls. Finally, control rats under the dietary deprivation period presented higher levels of uncoupling protein 2 mRNA and lower levels of leptin receptor mRNA compared with the reference control group. These results suggest that energy restriction is able to restore, at least in part, the impaired immune response commonly observed in overweight animals.

Dietary repletion can replenish reduced T cell subset numbers and lymphoid organ weight in zinc-deficient and energy-restricted rats.

The objective of the present study was to investigate the time course for recovery of lymphoid tissue and T cell subset numbers when Zn-deficient (ZD) or energy-restricted (ER) rats were repleted with control diet; in a second experiment, the link between the stress axis and lymphoid organs was explored. During the deficiency phase, rats were fed a ZD (<1 mg Zn/kg) or control diet (30 mg Zn/kg, nutritionally complete) either as pair-fed controls (ER) or ad libitum-fed controls (CTL) for 3 weeks. During the repletion phase, all rats were fed control diet ad libitum for 3, 7 or 23 d. After the deficiency phase, ZD and ER had lower T cell subset numbers in the thymus compared with CTL, and ZD had reduced T cell subset numbers in the spleen compared with both ER and CTL. T cell subset numbers and lymphoid organ weights recovered from dietary Zn deficiency and energy restriction by 7 d of repletion (except 23 d for thymus weight in ZD), while body weight required more than 23 d for recovery. At the end of the deficiency phase, ZD and ER had higher circulating corticosterone concentrations compared with CTL; plasma TNFalpha was not detectable and there were no differences in plasma haptoglobin, an acute-phase protein. In conclusion, Zn deficiency and energy restriction elevated circulating corticosterone and reduced T cell subset numbers in the thymus and spleen of growing rats. Repletion with a nutritionally complete diet allowed recovery of T cell subset numbers and lymphoid organ weight.

Adrenalectomy Does Not Block the Inhibition of Mammary Carcinogenesis by Dietary Energy Restriction in Rats

Dietary energy restriction (DER) has been shown to reproducibly inhibit chemically induced mammary carcinogenesis. The inhibitory activity of DER has been reported to be associated with an increase in circulating corticosterone as well as a decrease in insulin-like growth factor 1 (IGF-1). To determine whether the adrenal glands are required for cancer inhibitory activity, the effects of DER were investigated in adrenalectomized (ADX) rats. Female Sprague-Dawley rats, 29-31 per group, were injected with 0.05 g 1-methyl-1-nitrosourea/kg body wt at 21 d of age, sham operated (SHAM) or bilaterally ADX at 24 d of age, and after 3 d adapted to meal feeding during which rats ate ad libitum (AL) or were restricted to 60% of AL energy intake. ADX resulted in a marked reduction in serum corticosterone in both AL and DER rats. Whereas the carcinogenic response in the mammary gland was not statistically different in SHAM-AL and ADX-AL rats, ADX did not block the cancer inhibitory activity of DER. In fact, cancer inhibitory activity was greatest in ADX-DER rats. Circulating levels of glucose, insulin, IGF-1, and IGF binding protein 3 also were reduced in DER rats. Collectively, these findings indicate that adrenal glands are not required for manifestation of the cancer inhibitory activity of DER. If circulation-borne factors such as corticosterone or IGF-1 are involved in the inhibition of mammary carcinogenesis by DER, IGF-1 is likely to play a greater role than corticosterone.

Dietary protein deprivation decreases the serine phosphorylation of insulin receptor substrate-1 in rat skeletal muscle.

Evidence has shown that protein malnutrition tends to increase peripheral insulin sensitivity, but the molecular mechanism underlying this increase is not yet clear. Here we show that, in rat muscle, the state of insulin receptor (IR) substrate-1 (IRS-1), a pivotal component of the signaling pathway of the IR, changes drastically according to protein supply. After rats were fed a protein-free diet (PF) or a 12% casein diet for 1 week, their IR and IRS-1 states were analyzed by immunoblotting using various antibodies. PF slightly increased the amount of IR without affecting the state of IR tyrosine phosphorylation. In contrast, PF decreased the amount of IRS-1 and markedly increased phosphorylation of IRS-1 tyrosine residues after insulin injection. Moreover, IRS-1 in PF rats exhibited faster mobility in SDS-PAGE as well as far less phosphorylation of Ser612 and Ser307, indicating hypophosphorylation on its serine residues. Results of additional experiments using energy-restricted (pair-fed) rats and streptozotocin-induced diabetic rats suggest that dietary protein deficiency by itself alters serine phosphorylation of IRS-1, while the up-regulation of tyrosine phosphorylation requires other factors, such as a reduction in basal plasma insulin. The serine dephosphorylation followed by up-regulation of insulin-dependent IRS-1 tyrosine phosphorylation in skeletal muscle of PF rats in vivo is similar to a phenomenon observed in cultured cells under restriction of amino acids in the medium. With these findings, it could be inferred that the reduction of serine phosphorylation contributes to the sensitization of IRS-1 to IR tyrosine kinase under protein malnutrition.

Decreased serum leptin and muscle oxidative enzyme activity with a dietary loss of intra-abdominal fat in rats

The purpose of the present study was to investigate the relationship among intra-abdominal adipose storage, adaptation in the serum leptin concentration and skeletal muscle enzyme activity after a 4-week energy restriction (ER). Thirty-one male Wistar rats were divided into 40% energy restricted ( n=24) or ad libitum-fed control (CL) rats ( n = 7). The energy-restricted rats were grouped into the most fat (MF, n=7), medium ( n 10) and the least fat (LF, n=7) by their intra-abdominal fat pads mass (epididymal, mesenteric, and perirenal) after ER. A superficial portion of M. gastrocnemius tissue obtained before and after the diet period were analyzed to determine the activities of hexokinase (HK), β-hydroxyacyl CoA dehydrogenase (β-HAD) and citrate synthase (CS). Blood samples were also collected for a serum leptin assay. At the baseline, no difference was found in either the leptin concentration or the enzyme activities among LF, MF and CL. The serum leptin concentration was positively correlated with the muscle activities of β-HAD and CS, while it negatively correlated with HK/β-HAD. After ER, the activities of HK, β-HAD and CS were all significantly lower in LF than in CL. Among the energy-restricted rats, the intra-abdominal fat pad weight, leptin concentration and the activities of β-HAD, CS, β-HAD/CS all significantly correlated with one another. The changes in leptin and the activity of β-HAD were also positively correlated. These findings indicate that parallel decreases in the serum leptin and skeletal muscle enzyme activities with the energy restriction-induced intra-abdominal adipose reduction, thus may suggest the leptin to have a regulative effect on the muscle enzyme activity during ER.

Energy Restriction Reduces Fractional Calcium Absorption in Mature Obese and Lean Rats

Weight loss is associated with bone loss and the risk may be greater in lean than heavier individuals, but the mechanisms involved remain unclear. We hypothesized that energy restriction (EnR) would decrease true fractional Ca absorption (TFCA) and be mediated by Ca-regulating hormones, but differently in obese and lean rats. Rats were fed a high fat (47% energy) or low fat (16% energy) diet for 4 mo. At 6 mo of age, the resulting lean [284 +/- 28g (mean +/- SD, n = 18)] and obese (319 +/- 34g, n = 20) groups (P < 0.005) were divided into controls (CTL, ad libitum) and energy-restricted (40% restriction) groups. At baseline, bone resorption (urinary crosslinks) was higher and bone formation (serum osteocalcin) was lower in obese than in lean rats, whereas Ca balance components and Ca-regulating hormones did not differ. EnR for 10 wk reduced body weight by 25 +/- 7% compared with a 6 +/- 6% gain in CTL rats (P < 0.001). For both lean and obese rats, TFCA (5-d measurement, (45)Ca radioisotope) decreased from 30 +/- 9% to 24 +/- 9% with EnR, compared with 25 +/- 10% to 29 +/- 11% in controls (P < 0.05). Weight loss was directly correlated with the decrease in TFCA (r = 0.34, P < 0.05). Uterine weights indicated a reduced estrogenic activity in energy-restricted rats (P < 0.0001). In lean, but not obese rats, serum estradiol (E(2)) correlated with weight loss (r = 0.52, P < 0.05), and tended to correlate with the decrease in TFCA (r = 0.48, P = 0.06). At the end of the study, serum 25-hydroxyvitamin-D was lower and urinary Ca was higher in lean than obese energy-restricted rats. Distinct endocrine profiles during weight loss in obese and lean rats suggest that the susceptibility of bone and Ca metabolism to EnR could differ depending on initial body weight.