Effect of corticosterone and protein malnutrition on muscle protein breakdown in vivo in rats as measured by the urinary excretion of 3-methylhistidine

The role of corticosterone in regulating the rate of muscle protein breakdown was evaluated by measuring the urinary excretion of 3-methylhistidine (3-Mehis) during the administration of 0.0 (vehicle), 0.8 (physiological dose) and 10 (pharmacological dose) mg of the glucocorticoid/100 g body weight/day to adrenalectomized rats (AdX, AdX 0.8 and AdX 10 respectively). A fourth group of intact rats receiving only vehicle (In) was included as control. Rats were fed on either adequate protein and energy (Co) or low-protein (1-P) diets, for eight consecutive days. No differences were found between AdX and AdX 0.8 groups as compared to the In group in regard to body and liver weights. The AdX 10 group exhibited a significant reduction in body weight and a considerable increase in liver weight; these results were found in rats fed on the Co and 1-P diets, although rats on the 1-P diet showed a proportional decrease in those parameters as compared to the rats fed on the Co diet. Gastrocnemius, tibialis and E.D.L. muscle weights were significantly reduced in AdX 10 group, approximatley at the same extent in the two dietary groups. Soleus muscle weight increased in the AdX 10 group, at the same extent in the two dietary groups, as compared to the In group. Plasma corticosterone levels were significantly greater in the AdX 10 group in both dietary treatments, though restriction of protein in the diet induced a higher plasma hormone level than that of the Co group. Urea-N and creatinine outputs were significantly higher in the AdX 10 group. 3-Mehis excretion underwent an immediate and significant rise in the AdX 10 group, although rats fed on 1-P diet showed a more persistent rise than those fed on the Co diet. No differences were found among the other groups. It is concluded that high plasma corticosterone levels can accelerate muscle protein breakdown and that this action is not seriously affected by the protein content of the diet.     

Effect of a Low-Protein Diet Supplemented with Ketoacids on Skeletal Muscle Atrophy and Autophagy in Rats with Type 2 Diabetic Nephropathy

A low-protein diet supplemented with ketoacids maintains nutritional status in patients with diabetic nephropathy. The activation of autophagy has been shown in the skeletal muscle of diabetic and uremic rats. This study aimed to determine whether a low-protein diet supplemented with ketoacids improves muscle atrophy and decreases the increased autophagy observed in rats with type 2 diabetic nephropathy. In this study, 24-week-old Goto-Kakizaki male rats were randomly divided into groups that received either a normal protein diet (NPD group), a low-protein diet (LPD group) or a low-protein diet supplemented with ketoacids (LPD+KA group) for 24 weeks. Age- and weight-matched Wistar rats served as control animals and received a normal protein diet (control group). We found that protein restriction attenuated proteinuria and decreased blood urea nitrogen and serum creatinine levels. Compared with the NPD and LPD groups, the LPD+KA group showed a delay in body weight loss, an attenuation in soleus muscle mass loss and a decrease of the mean cross-sectional area of soleus muscle fibers. The mRNA and protein expression of autophagy-related genes, such as Beclin-1, LC3B, Bnip3, p62 and Cathepsin L, were increased in the soleus muscle of GK rats fed with NPD compared to Wistar rats. Importantly, LPD resulted in a slight reduction in the expression of autophagy-related genes; however, these differences were not statistically significant. In addition, LPD+KA abolished the upregulation of autophagy-related gene expression. Furthermore, the activation of autophagy in the NPD and LPD groups was confirmed by the appearance of autophagosomes or autolysosomes using electron microscopy, when compared with the Control and LPD+KA groups. Our results showed that LPD+KA abolished the activation of autophagy in skeletal muscle and decreased muscle loss in rats with type 2 diabetic nephropathy.     

Postnatal protein deprivation in rhesus monkeys

We studied the growth of 97 rhesus monkeys (M. mulatta) that from birth until 120 days of age were fed a diet containing 13.4%, 6.7%, 3.35% protein or a commercial simulated human-milk formula (SMA) affording 9% protein. After 120 days, all animals were fed SMA. Females fed the diet lowest in protein, but not the other diets, were moderately affected, gaining less weight than their well-fed counterparts did, but they recovered their deficit during the repletion period, so that at 240 days no group differences remained. Males fed the two lowest-protein diets were severely and profoundly affected, in keeping with the depth of their protein deficiency. Moreover, unlike the females, they recovered none of their weight deficit during the repletion period. Food intake during deprivation was lower in the animals eating the low-protein diets. During SMA repletion, intakes followed no simple rule, but they did not converge. Except for the males fed the 3.35% protein diet, relative food intake (ml formula/kg body weight) did not differ substantially between diets or sexes at any time during the deprivation and diminished as the animals got older. Those males' relative intakes did not decline. During repletion, the SMA females ate the most in proportion to weight and the 13.4% group the least. The lowest-protein males, in contrast, ate the most in proportion to their weight during both deprivation and repletion. Males fed the lowest-protein diet gained little for what they ate; those fed the highest-protein diet gained much. Females were more efficient than males were when eating the low-protein diet and less efficient when eating the high-protein diet. When fed SMA during repletion, males' food efficiencies (grams gained/liter of diet) were nearly equal; females still differed: the SMA group was the least efficient and the low-protein group most efficient. Its counterpart, protein efficiency, was greatest (during deprivation) for females eating the low-protein diet and least (among females) for those eating the high-protein diet. Males were least protein efficient if eating the low-protein diet. Evidently, a 4 month bout of protein deprivation had prolonged effects on the amount of food the animals needed to produce a given gain in weight.     

Muscle hypertrophy in rats fed on a buckwheat protein extract.

Growing rats were examined for the influence of a buckwheat protein diet on muscle weight and protein. In experiment 1, the rats were fed on a diet containing either casein or a buckwheat protein extract (BWPE) as the protein source (10%, 20% or 30%) for 5 wk. The relative weights (g per kg of body wt) of the gastrocnemius, plantaris and soleus muscles were higher in the BWPE-fed animals than in the casein-fed ones, but were unaffected by the dietary level of protein. These differences were not associated with growth. In experiment 2, the rats were fed on either a casein or BWPE diet at the 20% protein level for 5 wk. BWPE intake significantly elevated the gastrocnemius muscle weight, carcass protein and water, and reduced carcass fat. These results demonstrate that BWPE consumption causes muscle hypertrophy, elevates carcass protein and water, and reduces body fat.     

Selenium Supplementation Modulates Zinc Levels and Antioxidant Values in Blood and Tissues of Diabetic Rats Fed Zinc-Deficient Diet

Diabetes mellitus is associated to a reduction of antioxidant defenses that leads to oxidative stress and complications in diabetic individuals. The present study was undertaken to investigate the effect of selenium on blood biochemical parameters, antioxidant enzyme activities, and tissue zinc levels in alloxan-induced diabetic rats fed a zinc-deficient diet. The rats were divided into two groups; the first group was fed a zinc-sufficient diet, while the second group was fed a zinc-deficient diet. Half of each group was treated orally with 0.5 mg/kg sodium selenite. Tissue and blood samples were taken from all animals after 28 days of treatment. At the end of the experiment, the body weight gain and food intake of the zinc-deficient diabetic animals were lower than that of zinc-adequate diabetic animals. Inadequate dietary zinc intake increased glucose, lipids, triglycerides, urea, and liver lipid peroxidation levels. In contrast, serum protein, reduced glutathione, plasma zinc and tissue levels were decreased. A zinc-deficient diet led also to an increase in serum glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and liver glutathione-S-transferase and to a decrease in serum alkaline phosphatase activity and glutathione peroxidase. Selenium treatment ameliorated all the values approximately to their normal levels. In conclusion, selenium supplementation presumably acting as an antioxidant led to an improvement of insulin activity, significantly reducing the severity of zinc deficiency in diabetes.     

Effect of a selectiveβ 2-adrenergic agonist (Cenbuterol) on energy balance and body composition in normal and protein deficient rats

In rats fed a normal (22% protein) diet, injection of clenbuterol (1 mg/kg/d for 21 d) did not affect energy intake, energy expenditure or weight gain, but reduced energetic efficiency, and fat and energy gains and increased body protein content. Presenting a low-protein (8%) diet reduced energy intake, gain and efficiency, body protein content and the mass of the gastrocnemius muscle when compared to rats fed the control diet. Injection of the protein-deficient rats with clenbuterol (1 mg/kg/d for 21 d) caused hypophagia and reduced body weight and energy gains, energy expenditure and total body fat. However, the total body content of protein was not significantly reduced and the percentage of body protein in this protein deficient, clenbuterol-treated group was greater than that of untreated rats on both the high- and low-protein diets. The ratio of body protein to fat following clenbuterol treatment was increased by over 50% in both normal and protein-deficient rats. The results show that in protein deficient animals, clenbuterol treatment may help conserve body protein at the expense of fat, resulting in a smaller, but leaner body mass.     

Vitamin D Supplementation Modulates Blood and Tissue Zinc, Liver Glutathione and Blood Biochemical Parameters in Diabetic Rats on a Zinc-Deficient Diet

Previous studies suggest a protective effect of vitamin D3 on zinc deficiency-induced insulin secretion and on pancreas β-cell function. The aim of this study was to investigate the effect of vitamin D on blood biochemical parameters, tissue zinc and liver glutathione in diabetic rats fed a zinc-deficient diet. For that purpose, Alloxan-induced diabetic rats were divided into four groups. The first group was fed a zinc-sufficient diet while the second group was fed a zinc-deficient diet. The third and fourth groups received zinc-sufficient or zinc-deficient diets plus oral vitamin D3 for 27 days. At the end of the experiment, blood, femur, pancreas, kidney and liver samples were taken from all rats. The serum, femur, pancreas, kidney and liver zinc concentrations, liver glutathione, serum alkaline phosphatase activity, daily body weight gain and food intake were lower in the zinc-deficient rats in comparison with those receiving adequate amounts of zinc. These values were increased in the zinc-deficient group that was supplemented with vitamin D3. The serum total cholesterol, triglycerides, total protein, urea, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and blood glucose values were higher in rats fed a zinc adequate diet, but their concentrations were decreased by vitamin D3 supplementation. The serum total protein levels were not changed by zinc deficiency and vitamin D3 supplementation. These results suggest that vitamin D3 modulates tissue zinc, liver glutathione and blood biochemical values in diabetic rats fed a zinc-deficient diet.     

Depletion of delta 9 desaturase (EC 1.14.99.5) activity in lactating rat during protein restriction

The effects of protein restriction on the activity of delta9 desaturase (EC 1.14.99.5) were investigated in lactating rats. A control group was fed a balanced diet (20% casein) for 14 days, whereas the experimental groups were fed a low-protein diet (8% casein), supplemented with or without L-methionine (0.4%), for 14 days. The enzyme activity was measured by incubations of hepatic microsomal pellets with (1-14C) stearic acid. Results showed a decreased delta9 desaturase activity, after 2,7 and 14 days of depleted diet, of -50, -40 and -33% respectively, compared with control. The supplementation of the low-protein diet with 0.4% methionine, which favours food consumption as well as growth, did not improve the altered delta9 desaturase activity. Our data evidenced that delta9 desaturase activity is depleted by protein restriction during lactation, when the protein needs are high for the biosynthesis of animal tissues. This change has to be considered as a sign of depressed delta9 desaturase biosynthesis or modifications of enzymatic properties, or both.     

Activity and subcellular distribution of protein kinase C (PKC) in muscle and brain of force-fed zinc-deficient rats

The purpose of the present study was to investigate, in force-fed rats, whether alimentary zinc (Zn) deficiency affects the activity of the Zn-metalloenzyme protein kinase C (PKC). The in vivo activity of PKC was determined by measuring the subcellular distribution of the enzyme between the cytosolic and the particulate fraction in brain and muscle. For this purpose, 24 male Sprague-Dawley rats with an average live mass of 126 g were divided into 2 groups of 12 animals each. The Zn-deficient and the control rats received a semisynthetic casein diet with a Zn content of 1.2 and 24.1 ppm, respectively. All animals were fed four times daily by gastric tube in order to ensure that the depleted animals also received adequate nutrients and to synchronize the feed intake exactly. After 12 d, the depleted rats were in a state of severe Zn deficiency, as demonstrated by a 70% lower serum Zn concentration and a 66% reduction in the serum activity of alkaline phosphatase. Neither the cytosolic nor the particulate fraction of the thigh muscle showed any difference between the depleted and the control animals as regards PKC activity/g of muscle. The specific activity of PKC/mg of protein in the cytosolic fraction of the muscle was not affected by alimentary zinc deficiency, whereas the specific activity of PKC in the particulate fraction of the muscle was reduced by a significant 10% in Zn deficiency (150±12 vs 135±14 pmol P/min/mg protein). In the brain, neither the cytosolic nor the particulate fraction revealed any difference in PKC activity/g of fresh weight or in the specific activity/mg of protein between the control and the Zn-deficient rats.     

Restoration by dietary glutamine of reduced tumor necrosis factor production in a low-protein-diet-fed rat model

Tumor necrosis factor-alpha (TNF) production by peritoneal macrophages and its dietary modification were investigated by using rats fed on a low-protein diet. The rats were given a 20% casein (control) diet or a 3% casein diet for 21 days, and TNF production was measured in activated macrophages of these animals. TNF production was significantly lower in macrophages from rats fed on the low-protein diet than that in macrophages from rats fed on the control diet. Oral administration of a cabbage extract, a known modulator of TNF production, to the low-protein-diet-fed rats significantly enhanced TNF production by macrophages. Glutamine supplementation to the low-protein diet significantly enhanced TNF production as well as TNF mRNA expression. These results indicate that the 3%-casein-diet-fed rat would be useful as a model for reduced TNF production in protein malnutrition. These results also suggest that glutamine administration restored the reduced TNF production associated with protein malnutrition.     

Amino acid regulation of synthesis of ribonucleic acid and protein in the liver of rats

Weanling (23-day-old) rats were fed on either a low-protein diet (6% casein) or a diet containing an adequate amount of protein (18% casein) for 28 days. Hepatic cells from animals fed on the deficient diet were characterized by markedly lower concentrations of protein and RNA in all cellular fractions as compared with cells from control rats. The bound rRNA fraction was decreased to the greatest degree, whereas the free ribosomal concentrations were only slightly less than in control animals. A good correlation was observed between the rate of hepatic protein synthesis in vivo and the cellular protein content of the liver. Rates of protein synthesis both in vivo and in vitro were directly correlated with the hepatic concentration of individual free amino acids that are essential for protein synthesis. The decreased protein-synthetic ability of the ribosomes from the liver of protein-deprived rats was related to a decrease in the number of active ribosomes and heavy polyribosomes. The lower ribosomal content of the hepatocytes was correlated with the decreased concentration of essential free amino acids. In the protein-deprived rats, the rate of accumulation of newly synthesized cytoplasmic rRNA was markedly decreased compared with control animals. From these results it was concluded that amino acids regulate protein synthesis (1) by affecting the number of ribosomes that actively synthesize protein and (2) by inhibiting the rate of synthesis of new ribosomes. Both of these processes may involve the synthesis of proteins with a rapid rate of turnover.     

Effect of dietary protein on cholesterol homeostasis in diabetic rats

Normal and streptozotocin (STZ)-diabetic rats were studied in order to examine the effects of altering the type of dietary protein on cholesterol homeostasis. Rats were fed a non-purified or a purified diet containing either casein or soybean protein. The results obtained on the specific aspects of lipid metabolism were remarkably similar in control rats fed the non-purified (Purina Lab Chow) diet or the purified diet with the soybean protein. However, most of the findings obtained with the above two groups were different from those obtained with rats fed the purified diet containing casein. In the latter group, plasma cholesterol was elevated following a 15-day feeding period as compared to the other two dietary groups. The excess plasma cholesterol in the casein-fed group was found in two lipoprotein fractions with densities of 1.023-1.045 g/ml and 1.045-1.086 g/ml, respectively. The latter lipoprotein fraction was also enriched with apolipoprotein E. The casein-fed animals also showed a lower fractional rate of plasma cholesterol esterification and an abnormal accumulation of cholesterol in the body despite inhibition of cholesterol synthesis in the liver and in the intestines. Twelve to 15 days after the induction of diabetes, plasma cholesterol increased to a similar extent in the rats on all three diets. However, the distribution of cholesterol among the lipoprotein fractions was markedly different. The percentage of cholesterol in fractions of d less than 1.086 g/ml was increased while that carried in the fraction of d 1.086-1.161 g/ml decreased in the rats fed the nonpurified diet and the casein diet. In contrast, there was no change in the distribution of lipoprotein cholesterol between the diabetic and the control rats fed the soybean protein diet. The hepatic synthesis of cholesterol was unaltered in diabetic rats fed the nonpurified diet and the purified diet with soybean protein, but was increased 2.4-fold in diabetic rats fed casein. Intestinal cholesterol synthesis was increased in all three dietary groups. The increase was highest in the rats fed casein and lowest in rats fed soybean protein. The rate of sterol synthesis in the kidneys was not significantly affected by the diet or diabetes. In all three dietary groups diabetes led to an abnormal accumulation of cholesterol in the body. This accumulation was highest in the casein-fed rats and lowest in those fed the soybean protein diet. The cholesterol content of the kidneys was markedly increased by dietary casein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of diabetes and dietary manipulation on rat parotid gland secretory response to sympathetic nerve stimulation.

1. The effects of streptozotocin-diabetes, bulk-diet, low protein diet (8%) and protein-calorie malnutrition on parotid gland response to sympathetic nerve stimulation were studied in male Wistar rats. 2. Mean body weights were considerably less in diabetic and protein-calorie malnourished rats than in the other groups, but parotid gland weight was reduced only in animals placed on a low-protein diet. 3. Salivary flow rate (microliter/min/g tissue) and total protein output (mg secreted/g tissue) were reduced in diabetic rats. 4. Salivary composition was altered in diabetes and protein-calorie restriction, and the specific changes were unique to each condition. 5. Thus, with the possible exception of gland weight the effects of diabetes on parotid gland structure and function are not related to either hyperphagia or nutritional status.     

Dietary tyrosine supplementation enhances weight gain in streptozotocin-diabetic rats

The effect of dietary protein concentration and tyrosine supplementation on growth in streptozotocin (65 mg/kg, ip) diabetic rats was evaluated. When rats were fed diets ranging from 15 to 60% protein, weight gain and feed efficiency were greatest in rats fed the 45% protein diet. Adding tyrosine to this diet (8%, incorporated as a percentage of protein) did not promote further weight gain relative to nonsupplemented diabetic animals. In contrast, rats choosing 45% of total calories as protein by selecting from 10 and 60% protein diets supplemented with either 0, 4, or 8% tyrosine demonstrated a 35% (4% tyrosine) to 45% (8% tyrosine) increase in weight gain. Proximate analysis indicated similar body composition in tyrosine supplemented and nonsupplemented diabetic animals. Including tryptophan (1.45%) with tyrosine in the self-selection diet was without effect. Thus, tyrosine supplementation promoted a modest but consistent and specific increase in weight gained by self-selecting diabetic rats.     

The State of the Adenyl Nucleotide System in the Liver of Rats with Toxic Hepatitis under Conditions of Protein Deficiency

Adenyl nucleotide levels and activity of AMP catabolism enzymes in the cytosolic liver fraction of rats with acetaminophen-induced hepatitis have been studied under different dietary protein regimens. It was found that in animals with toxic hepatitis maintained on a diet rich in protein the ATP and ADP levels decreased, while AMP levels were similar to those in control animals. At the same time, in the cytosolic liver fraction of rats with acetaminophen-induced hepatitis kept under conditions of protein deficiency, ATP and AMP pools were depleted. Changes in the adenyl nucleotides content were accompanied by altered activity of AMP catabolism enzymes, such as 5′-nucleotidase and AMP deaminase. It was found that in rats with toxic hepatitis that were fed a complete diet, AMP deaminase activity increased in comparison to the control level along with 5′-nucleotidase stimulation. At the same time, in protein-restricted rats with toxic liver damage, AMP deaminase activity decreased, while 5′-nucleotidase activity was elevated in comparison to control values. These results indicate depletion of energy sources in the liver cells of rats with acetaminophen-induced hepatitis that were fed a low-protein diet. The observed changes in the activity of AMP catabolism enzymes may be considered as one of the mechanisms that regulate the cellular energy function.     

Dietary protein intake and skeletal-muscle protein metabolism in rats. Studies with the ammonium chloride-wash fraction from crude polyribosomes of well-nourished and protein-depleted rats

1. Crude polyribosomes from skeletal muscle of the hind leg of rats fed on a low-protein diet for 10 days are less active in cell-free protein synthesis than are polyribosomes obtained from well-nourished control rats. 2. The polyribosomes were salt-washed (0.5m-NH(4)Cl) and the wash extract was examined for its amino acid incorporating activity and for EF (elongation factor) 1 and EF 2 activities. 3. Compared with preparations from control rats, the salt-wash fraction from protein-depleted rats was less active and showed lower EF 1 and EF 2 activity. 4. The ribosomes were rendered equal in activity by salt-washing, but no inhibitor was detected in the salt wash. 5. Differences in the incorporating activity of crude polyribosomes from the diet groups persisted in the presence of saturating amounts of partially purified EF 1 and EF 2. 6. It is concluded that the lowered protein-synthetic activity of crude polyribosomes caused by restricted protein intake is not causally related to the lower activities of EF 1 and EF 2 in the polyribosome preparations. 7. The possible nature of the change in crude polyribosome activity due to low-protein feeding is discussed.     

The effects of maternal mild protein restriction on stroke incidence and blood pressure in stroke-prone spontaneously hypertensive rats (SHRSP)

The effect of maternal protein restriction during pregnancy on the offspring's blood pressure was assessed in stroke-prone spontaneously hypertensive rats (SHRSP) which are genetically predisposed to hypertension and stroke. After the confirmation of pregnancy, the control group was given a 20% casein diet, and the low-protein group was fed a 9% casein diet. After the confirmation of delivery, commercial feed was given to both of the groups. No differences were seen between the control and low-protein offspring in regard to body weight, blood pressure elevation, or life span. One percent saline solution was put in the control and low-protein groups after the age of 11 weeks. Blood pressure increased markedly in the low-protein group, on the blood pressure level in the low-protein group on week 2 after salt loading (242+/-6 mmHg) was significantly higher than that in the control group (223+/-9 mmHg; p<0.05). The survival duration was significantly shorter in the low-protein group (113+/-4 days) than in the control group (135+/-22 days; p<0.05). These results suggest that maternal protein malnutrition in SHRSP exerted a high salt sensitivity and a malignant influence on stroke incidence on offspring.     

L-Arginine improves multiple physiological parameters in mice exposed to diet-induced metabolic disturbances

L-Arginine (L-Arg) is a conditionally essential amino acid and a natural constituent of dietary proteins. Studies in obese rats and type 2 diabetic humans have indicated that dietary supplementation with L-Arg can diminish gain in white adipose tissue (WAT) and improve insulin sensitivity. However, the effects of L-Arg on glucose homeostasis, body composition and energy metabolism remain unclear. In addition, no studies have, to our knowledge, examined whether L-Arg has beneficial effects as a dietary supplement in the mouse model. In the present study, we investigated the effects of L-Arg supplementation to male C57BL/6 mice on an array of physiological parameters. L-Arg supplemented mice were maintained on a low-protein diet and body composition, appetite regulation, glucose tolerance, insulin sensitivity and energy expenditure were evaluated. A significant reduction in epididymal WAT was observed in L-Arg supplemented mice compared with mice fed an isocaloric control diet. Surprisingly, the L-Arg supplemented animals were hyperphagic corresponding to a highly significant decrease in feed efficiency, as body weight developed in a similar pattern in both experimental groups. Glucose homeostasis experiments revealed a major effect of L-Arg supplementation on glucose tolerance and insulin sensitivity, interestingly, independent of a parallel regulation in whole-body adiposity. Increased L-Arg ingestion also raised energy expenditure; however, no concurrent effect on locomotor activity, substrate metabolism or expression of uncoupling proteins (UCP1 and UCP2) in adipose tissues was displayed. In conclusion, dietary L-Arg supplementation substantially affects an array of metabolic-associated parameters including a reduction in WAT, hyperphagia, improved insulin sensitivity and increased energy expenditure in mice fed a low-protein diet.