Protein-deprived Rats Research Articles

Intestinal Permeability to Large Particles in Normal and Protein-Deficient Adult Rats

Intestinal absorption of ferritin and virus particles was assessed in control and protein-deficient rats using light and electron microscopic procedures. Rats weighing 100 to 130 grams were divided into two groups and fed either a 0.5% of 18% lactalbumin diet. At monthly intervals following diet initiation, three rats from each group were administered ferritin (MW-650,000) or adenovirus Type 5 (MW is greater than 1,000,000) via ligated jejunal loops either 15 or 30 minutes prior to loop excision and processing for electron microscopy. Morphological evaluation of jejunal tissues revealed that both control and protein-deficient rats absorbed ferritin and virus particles via pinocytosis and in both situations the exogenous particles were believed to be present in lysosmal bodies of typical absorptive cells. Neither ferritin molecules nor virus particles were identified elsewhere in the jejunal mucosa of control rats. Rats deprived of sufficient dietary protein for four months or longer, however, demonstrated exogenous tracer materials in intercellular spaces of the epithelium and lamina propria. Since these severely protein-deprived rats also demonstrated deterioration of apical intercellular junctions, the possibility exists that large particles moved directly between cells of the epithelial lining and subsequently into underlying connective tissue and vascular compartments for conveyance (as antigens) to other body regions.

Ammonia Intoxication: A Hazard during Rehabilitation of Protein-deprived Rats

The ammonia tolerance of weanling rats raised for 3 to 6 weeks on a protein-deficient diet was compared with that of a group of age-matched control animals raised from weaning on an optimal protein diet. The groups were challenged with a nitrogen load of either ammonium chloride or amino acids. The results of the loading studies indicate that severe protein deprivation increases vulnerability to ammonia poisoning. These observations suggest that ammonia-induced coma may pose a significant hazard during the initial intravenous rehabilitation of malnourished subjects, if replenishment is undertaken too vigorously.

Modulation of leucine transaminase activity by dietary means.

The specific activity of leucine transaminase was measured in supernatants of liver, skeletal muscle (gastrocnemius), and kidney homogenates obtained from fed, starved, and protein-deprived rats. After 12 h of starvation, there were slight reductions in leucine transaminase activity of both muscle and kidney tissues. When starvation was prolonged to 1 full day, the activity of this enzyme increased by approximately twofold in both muscle and kidney. Prolongation of fasting to 5 days resulted in an additional increase in specific activity of leucine transaminase in muscle. During the entire 5 days of starvation, leucine transaminase activity remained unaltered in liver of starved rats. Protein deprivation for 1 or 5 days resulted in significant reductions in specific activity of leucine transaminase in skeletal muscle. Protein deprivation did not produce a remarkable effect on the activity of this enzyme in kidney or liver tissue. The results of this study, together with those previously obtained, indicate that within our experimental conditions increased oxidation of leucine in skeletal muscle of starved rats is not initially related to an alteration in activity of leucine transaminase. When caloric deficiency is prolonged, the potential for transamination is also increased. These adaptive changes increase the ability of skeletal muscle to use leucine as a metabolic fuel.

Elevated serum growth hormone in protein-deprived rats, and decreased liver RNA after hypophysectomy.

Serum growth hormone was significantly higher in rats fed on a non-protein than on a 25% casein diet. In both dietary groups, hypophysectomy provoked a decrease in the RNA/DNA ratio of the liver, and, especially in the protein-deprived rats, a decrease in the RNA synthesis per liver cell, measured as the product of the RNA/DNA ratio, and the ratio of specific RNA activity to specific nucleotide activity, two hours after administration of [3H]orotic acid. In the protein-deprived rats, full reconstitution of these figures was obtained with a combination of growth hormone, hydrocortisone and triiodothyronine, but not quite with growth hormone alone. The possible role of growth hormone in protein deprivation is discussed.

Influence of experimental dietary conditions on hepatic enzymes of glutamic acid metabolism in rats.

The lowering levels of dietary protein induced a significant fall of some hepatic enzymes associated with glutamic acid metabolism. The changes were later normalised during dietary rehabilitation of the protein-deprived rats. The levels of these enzymes were found to be increased as compared to those observed after starvation, on feeding a carbohydrate-free, protein-rich diet or by the dietary supplementation by glutamic acid.

Changes in the activity of rat muscle AMP deaminase in relation to the proportion of dietary protein.

1. The purine nucleotide cycle has been proposed (Lowenstein, 1972) as an alternative scheme for amino acid deamination in tissues, such as skeletal muscle, having low concentrations of glutamate dehydrogenase (EC1.4.1.2).2. Activities of AMP deaminase (EC3.5.4.6), one of the enzymes of the cycle, have been measured in soleus, plantaris and extensor digitorum longus muscles of rats maintained for 18 d on diets providing 0, 0·035 or 0·10 net dietary protein energy (energy supplied by utilizable protein: total metabolizable energy, NDp:E), and in rats given the 0·10 NDp:E diet for 3 d after the 0 or 0·035 NDp:E regimens.3. Concentration of AMP deaminase in the different muscles from the control (0·10 NDp:E diet) rats appeared to bear an inverse relationship to the proportion of mitochondria-rich fibres (i.e. rich in glutamate dehydrogenase) in each muscle.4. Dietary protein deprivation (0 or 0·035 NDp:E) led to adaptive reductions in AMP-deaminase activity in the soleus and plantaris muscles, but in the extensor muscle the 0·035 NDp:E diet produced no change, while the 0 NDp:E diet caused an increase in activity.5. Refeeding the 0·10 NDp:E diet to the protein-deprived rats caused reductions of AMP-deaminase activity to lower levels in all three muscles, except in the instance of soleus in rats refed after the 0·035 NPp:E diet.6. In view of the different responses shown by the three muscles to the dietary treatments, the importance of specifying the particular muscles used in future nutritional studies is emphasized.7. The adaptive changes in AMP deaminase are discussed in terms of operation of the purine nucleotide cycle for amino acid deamination responding to the changes in amino acid catabolism known to be caused in muscle by these protein-deficient diets.

Acquired sensory preference for protein in diabetic and normal rats

Rats made diabetic by injection of streptozotocin, either after adaptation to a cafeteria or some weeks beforehand, increased their intake of protein relative to that of other macronutrients. Preference for an arbitrary odor added to the protein was increased. Normal rats were repeatedly given brief access to either protein-free or protein-containing diet while maintenance chow was withheld for 10 h. They came to prefer the odor-taste combination included in the protein diet to the flavor included in the protein-free diet. It is suggested that, not only in diabetic and protein-deprived rats, but also in normal rats not long since the end of amino acid absorption, the supply of amino acids establishes a relative conditioned attraction for associated food flavors.

The effect of a protein-free diet on the immune response of rats to sheep red blood cells. Role of the thymus

Following immunization with sheep red blood cells (either by a single i.p. injection without adjuvant (A) or by two injections at one week's interval, the first of them combined with complete Freund's adjuvant (B)) rats kept on a protein-free diet showed lower levels of serum hemagglutinins and hemolysins than control rats. The effect of protracted protein depletion on the number of spleen cells forming hemolytic plaques (PFC) or rosettes (RFC) depended on the immunization schedule: with schedule A, the number of PFC per 10 6 lymphocytes decreased markedly, whereas an increase in the number of RFC was observed in most cases. With schedule B, the proportion of PFC remained unchanged while that of the RFC increased to a much greater extent than with schedule A, due to the concomitant drop in the number of other cell types in the spleen. A comparison of the findings obtained in intact rats and in thymectomized rats indicated that involution of the thymus caused by protein deprivation was a major factor contributing to decreased serum antibody levels, in the absence of adjuvant. However, this involution contributed little to quantitative changes of the PFC and RFC brought about by the deficient diet. Apparently, therefore, the number of residual T-lymphocytes in protein-deprived rats is still sufficient to cooperate in the production of PFC and ion particular of RFC.

Plasma Insulin and Amino Acid Concentrations in Rats Given an Adequate or Low Protein Diet

Plasma insulin and free amino acid levels were measured in young male rats given inadequate amounts of dietary protein. In experiment 1, rats were fed an adequate (18% lactalbumin) or low protein (0.5% lactalbumin) diet for 5 weeks; some protein-depleted rats were then refed with the adequate diet for 1 week. After 1, 3 and 5 weeks of protein deprivation and after refeeding, groups of rats from each diet treatment were fasted overnight and force-fed a test dose of one of the experimental diets; plasma insulin, glucose and amino acid levels were measured after 30 and/or 60 minutes. Basal insulin levels rose after the oral load; after the 3- and 5-week periods, however, the increment was significantly less in protein-deprived rats than in well-nourished controls. The response of plasma amino acid levels to force-feeding suggested an impaired uptake of amino acids by muscle in rats deprived of protein for 3 or 5 weeks. In experiment 2, rats were given the experimental diets for 1 week and then groups of 10 were killed at 3-hour intervals throughout the day, beginning at 1200 hours, while they were fed their usual low protein (group A) or adequate protein (group B) diet. Another group of protein-depleted rats (group C) was fed the adequate protein diet, from 1200 hours. Plasma insulin and essential amino acid levels were lower at all times in the low protein group (group A) than in the controls (group B); they decreased further still at the beginning of the dark period and then rose gradually during the next 12 hours. In contrast, plasma amino acid concentrations (from group B) rose at the beginning of the dark period and peaked at 2400 to 0600 hours. In group C, insulin levels rose immediately after refeeding and remained high throughout the 24-hour day; amino acid levels also rose immediately after refeeding. The pattern of daily fluctuation in amino acid levels for group C was comparable to that of well-nourished rats. The results are discussed in relationship to the importance of both insulin and amino acids as factors associated with the altered rates of muscle protein synthesis during dietary protein restriction and refeeding.

Bone metabolism and composition in the protein-deprived rat.

1. Bone and calcium metabolism in protein-deprived 5-week-old male rats who exhibited no biochemical or histological features of rickets were compared with age-and weight-matched control animals. 2. Intestinal calcium absorption was significantly lower in protein-deprived rats. This was associated with a smaller exchangeable calcium pool and lower bone formation as measured by calcium accretion and proline incorporation into hydroxyproline. However, calcium resorption from bone barely changed in this group and a disproportionately high fraction of the calcium pool continued to be excreted. 3. As a consequence, retarded bone growth occurred in the protein-deprived rat, leading to bones which were shorter and lighter than those of age-matched control animals, the epiphyseal regions appearing to bear the brunt of the growth retardation. This was not accompanied by a change in quality of bone, as the percentage ash content of extirpated bone remained normal. 4. Though alternative hypotheses are possible, the data are consistent with a primary disturbance of intestinal, and possibly renal tubular, calcium transport, perhaps associated with impaired synthesis of calcium-binding protein. An independent depression of collagen synthesis is likely.

Decreased half-life of the RNA of free and membrane-bound ribosomes in the liver of protein-deprived rats.

Decreased Half-Life of the RNA of Free and Membrane-Bound Ribosomes in the Liver of Protein-Deprived Rats

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.

Albumin synthesis by the perfused rat liver. A comparison of methods with special reference to the effect of dietary protein deprivation.

1. Isolated perfused rat livers were used to study synthesis of albumin after donors had been fed on normal or protein-free diets. 2. Methods of determining the liver's ability to produce albumin included incorporation of [(14)C]carbonate, [(3)H]lysine and [(14)C]arginine, as well as a direct method based on a heterologous perfusing system of rat erythrocytes and rabbit plasma. 3. Livers from protein-deprived rats were found to form extremely little urea and not to incorporate (14)CO(2) into [(14)C]urea, but they were capable of producing [(14)C]urea from [(14)C]arginine and of incorporating the latter and [(3)H]lysine into albumin. 4. By immunological means these lives were found to synthesize less albumin than normal, but their ability was only slightly impaired when related to body weight or liver weight. 5. These findings are consistent with a block in urea-cycle enzymes with relative integrity of arginase activity and of amino acid activation.

Food preferences acquired by association with variations in amino acid nutrition.

Protein-deprived rats were given, on one day, a balanced mixture of amino acids followed by access to protein-free food having a distinctive odour. On another day, an imbalanced (histidine-free) amino acid mixture was given just before food having another odour. The rats afterwards preferred the balance-paired odour to the imbalance-paired odour. The preference was acquired whether the duration of odour presentation, or the amount of odourised food taken, was kept constant on the two conditioning days. Retention of the preference seemed unattenuated after 4 weeks. An attraction to the balance-paired odour (relative to odours paired with a water load) contributed to the acquired preference. There was also a relative aversion to unfamiliar odours when they had been paired with imbalance. Such acquired chemosensory control of preferences, together with an anorexigenic effect of imbalanced amino acid mixtures, can account for characteristics of feeding behaviour under conditions in which the diet is deficient in an essential amino acid.

Liver ultrastructure and RNA labelling after partial hepatectomy in protein-fed and protein-deprived rats.

Male rats were pair-fed on a protein-free or a 25% casein diet for 14-days and then submitted to partial hepatectomy or sham operation. As seen in the electron microscope, both dietary groups of hepatectomized rats showed about the same degree of regeneration of rough-surfaced endoplasmic reticulum and polysomes, and the same enlargement of nucleoli. The nucleoli of the regenerating livers of protein-deprived rats often showed an increase in the granular component. The RNA labelling after cytidine-3H administration, studied by radioautography and by polyacrylamide-agarose gel electrophoresis of total liver and a cytoplasmic fraction, was heavier in all parts of the liver cell in protein-deprived rats as compared to protein-fed rats and heavier in hepatectomized than in sham-operated rats. There was no delay in the appearance of 29 and 18 S ribosomal RNA in the cytoplasm in protein-deprived and hepatectomized rats. The results suggest that the protein-deprived rats have a good ability to regenerate liver cell structures after partial hepatectomy, that their enlarged liver cell nucleoli have a high synthesis of RNA, and that the processing and delivering of nucleolar RNA to the cytoplasm proceeds in a normal way.

Protein Turnover in Skeletal Muscle. II. The Effect of Starvation and a Protein-Free Diet on the Synthesis and Catabolism of Skeletal Muscle Proteins in Comparison to Liver

1. Rates of synthesis and catabolism of liver and muscle sarcoplasmic and myofibrillar protein have been measured in young control, starved and protein (deprived) rats using [14C]Na2CO3 to label protein. 2. Half-lives for synthesis of 1·35, 2·8 and 7·2 days for liver, sarcoplasmic and myofibrillar proteins respectively were obtained, whilst half-lives for catabolism were 1·55, 3·6 and 15·6 days in each case in the control animals. 3. The protein free diet for 3 days caused a small decrease in the rate of synthesis of liver and muscle proteins. The catabolic rate of liver protein was increased by 20% whilst there was a smaller increase in the catabolic rate of myofibrillar proteins. 4. Starvation for 3 days caused a 20% reduction in the rate of liver protein synthesis whilst there were greater reductions in muscle protein synthesis. The catabolic rate of liver protein was only slightly increased whereas there was a 75% increase in the rate of myofibrillar protein breakdown. 5. The total amount of protein synthesis and catabolism in liver and the two muscle protein fractions over the first 3 days of the three regimes were calculated. Muscle protein turnover, in particular myofibrillar, was shown to be very sensitive to dietary protein and/or calorie deficiency. 6. These results are discussed in terms of the mobility and therefore importance of muscle protein metabolism in the economy of the whole animal.

Maintenance of Pregnancy in Protein-deprived Rats by Transitory Protein Supplements during Early Gestation

A study was made to determine the relationship between dietary protein and maintenance of pregnancy in protein-deprived rats at 2 stages of gestation, namely, after mating (day zero) and after implantation of the blastocyst (day 5). By means of transitory feeding, it was shown that there was a critical need for protein at both stages, and that protein supplements supported pregnancy at either stage. Particularly striking was the observation that supplements of protein given 2 or 3 days after inception of pregnancy were sufficient to sustain reproduction in rats deprived of dietary protein at the same high level as in protein-fed controls. Litter size was unaffected by protein deficiency, but fetal growth was retarded. Food consumption of rats receiving a protein-free diet throughout gestation was less than that of rats receiving 20% protein, and the reduction in food intake was greater in protein-deprived rats carrying young than in those failing to litter. The results of the present experiments correspond to those reported previously with a restricted stock diet, and suggest that the effects of restriction were attributable to protein deficiency.

Protein Metabolism and Erythropoiesis. II. Erythropoietin Formation and Erythroid Responsiveness in Protein-deprived Rats

Abstract (1) Rats on a normal and non-protein diet were hypertransfused to suppress their endogenous erythropoietin formation. Injection of erythropoietin elicited nearly identical increases in radioiron incorporation in the two dietary groups. (2) Normal and protein-starved rats were exposed to lowered atmospheric pressure. At each level of hypoxia the erythropoietin titer in the plasma of protein-starved rats were significantly lower than those in normal diet groups. (3) Re-proteinization resulted in significant increases in erythropoietin level. (4) It is concluded that protein deficiency does not affect cytoplasmic protein synthesis in erythroid precursors directly, and the depression of erythropoiesis is attributed to a diminished formation of erythropoietin.

Interferometric determinations of the ribose nucleic acid concentration in liver nucleoli of protein-fed and protein-deprived rats

Liver nucleoli of rats fed on a non-protein diet were considerably larger than those of rats fed on a 25 per cent casein diet. The concentration of ribose nucleic acid (RNA) of the nucleoli in Carnoy-fixed sections was determined by interferometric estimation of the dry matter before and after digestion with ribonuclease. The RNA concentration was 14 per cent of the nucleolar dry matter in both groups, corresponding to 5 per cent of the total nucleolar mass. The results indicate absolute increase of RNA in the large nucleoli of protein-deprived animals in comparison with protein-fed animals. It is concluded that there is intense nucleic acid metabolism in these enlarged nucleoli. The liver nucleoli of protein-fed animals are also thought to be metabolically active.