Dietary protein intake and skeletal-muscle protein metabolism in rats. Studies with salt-washed ribosomes and transfer factors

1. Aspects of skeletal muscle protein synthesis in vitro were studied in young rats given a low-protein diet for up to 10 days and during re-feeding with an adequate diet. 2. Partially purified muscle transfer factors (transferases I and II), crude and purified (NH(4)Cl-washed) ribosomes and a pH5 enzyme fraction were prepared for this purpose. 3. A marked decrease in the capacity of crude ribosomes to carry out cell-free polypeptide synthesis occurred within 4 days of feeding the low-protein diet. 4. The capacity of salt-washed ribosomes to promote amino acid polymerization, in the presence of added transfer factors and aminoacyl-tRNA, was only slightly decreased by the dietary treatment. 5. However, the capacity of salt-washed ribosomes to bind (14)C-labelled aminoacyl-tRNA was decreased by feeding the low-protein diet. 6. The capacity of the pH5 enzyme fraction to promote amino acid incorporation in a complete cell-free system was decreased within 2 days of feeding the low-protein diet. There is no evidence that the change is associated with aminoacyl-tRNA synthetase or binding enzyme activities of the pH5 fractions. 7. These changes are discussed in relation to the diminished rate of protein synthesis in the intact muscle cell when rats are given a low-protein diet.     

Dietary whey protein decreases food intake and body fat in rats

We investigated the effects of dietary whey protein on food intake, body fat, and body weight gain in rats. Adult (11-12 week) male Sprague-Dawley rats were divided into three dietary treatment groups for a 10-week study: control. Whey protein (HP-W), or high-protein content control (HP-S). Albumin was used as the basic protein source for all three diets. HP-W and HP-S diets contained an additional 24% (wt/wt) whey or isoflavone-free soy protein, respectively. Food intake, body weight, body fat, respiratory quotient (RQ), plasma cholecystokinin (CCK), glucagon like peptide-1 (GLP-1), peptide YY (PYY), and leptin were measured during and/or at the end of the study. The results showed that body fat and body weight gain were lower (P < 0.05) at the end of study in rats fed HP-W or HP-S vs. control diet. The cumulative food intake measured over the 10-week study period was lower in the HP-W vs. control and HP-S groups (P < 0.01). Further, HP-W fed rats exhibited lower N(2) free RQ values than did control and HP-S groups (P < 0.01). Plasma concentrations of total GLP-1 were higher in HP-W and HP-S vs. control group (P < 0.05), whereas plasma CCK, PYY, and leptin did not differ among the three groups. In conclusion, although dietary HP-W and HP-S each decrease body fat accumulation and body weight gain, the mechanism(s) involved appear to be different. HP-S fed rats exhibit increased fat oxidation, whereas HP-W fed rats show decreased food intake and increased fat oxidation, which may contribute to the effects of whey protein on body fat.     

Studies on an antineoplastic fraction from human urine. Characterization of the major protein in this fraction.

A fraction has been isolated from human urine which exhibits antiproliferative activity against human tumour cell lines without affecting the growth of several normal diploid cell lines or tumour cells of mouse or hamster origin. The major protein present in this fraction has been characterized and tentatively designated antineoplastic urinary protein (ANUP). An S020,W value of 3.69 S was obtained by sedimentation velocity analysis, and a subunit molecular mass of 16 300 Da was obtained by sedimentation equilibrium and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Centrifugation data also indicated that the protein self-associates. The amino acid analysis of ANUP was consistent with its low pI (4.2) as determined by chromatofocusing analysis. Furthermore, the amino acid composition exhibited some features similar to collagen, as shown by high levels of proline and glycine, the absence of cysteine, and the presence of low levels of hydroxyproline.     

Studies on the metabolism of retinol-binding protein by primary hepatocytes from retinol-deficient rats

Studies were conducted to explore the regulation of retinol-binding protein (RBP) metabolism in cultured primary hepatocytes from retinol-deficient rats. Newly isolated hepatocytes from retinol-deficient rats contained elevated levels (3.4-fold) of RBP, compared to hepatocytes from normal (retinol-adequate) rats. Addition of retinol to retinol-depleted hepatocytes stimulated RBP secretion by the cells in a concentration-dependent manner. Maximal stimulation of RBP secretion was seen with a retinol level of 0.3 micrograms/ml. The effect of retinol was quite rapid, and was evident by 20 minutes after addition of retinol to the medium. Stimulation of RBP secretion was only seen during the first few hours after retinol addition. The effect of retinol was specific for RBP; thus, retinol had no effect on the secretion rates of transthyretin or albumin. Addition of retinoic acid also stimulated RBP secretion by retinol-deficient hepatocytes. Addition of dexamethasone to retinol-deficient cells did not maintain the initial rate of RBP secretion. Dexamethasone also had no effect on the secretion of transthyretin or albumin by these cells. The effects of retinol and of dexamethasone seen here with retinol-depleted cells differed dramatically from effects seen in other studies with normal (retinol-adequate) hepatocytes. Thus, with normal cells, dexamethasone maintains RBP, TTR, and albumin production and secretion rates close to initial rates. Also in normal hepatocytes, with ample retinol available within the cell, addition of exogenous retinol does not appear to influence RBP secretion. In contrast, and as shown previously in intact rats, in retinol deficiency the availability of retinol specifically regulates the secretion of RBP by hepatocytes.     

Characterization of glycyrrhizin-binding protein kinase from the crude membrane fraction of rat liver

Using GL-affinity column chromatography, a casein phosphorylating protein kinase was purified selectively from the crude membrane fraction of rat liver. The biochemical characteristics of the purified kinase (approximately Mr 210 kDa) are very similar to those reported for polypeptide-dependent protein kinase (kinase P). Moreover, low doses of GL selectively inhibit phosphorylation of Mr 35-36 kDa polypeptides (which are cross-reacted with anti-lipocortins I and II) by the kinase in vitro. These results suggest that the anti-inflammatory activity of GL may involve the impairment of the physiological functions of lipocortins through their specific modification by the kinase at the cell membrane level.     

Dietary soybean protein moderates the deleterious disturbance of lipid metabolism caused by exogenous oxidized cholesterol in rats.

Effects of dietary protein on oxidized cholesterol-induced disturbance of lipid metabolism were examined in 4 week old male Sprague-Dawley rats, using casein and soybean protein as dietary protein source. The rats were given one of the two proteins in 0. 078% cholesterol (control), 0.25% cholesterol or 0.25% oxidized cholesterol mixture (containing 0.078% cholesterol) diets. Dietary oxidized cholesterol, compared to cholesterol, tended to inhibit hepatic sterol biosynthesis in casein-fed rats, whereas this inhibitory action was slightly moderated by intake of soybean protein. As a result, the hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity was rather higher in the rats fed oxidized cholesterol than in those fed cholesterol in the soybean protein-fed group. The hepatic cholesterol 7alpha-hydroxylase activity tended to be higher in the rats fed oxidized cholesterol than in those fed control diet in the soybean protein-fed group, despite the fact that oxidized cholesterol lowered the hydroxylase activity in the casein-fed group. On the other hand, dietary oxidized cholesterol tended to slightly enhance the hepatic Delta6 desaturase activity in the casein-fed group; however, this observation was not shown in the soybean protein-fed group. Moreover, dietary soybean protein facilitated fecal oxidized cholesterol excretion and simultaneously inhibited the accumulation of oxidized cholesterol in serum and liver. In conclusion, dietary soybean protein alleviated the deleterious actions of exogenous oxidized cholesterol on hepatic cholesterol and linoleic acid metabolism, although these efficacies were not necessarily significant. A great part of these moderations may be exerted by the specific hypocholesterolemic function of soybean protein, such as the stimulation of fecal oxidized cholesterol excretion, the change of hormonal release and modulation of lipoprotein catabolism.     

The effect of Echis carinatus crude venom and purified protein fractions on carbohydrate metabolism in rats

Echis carinatus crude venom was fractionated into 11 protein fractions by preparative native polyacrylamide gel electrophoresis (PAGE). All fractions except fractions 5 and 10 appeared as a single band on analytical native PAGE. Purified venom fractions 1, 4, 8, 10 and 11 appeared as single bands on SDS-PAGE whereas fractions 2, 3 and 7 contained two bands and fraction 6 contained three bands. Fractions 1 and 3 exhibited basic pI (7.3 and 7.6) respectively, while fractions 2, 4, 6, 8, 10 and 11 showed an acidic pI. Amino acid analysis also showed that crude venom is rich in acidic amino acids. A significant hyperglycaemia was produced by i.p. injection of E. carinatus crude venom, after 15 min of envenomation which persisted even after 24 h. Along with hyperglycaemia there was a significant decrease of liver glycogen at 15 min and 1, 12 and 24 h. A significant decrease of plasma [pyr + lac] levels was found from 15 min to 24 h. The liver [pyr + lac] levels increased significantly after 24 h. Skeletal muscle [pyr + lac] level was significantly decreased after 24 h of envenomation. Fractions 2 and 6 produced the highest increase in plasma glucose after 12 h and fraction 7 after 24 h. The plasma insulin level was significantly decreased by these three fractions (2, 6 and 7). So it can be hypothesized that the hyperglycaemia may result from a direct effect of a venom component on plasma insulin. Fractions 7, 8 and 11 caused the highest decrease in plasma [pyr + lac] while fractions 1, 2, 3, 4 and 8 produced the most significant decrease in liver [pyr + lac]. The most significant increase in lactate dehydrogenase level was also produced by fractions 1, 2, 3, 4 and 8.     

Dietary soy isoflavone-aglycone lowers food intake in female rats with and without ovariectomy

Objective: Estrogens downregulate eating behavior, and soy isoflavones are known to be estrogenic agents. We aimed to examine whether the estrogenic property of soy isoflavones can affect food intake and body weight. Methods and Procedures: Seven‐week‐old male, female, and ovariectomized (OVX) Sprague‐Dawley rats were given free access to a diet containing 100–300 mg total isoflavone/kg diet, or to a control diet, either with or without concurrent administration of estradiol by subcutaneous implantation. Results: Dietary soy isoflavone was shown to lower food intake in female rats, whether or not the animals had undergone ovariectomy. Administration of estradiol lowered the food intake in male rats and in OVX female rats. The decrease in weekly food intake in female rats led to a reduction in their weekly gain in body weight. Dietary soy isoflavone significantly increased the concentration of serum isoflavones, especially equol (a metabolite of daidzein), regardless of gender or ovariectomy. Dietary soy isoflavone did not affect either serum estradiol concentration or uterine and didymus weights, but estradiol administration improved the uterine atrophy in OVX rats, and decreased the didymus weight in male rats. Discussion: Soy isoflavone lowers the food intake in female rats, but not in the male animals. Contrary to the hypothesis currently in vogue, the reduction in food intake caused by soy isoflavone may not be a purely estrogenic effect. This follows from the finding that the effects of soy isoflavones on food intake and on the reproductive organs differ from the corresponding effects produced by estrogen.     

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats.

1. The effect of phenobarbitone on the rate of protein synthesis and on the sedimentation patterns of various liver subcellular fractions containing ribosomes was studied in rats. 2. Phenobarbitone treatment increased the incorporation of [114C]leucine into protein by all preparations, provided they had not been subjected to preliminary treatment with Sephadex G-25. The phenobarbitone-induced effect on incorporation was associated with a gain in liver weight and a higher degree of polyribosomal aggregation. 3. Preparations that were treated with Sephadex G-25 incorporated more radioactivity into protein, but did not show the response to phenobarbitone treatment. 4. When the influence of starvation and phenobarbitone was studied separately on membrane-bound and membrane-free polyribosomes, it was shown that whereas both classes of polyribosomes were affected by starvation, apparently only the former class was susceptible to phenobarbitone stimulation of protein synthesis. 5. The decreased capacity for protein synthesis of polyribosomes from starved rats was independent of their association with the membranes of the endoplasmic reticulum, but resulted from polyribosomal disaggregation, from an intrinsic defect of the polyribosomes themselves and from changes in composition of the cell cap. 6. The results are discussed in relation to the problem of the control of protein biosynthesis and of the functional separation of membrane-bound and membrane-free polyribosomes.     

Dietary carbohydrates affect caecal fermentation and modify nitrogen excretion patterns in rats II. Studies with diets differing in protein quality

In 2 two‐factorial experiments, each conducted on 80 growing male rats, the effects of substituting 10% raw potato starch (PS), pectins (PEC), or cellulose (CEL) for wheat starch (WS) and the addition of tannic acid to WS (WSTA) were studied using diets differing in protein quality. Casein unsupplemented or supplemented with DL‐methionine and gluten unsupplemented or supplemented with lysine, methionine and tryptophan were used as protein sources in Experiment 1 and 2, respectively. Parameters indicative of caecal fermentation intensity (pH, acetic, propionic and butyric acid contents, digesta and tissue weight) and of protein metabolism (urea blood concentration, faecal and urinary nitrogen excretion) were determined. Ten‐day balance experiments were preceded by a 10‐day adaption period to respective carbohydrates given in a diet containing balanced protein.

In both experiments the type of carbohydrates affected the caecal concentration of individual and total SCFA and other parameters of fermentation intensity. Pectins and potato starch were fermented more intensively than cellulose. Faecal N excretion was increased by all carbohydrates substituted for cereal starch, and by tannic acid. Urinary excretion was greater on CEL than on PEC and WSTA containing casein and on other diets containing gluten. In both experiments urinary N excretion was the lowest on PEC diets. Protein quality had the greatest effect on apparent biological value and net protein utilization but all indices of protein utilization were also affected by carbohydrates. It is concluded that not only the amount of N excreted in faeces but also in urine is affected by the type and fermentability of carbohydrates.