Inhibition by ethanol of cardiac protein synthesis in the rat

Protein synthesis and degradation were measured in the hearts of rats fed on diets containing 27% of calories as ethanol. Feeding of ethanol decreased the rate of synthesis of mixed cardiac proteins but was without effect on the rate of breakdown of myofibrillar and sarcoplasmic proteins. Concentrations of RNA in the hearts were not altered by ethanol feeding, indicating a decrease in RNA activity for protein synthesis.     

The effect of branched chain amino acids on protein synthesis in two skeletal muscles of Japanese quail.

Two different isolated skeletal muscles of Japanese quail were used. The influence of branched chain amino acids on the fractional rate of protein synthesis (FSR) was evaluated using 14C-tyrosine. The addition of 0.5 mM valine, leucine or isoleucine to the incubation medium significantly decreased (P<0.05) the value of FSR in extensor metacarpalis radialis. In the ambiens muscle only the application of leucine increased the FSR significantly while valine and isoleucine were without any effect.     

Changes in the concentrations of branched chain amino acids in the blood in response to alterations in the composition of a hypocaloric diet.

This report describes an investigation of the effect of an alteration in the amount of protein, carbohydrate and fat in hypocaloric diets on the concentration ratios of valine/(isoleucine + leucine) in the morning fasting blood in healthy men: in some cases the effect of a higher caloric diet was also analysed. During the nutrition periods, each over 10 days, the concentrations of branched chain amino acids and alanine, blood levels of insulin, glucose and free fatty acids and the nitrogen balance were estimated daily. With hypocaloric diets, as well as with hypercaloric diets, supplementation of food protein from 0.6 or 0.8 up to 2.0 g protein per kg body weight (isocalorically) induced a rise of the molar ratios of valine/(isoleucine + leucine) in blood from 0.98 to 1.25 (4600 kJ/d) or 1.06 to 1.45 (14600 kJ/d) respectively. A correlation of molar ratios of valine/(isoleucine + leucine) in the blood and the nitrogen balance could not be established. However, it is suggested that ratios above a limiting value exclude the possibility of a distinct negative nitrogen balance during 7 or 10 days of observation.     

Changes in the exercise-induced hormone response to branched chain amino acid administration.

It was the aim of the present experiment to detect possible effects of branched-chain amino acids (BCAA) on the endocrine response to 1 h of continuous running. Blood samples were collected from 14 long-distance runners (age 24-42 years) in two different trials performed at 1-week intervals. In both trials (E and P) blood samples were collected at the following times: 9 a.m. (basal values sample), 10.30 a.m. (sample 90), 11.30 a.m. (sample 150), 12.30 p.m. (sample 210); the athletes performed 1 h of running at a constant predetermined speed between samples 90 and 150. Following the basal sample a mixture containing BCAA (E trial), or not containing BCAA (P trial) was ingested. In both trials no hormone basal concentrations, except insulin, were changed before exercise. In P trial, following exercise (sample 150), human growth hormone (HGH), prolactin (PRL), adrenocorticotropic hormone (ACTH) and cortisol (C) increased, while testosterone (T) decreased. In sample 210, after 1 h of rest, while ACTH, PRL and HGH had recovered to basal concentrations, C remained elevated and T displayed a further decrease. In the E trial a similar pattern of change was observed in sample 150 for HGH, PRL, ACTH and C; in sample 210 HGH and PRL displayed significantly lower values than in the corresponding P trial samples. The T was not modified by the running exercise and increased during the recovery period. It is, therefore, suggested that BCAA administration before exercise affects the response of some anabolic hormones, mainly HGH and T.     

Role of mitochondrial transamination in branched chain amino acid metabolism

Oxidative decarboxylation and transamination of 1-14C-branched chain amino and alpha-keto acids were examined in mitochondria isolated from rat heart. Transamination was inhibited by aminooxyacetate, but not by L-cycloserine. At equimolar concentrations of alpha-ketoiso[1-14C]valerate (KIV) and isoleucine, transamination was increased by disrupting the mitochondria with detergent which suggests transport may be one factor affecting the rate of transamination. Next, the subcellular distribution of the aminotransferase(s) was determined. Branched chain aminotransferase activity was measured using two concentrations of isoleucine as amino donor and [1-14C]KIV as amino acceptor. The data show that branched chain aminotransferase activity is located exclusively in the mitochondria in rat heart. Metabolism of extramitochondrial branched chain alpha-keto acids was examined using 20 microM [1-14C]KIV and alpha-ketoiso[1-14C]caproate (KIC). There was rapid uptake and oxidation of labeled branched chain alpha-keto acid, and, regardless of the experimental condition, greater than 90% of the labeled keto acid substrate was metabolized during the 20-min incubation. When a branched chain amino acid (200 microM) or glutamate (5 mM) was present, 30-40% of the labeled keto acid was transaminated while the remainder was oxidized. Provision of an alternate amino acceptor in the form of alpha-keto-glutarate (0.5 mM) decreased transamination of the labeled KIV or KIC and increased oxidation. Metabolism of intramitochondrially generated branched chain alpha-keto acids was studied using [1-14C]leucine and [1-14C]valine. Essentially all of the labeled branched chain alpha-keto acid produced by transamination of [1-14C]leucine or [1-14C]valine with a low concentration of unlabeled branched chain alpha-keto acid (20 microM) was oxidized. Further addition of alpha-ketoglutarate resulted in a significant increase in the rate of labeled leucine or valine transamination, but again most of the labeled keto acid product was oxidized. Thus, catabolism of branched chain amino acids will be favored by a high concentration of mitochondrial alpha-ketoglutarate and low intramitochondrial glutamate.     

Effect of leucine and metabolites of branched chain amino acids on protein turnover in heart.

Leucine, but not isoleucine or valine, inhibited protein degradation and accelerated protein synthesis in hearts perfused with buffer that contained glucose (15 mM) and normal plasma levels of other amino acids, except for the branched chain compounds. Products of leucine, isoleucine, and valine metabolism also inhibited protein degradation and stimulated protein synthesis. These compounds included the transamination and decarboxylation products, as well as acetate, acetoacetate, and propionate. In some, but not all instances, inhibition of degradation and acceleration of synthesis were accompanied by an increase in intracellular leucine. When insulin was added to the perfusate, the rate of degradation was reduced by 40%, but addition of leucine was ineffective in the presence of the hormone. Insulin, leucine (2 mM) and a mixture of branched chain amino acids at normal plasma levels increased latency of cathepsin D in hearts that were perfused with buffer containing glucose. A combination of leucine and insulin increased latency more than either substance alone. These studies indicate that leucine as well as a variety of substrates that are oxidized in the citric acid cycle are involved in regulation of protein turnover in heart muscle.     

The induction of a dehydrogenase activity for branched chain amino acids in Bacillus subtilis

Summary In Bacillus subtilis a dehydrogenase activity for branched chain amino acids was induced twelvefold in glucose medium by isoleucine. To a lesser degree this activity was induced by metabolically related amino acids with the exception of leucine which hardly induced. The induced enzyme actvity is different from alanine dehydrogenase. The presumable role of this inducible enzyme in anteiso fatty acid biosynthesis is discussed.     

Calcium-dependent inhibition of protein synthesis in rat parotid gland.

1. Protein synthesis in the rat parotid gland in vitro was studied by measuring the incorporation of [3H]phenylalanine into trichloroacetic acid-insoluble proteins. In the unstimulated gland, the rate of incorporation was dependent on the phenylalanine concentration in the medium and proceeded linearly for up to 3h. 2. Adrenaline, carbamoylcholine, phenylephrine and ionophore A23187 inhibited the incorporation of [3H]phenylalanine into acid-insoluble protein; isoprenaline, dibutyryl cyclic AMP and 8-bromo-cyclic GMP were inactive. 3. Inhibition by adrenaline and carbamoylcholine but not by ionophore A23187 required extracellular Ca2+. 4. Both adrenaline and carbamoylcholine increased the magnitude of the acid-soluble [3H]phenylalanine pool at 10 micrometer extracellular phenylalanine, but had no effect if the phenylalanine concentration was increased to 200 micrometer. 5. There was no correlation between cellular ATP content and the observed inhibition of protein synthesis. 6. Our results suggest that both alpha-adrenergic and cholinergic receptors may play a role in the regulation of protein synthesis in the rat parotid gland, and that their effects are mediated by a rise in intracellular free Ca2+.     

Mechanism of the inhibition of protein synthesis by vasopressin in rat liver

A recent study reported that protein synthesis was inhibited in rat livers perfused with medium containing vasopressin (Chin, K. -V., Cade, C., Brostrom, M. A., and Brostrom, C. O. (1988) Int. J. Biochem. 20, 1313-1319). The inhibition of protein synthesis caused by vasopressin was associated with a disaggregation of polysomes, suggesting that peptide chain initiation was slowed relative to elongation. In contrast, Redpath and Proud (Redpath, N. T., and Proud, C. G. (1989) Biochem. J. 262, 69-75) recently reported an inhibition of peptide chain elongation by a calcium/calmodulin-dependent mechanism. Therefore, the question remained whether only peptide chain initiation was inhibited or both initiation and elongation were affected by vasopressin. In the present study, vasopressin was found to inhibit protein synthesis in both perfused rat livers and isolated rat hepatocytes. Ribosomal half-transit times in isolated hepatocytes averaged 1.9 +/- 0.1 min with or without vasopressin present in the media, demonstrating that the rate of peptide chain elongation was unaffected by vasopressin. Instead, the inhibition of protein synthesis induced by vasopressin was manifested at the level of peptide chain initiation. Vasopressin treatment resulted in both a 2-fold increase in the number of free ribosomal particles and a greater than 50% decrease in the amount of [35S]methionine bound to 43 S preinitiation complexes. In addition, the activity of eukaryotic initiation factor (eIF) 2B in crude extracts from perfused livers was reduced to 53% of the control value in response to vasopressin. The inhibition of eIF-2B activity was associated with an increase in the proportion of the alpha-subunit of eIF-2 in the phosphorylated form from 9.6% in control livers to 30.7% in livers perfused with medium containing vasopressin. The results demonstrate the novel finding that the inhibition of protein synthesis in vasopressin-treated livers is caused by a reduction in eIF-2B activity due to an increase in phosphorylation of eIF-2 alpha.     

The effects of a low protein diet on amino acids and enzymes in the serine synthesis pathway in mice

Branched-chain amino acids, especially leucine, exert regulatory influences on protein and carbohydrate metabolism, ribosome biogenesis and gene expression. This study investigated the effects of leucine in fibroblastic cells analysing viability, proliferation, morphology, proteolysis enzymes activities and protein turnover. After exposure to culture medium enriched with 25 or 50 μM leucine for 24, 48 and 72 h, Vero cells have no alterations on viability and morphology. Leucine-treated cells showed increase on alkaline phosphatase activity and proliferation. The protein synthesis was slightly increased, whereas the protein degradation showed a deep reduction after leucine incubation. The chymotrypsin-like, cathepsin B and H and calpain activities were decreased in leucine-treated cells. In conclusion, the proteolytic pathways and the total protein degradation were modulated by leucine in Vero cells. Our observations support the concept that Vero cells may represent a new model for protein turnover study.     

Nutritional control of branched chain alpha-ketoacid dehydrogenase in rat hepatocytes

Branched chain alpha-ketoacid dehydrogenase (EC 1.2.4.4) complex, the rate-limiting enzyme of branched chain amino acid catabolism in most tissues, is subject to regulation by covalent modification, with phosphorylation inactivating and dephosphorylation activating the complex. The enzyme complex from liver of chow-fed rats is mainly in the active form but that from liver of rats fed a low-protein diet is mainly in the inactive form. Isolated hepatocytes were used to identify factors that affect interconversion of branched chain alpha-ketoacid dehydrogenase. The enzyme present in hepatocytes of rats fed a low-protein diet appears much more responsive to regulation by covalent modification than the branched chain alpha-ketoacid dehydrogenase present in hepatocytes of normal chow-fed rats. alpha-Chloroisocaproate, a specific inhibitor of the kinase responsible for phosphorylation and inactivation of the complex, greatly stimulates oxidation of alpha-keto[1-14C]isovalerate by hepatocytes prepared from rats fed a low-protein diet but not from normal chow-fed rats. Oxidizable substrates are also much more effective inhibitors of branched chain alpha-ketoacid oxidation with hepatocytes from rats fed a low-protein diet than from normal chow-fed rats. Activity measurements with cell-free extracts suggest that changes in flux through the dehydrogenase with intact hepatocytes prepared from rats fed a low-protein diet are explained in large part by changes in the proportion of the enzyme in the active, dephosphorylated form. Regulation of liver branched chain alpha-ketoacid dehydrogenase by covalent modification functions to conserve branched chain amino acids for protein synthesis during periods of restricted dietary protein intake.     

Purification of rat brain succinate-semialdehyde dehydrogenase and study of its inhibition by branched chain fatty acids]

Rat brain succinic semialdehyde deshydrogenase has been purified 1300 fold. This enzyme is inhibited non competitively by the same branched chain fatty acids which inhibit GABA-transaminase competitively with respect to GABA. The respective activities of GABA-T and SSADH found in rat brain indicate that at anticonvulsant doses, the acids dipropylacetic and 2-methyl 2-ethyl caproic preferentially inhibit GABA-transaminase thus inducing a rise in cerebral GABA level. This increase is therefore not due to metabolism of the succinic semialdehyde by GABA-T.     

Compartmentation of free amino acids for protein synthesis in rat liver

The concept that a general intracellular pool serves as the sole precursor of amino acids for protein biosynthesis has been vigorously debated in recent years. To help resolve this controversy, we followed the distribution of intraperitoneally administered [(3)H]valine in the tRNA and the extracellular and intracellular compartments of rat liver. The specific radioactivity of the valine released from isolated tRNA was 2-3 times higher than that of intracellular valine, suggesting that the intracellular pool cannot be the sole precursor of amino acids used for charging tRNA. In addition, the specific radioactivity of the tRNA was only half that of the extracellular valine. Therefore it is unlikely that the valyl-tRNA is charged exclusively with amino acids derived from the extracellular pool. A model is proposed which stipulates that both extracellular and intracellular amino acids contribute to a restricted compartment that funnels amino acids towards protein biosynthesis.     

Fatty acids reverse the cyclic AMP inhibition of triacylglycerol and phosphatidylcholine synthesis in rat hepatocytes.

The influence of cyclic AMP analogues and fatty acids on glycerolipid biosynthesis in monolayer cultures of rat hepatocytes was investigated. Chlorophenylthio-cyclic AMP and adenosine 3':5'-cyclic phosphorothioate inhibited the rate of triacylglycerol synthesis from [1(3)-3H]glycerol, and phosphatidylcholine synthesis from [Me-3H]-choline. Supplementation of the hepatocytes with palmitate (1 mM) reversed chlorophenylthio-cyclic AMP inhibition of triacylglycerol synthesis. Similarly, cyclic AMP analogue-inhibition of phosphatidylcholine synthesis was abolished when the cells were simultaneously incubated with oleate (3 mM). Reactivation of phosphatidylcholine synthesis in chlorophenylthio-cyclic AMP-supplemented cells with oleate was accompanied by conversion of CTP: phosphocholine cytidylyltransferase into the membrane-bound form, since these cells released the enzyme more slowly after treatment with digitonin. The opposing actions of cyclic AMP and fatty acids are discussed in relation to the regulation of glycerolipid biosynthesis during starvation, diabetes and stress.     

Thyroid hormone inhibition of synaptosome amino acid uptake and protein synthesis.

Abstract— 3,3′,5-Triiodothyronine (T₃) inhibited L-[¹⁴C]leucine uptake into synaptosomes. Inhibition was competitive with a K_i of 3.1 × 10^?5m . Hofstee plot revealed an inverted hyperbolic curve suggestive of a two carrier or carrier plus diffusion mediated system for amino acid uptake. Both the carrier mediated and diffusional components were inhibited by thyroid analogues. l -Thyroxine and analogues inhibited the incorporation of l -[¹⁴C] leucine into cerebral synaptosome protein. At 50 μm , the triiodo-compounds were more inhibitory than tetraiodo->3,5-triiodo-l -thyronine >3,3′,5-triiodothyropro-pionic> l -thyroxine >3,5-diiodo-l -tyrosine. Thyroid analogue inhibition was not seen in liver or brain mitochondrial protein synthesis. 3,3′,5-Triiodothyronine had no effect on respiratory control or 2,4-DNP stimulated synaptosome respiration supported by malate plus pyruvate. Ouabain did not inhibit [¹⁴C]leucine uptake into adult synaptosomes. There was synergistic inhibition of synaptosome protein synthesis by thyroid analogues in the presence of 0.2 mm -ouabain. 3,3′,5-Triiodothyronine had no effect on synaptosome fraction ATPase or Na-K ATPase. Addition of T₃ induced further inhibition of synaptosome protein synthesis in the presence of either chloramphenicol (100μm ) or cycloheximide (50μg/ml). [¹⁴C]Glycine uptake and incorporation into synaptosome protein was inhibited by 3,3′,5-triiodothyronine. There was no inhibition of [¹⁴C]proline uptake or incorporation. The above evidence and kinetic data strongly favor a selective competitive block in amino acid transport at the synaptosome membrane leading to a decreased rate of protein synthesis.