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The protein anabolic effect of branched chain amino acids was studied in isolated quarter diaphragms of rats. Protein synthesis was estimated by measuring tyrosine incorporation into muscle proteins in vitro. Tyrosine release during incubation with cycloheximide served as an index of protein degradation. In muscles from normal rats the addition of 0.5 mM leucine stimulated protein synthesis 36--38% (P less than 0.01), while equimolar isoleucine or valine, singly or in combination were ineffective. The three branched chain amino acids together stimulated no more than leucine alone. The product of leucine transamination, alpha-keto-isocaproate, did not stmino norborane-2-carboxylic acid (a leucine analogue) were ineffective. Leucine and isoleucine stimulated protein synthesis in muscles from diabetic rats.Leucine, isoleucine, valine and the norbornane amino acid but not alpha-ketoisocaproate or beta-hydroxybutyrate decreased the concentration of free tyrosine in tissues during incubation with cycloheximide; tyrosine release into the medium did not decrease significantly. Leucine caused a small decrease in total tyrosine release, (measured as the sum of free tyrosine in tissues and media), suggesting inhibition of protein degradation. The data suggest that leucine may be rate limiting for protein synthesis in muscles. The branched chain amino acids may exert a restraining effect on muscle protein catabolism during prolonged fasting and diabetes.

Incorporation of radiolabeled precursors into muscle proteins was studied in isolated rat hemidiaphragms. A mixture of three branched-chain amino acids (0.3 mM each) added to media containing glucose stimulated the incorporation of [14C]lysine into proteins. When tested separately, valine was ineffective, isoleucine was inhibitory, but 0.5 mM leucine increased the specific activity of muscle proteins during incubation with [14C]lysine or [14C]acetate in hemidiaphragms from fed or fasted rats incubated with or without insulin. Preincubation with 0.5 mM leucine increased the specific activity of muscle proteins during a subsequent 30- or 60-min incubation with [14C]lysine or [14C]pyruvate without leucine. Preincubation with other amino acids (glutamate, histidine, methionine, phenylalanine, or tryptophan) did not exert this effect. When hemidiaphragms were incubated with a mixture of amino acids at concentrations found in rat serum and a [14C]lysine tracer, the specific activity of muscle proteins increased when leucine in the medium was raised from 0.1 to 0.5 mM. Experiments with actinomycin D and cycloheximide suggested that neither RNA synthesis nor protein synthesis are required for the initiation of the leucine effect. Leucine was not effective when added after 1 h preincubation without leucine. The concentration of lysine in the tissue water of diaphragms decreased during incubation with 0.5 mM leucine in the presence or absence of cycloheximide, suggesting that leucine inhibited protein degradation. During incubation with [3h]tyrosine (0.35 mM) the addition of 0.5 mM leucine increased the specific activity of muscle proteins, while the specific activity of intracellular tyrosine remained constant and its concentration decreased, suggesting that leucine also promoted protein synthesis. The concentration of leucine in muscle cells or a compartment thereof may play a role in regulating the turnover of muscle proteins and influence the transition to negative nitrogen balance during fasting, uncontrolled diabetes, and the posttraumatic state. Leucine may play a pivotal role in the protein-sparing effect of amino aicds.

The in vitro incorporation of lucine, isoleucine and pyruvate into lipids was compared and the possibility that leucine might serve as an in situ precursor to the corresponding iso fatty acids in the rat sciatic nerve was studied. The relative incorporation of 14C from leucine into lipids vs. nonlipids was 20%, and the incorporation of label into total lipids from leucine was one-halp that from pyruvate. The incorporation of label from leucine and pyruvate into sterols was nearly equivalent, but the incorporation of label into all other lipid classes from leucine was less than that from pyruvate, and the incorporation of label from isoleucine into lipids was much less in all cases. No detectable label from leucine was incorporated into brached chain fatty acids. It is concluded that leucine may be a substantial in vitro precursor to all major lipids in peripheral nerve, espeically sterols. The possibility and significance of a leucine catabolic pathway in the cytosol in relation to availability of 3-hydroxy-3-methylglutaryl CoA for sterol biosynthesis is discussed.

The in vitro conversion of 14C-labeled leucine, isoleucine, and pyruvate to specific lipids was compared in rat aorta, diaphragm, anf fat pad. Total lipid specific radioactivity from all precursors was greatest in aorta. The ratio of label incorporated into polar lipids vs. neutral lipids by aorta was generally several-fold that incorporated by muscle and fat pad. The labeling of sterols in the aorta from 14C-leucine and pyruvate was equivalent. It is concluded that leucine may be a substantial precursor to polar lipids and to sterols in rat aorta.

The oxidation of leucine by hemidiaphragms of control and diabetic rats was studied in vitro. Rats were rendered diabetic with streptozotocin. Hemidiaphragms of diabetic rats produced approximately 50% more 14CO2 during incubation with 0.1 mM [1-14C]leucine than did control muscles. This was observed during incubation with or without glucose and in the presence or absence of a full complement of plasma amino acids. The concentration of leucine in the tissue water of hemidiaphragms from diabetic rats was greater than that in the control muscles before incubation. The specific activity of leucine at the end of 60 min incubation was not significantly different in diabetic and control muscles, indicating that the increased 14CO2 production represented stimulation of leucine oxidation. Hemidiaphragms of diabetic rats released more leucine into the medium during incubation than did control muscles. The stimulating effect of diabetes on leucine oxidation in vitro was reversible by insulin therapy prior to sacrifice. The addition of 5 mM pyruvate to a medium containing glucose inhibited 14CO2 production from [14C]leucine in control muscles, but stimulated leucine oxidation by hemidiaphragms of diabetic rats. Leucine oxidation by hemidiaphragms of diabetic rats was markedly stimulated by the addition of an electron acceptor, 0.02 mM methylene blue, suggesting that the NADH/NAD ratio may be rate-limiting for branched chain amino acid oxidation in muscles of diabetic rats, but not in muscles of controls. We suggest that the accelerated oxidation of branched chain amino acids by muscles may play a role in the acceleration of the muscle protein catabolism and gluconeogenesis which develop during insulin deficiency. The restraining effect of the cellular redox potential on branched chain amino acid oxidation may play a role in the eventual deceleration of protein catabolism during a prolonged fast.

The response of rat skeletal muscle branched-chain alpha-keto acid dehydrogenase to administration of branched-chain amino acids in vivo was determined using a soluble preparation of the enzyme. After detergent extraction of the complex in the presence of kinase and phosphatase inhibitors, initial in vivo activity was typically 1 nmol X min-1 X g muscle-1, with 0.1 mM alpha-[1-14C]ketoisocaproate as substrate. Total activity of the dephosphorylated complex, measured after preincubation with 15 mM Mg2+, typically reached a maximum of 29 nmol X min-1 X g-1. Thus in overnight-fasted rats the complex was 2-3% active. Initial activity increased three- to fivefold after leucine or isoleucine (at higher concentrations) but not valine administration in vivo. After intravenous leucine injection (0.25 mmol/kg) initial muscle enzyme activity increased rapidly and subsequently decreased, paralleling plasma leucine concentrations, while plasma valine and isoleucine decreased. In conclusion, muscle branched-chain alpha-keto acid dehydrogenase complex is rapidly activated when circulating leucine is increased to concentrations that may occur after meals. During hyperleucinemia accelerated valine and isoleucine degradation by muscle may account for the observed "antagonism" among the branched-chain amino acids.