We investigated the effects of the branched-chain amino acids—valine, leucine and isoleucine—or their keto analogs, the branched-chain keto acids—α-ketoisovaleric acid, α-ketoisocaproic acid and α-keto-β-methylvaleric acid—on protein synthesis and secretion by monolayers of rabbit hepatocytes incubated with [35S] methionine in pulse-chase and steady-state experiments. The branched-chain amino acids (2.0 mM or 1.0 mM), in the presence or absence of insulin (2 × 10−4 IU per dish) and in both types of experiments, reduced the trichloroacetic acid-precipitable 35S-protein secreted into the medium. The branched-chain keto acids (2.0 mM or 1.0 mM) had a stimulatory effect on secreted trichloroacetic acid-precipitable 35S-protein which was observed only by the pulse-chase technique in the presence of insulin. Immunoaffinity chromatography of medium demonstrated a slight inhibition by branched-chain amino acids and a slight stimulation by branched-chain keto acids on secretion of 35S-albumin and no effect of either treatment on secretion of 35S-fibrinogen. ELISA analysis of total (i.e., 35S-labeled and unlabeled) secreted albumin revealed an inhibitory effect of the branched-chain amino acids in both pulse-chase and steady-state experiments, and a small stimulatory effect, in steady-state experiments, of the branched-chain keto acids; both effects were insulin-dependent. Total secreted fibrinogen, under steady-state conditions, was increased by the branched-chain keto acids in the presence of insulin, while transferrin production was unaffected by any treatment. Intracellular trichloroacetic acid-precipitable 35S-protein was increased by the branched-chain keto acids in the presence of insulin and was reduced by the branched-chain amino acids in the presence or absence of insulin, suggesting that the effects of these agents may have been related to synthesis and not to effects on secretion.
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