Insulin and branched-chain amino acids are known to stimulate protein synthesis in skeletal muscle. Extracts prepared from rat diaphragms after incubation in balanced salt solution and glucose alone yielded heat- and acid-stable, TCA-precipitable, nondialyzable factor(s) that inhibit protein synthesis when added to rabbit reticulocyte lysates. Polyribosomal profiles of inhibited lysates were consistent with a defect in peptide-chain initiation. Addition of insulin and amino acids to the diaphragm incubation media partially removed the inhibition seen with the muscle extract and was accompanied by an increase in polysomes and decreased subunits. Similarly, extracts prepared from rat hindlimb muscle 48 h after induction of diabetes were much more inhibitory in rabbit reticulocyte lysates than extracts from control rats. Polyribosomal profiles were consistent with defective peptide-chain initiation. Trypsin treatment before assay abolished the inhibitory activity of muscle extracts from diabetic rats. Because translation-inhibiting peptide(s) appear to be under metabolic and/or hormonal control, their possible role in muscle protein homeostasis warrants further study.

In the eluted fractions of histone-treated crude extracts separated by Sephadex G-200 filtration, multiple protein kinase (PK) activities, including three from brain and two from skeletal muscle, were augmented by both S-100 protein and parvalbumin on the phosphorylation of endogenous substrates. One additional PK activity suppressed by both S-100 and parvalbumin was also found in muscle. In comparison, phosphoprotein phosphatases (PPase), which were also prepared by the same procedure of initial step of histone-treatment followed by the steps of Bio-Gel P-6DG for brain and DNA-cellulose for muscle, were all activated by S-100 while inhibited by parvalbumin and phosphatidylserine.

A heat- and acid-stable protein fraction that inhibited peptide chain initiation in rabbit reticulocyte lysates was extracted from frozen, powdered rat skeletal muscles by stepwise trichloroacetic acid precipitation. Streptozotocin-induced diabetes increased the inhibitory activity; this was prevented by insulin therapy. Size-exclusion high-performance liquid chromatography resolved four inhibitory fractions; only one was consistently increased (approximately 2-fold) in muscle extracts from diabetic rats. Polysome profiles of lysates incubated with this fraction indicated peptide chain initiation inhibition. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified inhibitory fraction migrated with apparent Mr 30 and 32 kDa, which on Western blot immunostained with antisera against histone H1/H1(0). Perchloric acid extraction of muscle homogenates yielded approximately twofold more H1 from diabetic than from control rats; yield from diabetics decreased to control values 5 h after subcutaneous insulin injection. Inclusion of detergent during homogenization increased H1 yield more from muscles of control than from diabetic rats and abolished the difference between them. Because H1 affects several biochemical reactions, its facilitated extraction from insulin-deprived tissues can bias interpretation of studies of insulin action.

The effect of chronic tumor necrosis factor-a (TNF-a) and cortisone treatment on the concentration of translational initiation factor eIF-4E in rat skeletal muscle was evaluated. Crude muscle extracts from control and experimental groups (TNF-a s.c. @ 50 microg/kg body wt each day for 5 days or cortisone s.c. @ 100 mg/kg body wt for 5 days) were used to purify eIF-4E by immunoprecipitation and polyacrylamide gel electrophoresis (PAGE) followed by Western blot analysis. Quantification of eIF-4E was done by densitometry. Both TNF-a and cortisone induced a marked decline in the concentration of eIF-4E in rat skeletal muscle. There was no difference in the ratio of phosphorylated to unphosphorylated eIF-4E after TNF-a treatment. These findings suggest that both TNF-a and cortisone inhibit peptide chain initiation in skeletal muscle cells by decreasing the expression of eIF-4E.

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