Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis

Abstract

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 microg x kg(-1) x day(-1)) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P<0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P<0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1.eIF4E complex association, and increased active eIF4E.eIF4G complex formation (P<0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.

Fig. 1.

Pancreatic-glucose-amino acid clamp protocol. Overnight-fasted diluent and somatotropin (pST)-treated pigs were injected with diluent or pST, respectively, 60 min before infusion. Fifteen minutes [time (t) = −15 min] before infusion, a primed, constant infusion of somatostatin was initiated. At t = 0 min, infusions of replacement glucagon and insulin commenced. Insulin was infused to reproduce fasting (5 μU/ml) or fed pST-treated (50 μU/ml) levels, amino acid concentrations were clamped at fasting [500 nmol branched-chain amino acid (BCAA)/ml] or fed (1,000 nmol BCAA/ml) levels, and glucose was clamped at baseline fasting levels. Fractional rates of protein synthesis were measured using a flooding dose of [³H]phenylalanine. Blood samples were collected at 5-min intervals to clamp glucose and amino acids, with additional samples taken at baseline and 2 h for hormone and substrate analysis.

Fig. 2.

Whole body net glucose disposal rates during pancreatic-glucose-amino acid clamps in diluent and pST-treated pigs. Pigs were infused with insulin to reproduce fasting (5 μU/ml) and fed (50 μU/ml) levels, and amino acids were clamped at either fasting (500 nmol BCAA/ml) or fed (1,000 nmol BCAA/ml) levels, whereas glucose was clamped at baseline fasting levels. Glucose disposal rates were calculated from the average rate of infusion of a 50% dextrose solution over the last hour of infusion. ANOVA indicated both a treatment [fasting, fed amino acid (AA), or fed insulin + AA] (P < 0.001) and pST (P < 0.05) effect. *Results of Tukey's multiple-comparison test: response to treatment with amino acids and/or insulin different from control group. Values are means ± SE; n = 6–8 pigs/group.

Fig. 3.

Fractional rate of protein synthesis (K_s) in skeletal muscle of pigs during pancreatic-glucose-amino acid clamps in diluent and pST-treated pigs. Pigs were infused with insulin to reproduce fasting (5 μU/ml) and fed (50 μU/ml) levels, and amino acids were clamped at either fasting (500 nmol BCAA/ml) or fed (1,000 nmol BCAA/ml) levels, whereas glucose was clamped at baseline fasting levels. ANOVA indicated a treatment (fasting, fed AA, or fed insulin + AA) (P < 0.001) and a pST (P < 0.001) effect and an interaction (P < 0.05). Results of Tukey's multiple-comparison test: response to treatment with amino acids and/or insulin different from control group (*) and effect of pST-treatment different from diluent (†). Values are means ± SE; n = 6–8/group.

Fig. 4.

Ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1) phosphorylation in skeletal muscle during pancreatic-glucose-amino acid clamps in diluent and pST-treated pigs. Pigs were infused with insulin to reproduce fasting (5 μU/ml) and fed (50 μU/ml) levels, and amino acids were clamped at either fasting (500 nmol BCAA/ml) or fed (1,000 nmol BCAA/ml) levels, whereas glucose was clamped at baseline fasting levels. ANOVA indicated a treatment (fasting, fed AA, or fed insulin + AA; P < 0.02) effect for both factors. *Results of Tukey's multiple-comparison test: response to treatment with amino acids and/or insulin different from control group. Values are means ± SE; n = 6–8/group.

Fig. 5.

Association of the 4EBP1·eIF4E and eIF4G·eIF4E complexes in skeletal muscle during pancreatic-glucose-amino acid clamps in diluent and pST-treated pigs. Pigs were infused with insulin to reproduce fasting (5 μU/ml) and fed (50 μU/ml) levels, and amino acids were clamped at either fasting (500 nmol BCAA/ml) or fed (1,000 nmol/BCAA ml) levels, whereas glucose was clamped at baseline fasting levels. ANOVA indicated a treatment (fasting, fed AA, or fed insulin + AA) (P < 0.004) effect for both complexes. *Results of Tukey's multiple-comparison test: response to treatment with amino acids and/or insulin different from control group. Values are means ± SE; n = 6–8/group.

Fig. 6.

eIF4G phosphorylation in skeletal muscle during pancreatic-glucose-amino acid clamps in diluent and pST-treated pigs. Pigs were infused with insulin to reproduce fasting (5 μU/ml) and fed (50 μU/ml) levels, and amino acids were clamped at either fasting (500 nmol BCAA/ml) or fed (1,000 nmol BCAA/ml) levels, whereas glucose was clamped at baseline fasting levels. ANOVA indicated a treatment (fasting, fed AA, or fed insulin + AA) effect (P < 0.01). *Results of Tukey's multiple-comparison test: response to treatment with amino acids and/or insulin different from control group. Values are means ± SE; n = 6–8/group.