High‐protein diets (HPDs) promote weight loss but other studies implicate these diets and their constituent amino acids (AAs) in insulin resistance. We hypothesized that AA‐induced insulin resistance is a temporal and reversible metabolic event. L6 myotubes were serum deprived for 4 h and then incubated in AA and/or insulin (100 nmol/L). Another group of cells was incubated overnight in AA + insulin, starved again, and then reincubated with AA and insulin. Mammalian (mechanistic) target of rapamycin complex 1 (mTORC1) signaling and glucose uptake were then measured. Healthy or insulin‐resistant rats were gavaged with leucine (0.48 g/kg) and insulin sensitivity was examined. In myotubes, incubation with AA and insulin significantly (P <0.05) increased the phosphorylation of the mTORC1 substrate ribosomal protein S6 kinase 1 (S6K1, T389) and of insulin receptor substrate 1 (IRS‐1, serine residues), but suppressed insulin‐stimulated glucose uptake by 40% (P <0.01). These modifications were mTORC1‐dependent and were reversible. In vivo, leucine gavage reversibly increased S6K1 phosphorylation and IRS‐1 serine phosphorylation 5‐ to 12‐fold in skeletal muscle and impaired insulin tolerance of glucose (P <0.05) in lean rats. In insulin‐resistant rats, the impairment of whole blood glucose and AA metabolism induced by leucine gavage (0.001 < P <0.05) was more severe than that observed in lean rats; however, the impairment was reversible within 24 h of treatment. If these data are confirmed in long‐term studies, it would imply that the use of leucine/HPD in treating metabolic diseases is unlikely to have lasting negative effects on insulin sensitivity.

. Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats. Am J Physiol Endocrinol Metab 300: E986 -E992, 2011. First published March 15, 2011; doi:10.1152/ajpendo.00642.2010.-Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (ϳ100 g; n ϭ 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser 67 phosphorylation of PDCD4 (Ϫ50%) but increased PDCD4 abundance (P Ͻ 0.05); refeeding reversed these changes (P Ͻ 0.05). Consistent with these effects being regulated by S6K1, activation of this kinase was suppressed by FD (Ϫ91%, P Ͻ 0.05) but was increased by refeeding. Gavaging rats subjected to FD with a mixture of amino acids partially restored muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid-and serumfree medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P Ͻ 0.0001). Thus feeding stimulates fractional protein synthesis in skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor. programmed cell death 4; protein metabolism; ribosomal protein S6 kinase; mRNA translation; mammalian target of rapamycin complex 1 OPTIMAL SKELETAL MUSCLE MASS AND METABOLISM are critical to the regulation of whole body substrate homeostasis and health. Indeed, in several diseases, including obesity, diabetes, and cancer, defects in muscle metabolism underlie or exacerbate the metabolic outcomes (21,24,41).The growth factor-and nutrient-sensitive kinase complex mammalian target of rapamycin complex 1 (mTORC1) is now recognized as a master regulator of skeletal muscle mass (6,7,42,43). Upon activation, this complex phosphorylates two principal substrates, the ribosomal S6 protein kinase 1 (S6K1) and the eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1) (20,40). Phosphorylation of S6K1 leads to its activation, whereas 4E-BP1, an inhibitor of cap-dependent mRNA translation initiation, is inhibited by phosphorylation. S6K1 mediates the growth-promoting action of mTORC1, whereas inhibition of 4E-BP1 increases cell proliferation (9, 16).S6K1 is critical to the regulation of skeletal muscle mass because mice lacking this enzyme have reduced muscle mass (3, 38). In several stu...

Aims/hypothesis Poorly controlled type 1 diabetes mellitus can cause reduced skeletal muscle mass and weakness during adolescence, which may affect long-term management of the disease. The aim of this study was to determine whether regular voluntary physical activity and leucine feeding restore rates of protein synthesis and deficits in skeletal muscle mass in a young, hypoinsulinaemic/hyperglycaemic rat model of diabetes. Methods Four-week-old male Sprague-Dawley rats were partially pancreatectomised (Px) to induce hypoinsulinaemia/hyperglycaemia and housed with/without access to running wheels for 3 weeks (n=12-14/group). Sham surgery rats (shams) served as sedentary controls (n=18). Protein synthesis and markers of protein anabolism were assessed in the fasted state and following leucine gavage.Fibre type and cross-sectional areas of the gastrocnemius muscle were measured using a metachromatic ATPase stain. Results Compared with sedentary behaviour, regular activity lowered fasting glycaemia and reduced fed hyperglycaemia in Px rats. Active-Px rats, which ran 2.2±0.71 km/night, displayed greater muscle mass and fibre areas similar to shams, while sedentary-Px rats displayed a 20-30% loss in muscle fibre areas. Muscle protein synthesis (basal and in response to leucine gavage) was impaired in sedentary-Px (by~65%), but not in active-Px rats, when compared with shams. Following leucine gavage, the phosphorylation status of eIF4E binding protein 1 (4E-BP1) and ribosomal S6 kinase 1 (S6K1), markers of mammalian target of rapamycin complex 1 (mTORC1) signalling, increased in shams (by two-and ninefold, respectively) and in active-Px (1.5-and fourfold, respectively) rats, but not in sedentary-Px rats. Conclusion/interpretation Moderate physical activity in young Px rats normalises impairments in skeletal muscle growth and protein synthesis. These findings illustrate the critical compensatory role that modest physical activity and targeted nutrition can have on skeletal muscle growth during periods of hypoinsulinaemia in adolescent diabetes.

Optimal skeletal muscle mass is vital to health as defects in muscle protein metabolism underlie or exacerbate human diseases. The mTORC1 is a critical regulator of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined the expression of PDCD4 in skeletal muscle and its regulation by nutrition. Male rats (~100g, n = 6) were subjected to feed deprivation (FD) for 48 h; some rats were re‐fed for 2 h. FD suppressed muscle protein synthesis and serine 67 phosphorylation of PDCD4 (−50%) but increased PDCD4 abundance (P<0.05); re‐feeding reversed these changes (P<0.05). Consistent with these effects being regulated by S6K1, activation of this kinase was suppressed by FD (−91%, P<0.05) but was increased by re‐feeding. Gavaging rats subjected to FD with a mixture of amino acids (AA) restored muscle protein synthesis and reduced PDCD4 abundance relative to FD (P<0.05). Finally, when myoblasts were grown in AA‐ and serum‐free medium, rates of proteins synthesis in cells depleted of PDCD4 more than doubled the values in cells with a normal level of this protein (P<0.0001). Thus, AA stimulate protein synthesis in skeletal muscle in parallel with the reduction of the abundance of PDCD4.