In this study, food-deprived (18 h) control rats and rats with alloxan-induced diabetes were orally administered saline or the amino acid leucine to assess whether it regulates protein synthesis independently of a change in serum insulin concentrations. Immediately after leucine administration, diabetic rats were infused with insulin (0.0, 4.0, or 20 pmol ⅐ min -1 ⅐ kg -1 ) for 1 h to examine the role of the hormone in the protein synthetic response to leucine. In control rats, leucine stimulated protein synthesis by 58% and increased phosphorylation of the translational repressor, eukaryotic initiation factor (eIF) 4E-binding protein (BP)-1, 4E-BP1, fivefold. Consequently, association of the mRNA cap-binding protein eukaryotic initiation factor (eIF)4E with 4E-BP1 was reduced to 50% of control values, and eIF4G•eIF4E complex assembly was increased 80%. Furthermore, leucine increased the phosphorylation of the 70-kDa ribosomal protein S6 (rp S6) and the ribosomal protein S6 kinase (S6K1). Diabetes attenuated protein synthesis compared with control rats. Nonetheless, in diabetic rats, leucine increased protein synthesis by 53% without concomitant changes in the phosphorylation of 4E-BP1 or S6K1. Skeletal muscle protein synthesis was stimulated in diabetic rats infused with insulin, but rates of synthesis remained less than values in nondiabetic controls that were administered leucine. Phosphorylation of 4E-BP1 and S6K1 was increased in diabetic rats infused with insulin in a dose-dependent manner, and the response was enhanced by leucine. The results suggest that leucine enhances protein synthesis in skeletal muscle through both insulin-dependent and -independent mechanisms. The insulin-dependent mechanism is associated with increased phosphorylation of 4E-BP1 and S6K1. In contrast, the insulin-independent effect on protein synthesis is mediated by an unknown mechanism. Diabetes 51: -936, 2002A fter consumption of a protein-containing meal, the fractional rate of protein synthesis of total mixed proteins in skeletal muscle of growing animals is upregulated. Two vital components of this response are elevations in circulating concentrations of the hormone insulin and an increase in amino acid supply. The relative importance of each of these components in regulating protein synthesis continues to be a topic of investigation and controversy. Several reports indicate that physiological increases in circulating insulin concentrations are not sufficient to stimulate protein synthesis in food-deprived rats (1-4). When postabsorptive rats are administered an oral bolus of carbohydrate alone, no change in protein synthesis is observed. In contrast, when food-deprived rats are administered an isocaloric macronutrient-mixed meal, rates of protein synthesis are stimulated (1). Plasma insulin concentrations in rats administered either meal are similar; hence, the enhanced rate of recovery in rats administered a mixed meal cannot be attributed to a differential insulin response between the groups. Likewise, when fasted rats are...

The goal of these studies was to investigate the mechanisms by which amino acid supply regulates global rates of protein synthesis as well as the translation of ribosomal protein (rp) mRNAs in liver. In the experiments conducted, male weanling rats were trained over a 2-wk period to consume their daily food intake within 3 h. On day 14, rats were fed the control diet or an isocaloric, isonitrogenous diet lacking glycine, tryptophan, leucine, or the branched-chain amino acids (BCAA) for 1 h. Feeding Trp-, Leu-, or BCAA-deficient diets resulted in significant reductions in serum insulin, hepatic protein synthesis, eukaryotic initiation factor 2B (eIF2B) activity, and phosphorylation of eIF4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K1). Phosphorylation of eIF2alpha was inversely related to eIF2B activity under all conditions. Alterations in the hepatic synthesis of rp were assessed by changes in the distribution of rp (S4, S8, L26) mRNAs across sucrose density gradients and compared with non-rp (beta-actin, albumin) mRNAs. In all dietary treatments, non-rp mRNAs were mostly polysome associated. Conversely, the proportion of rp mRNAs residing in polysomes was two- to fivefold less in rats fed diets lacking tryptophan, leucine, or BCAA compared with rats fed the control diet. Total hepatic abundance of all mRNAs examined did not differ among treatment groups. For all parameters examined, there were no differences between rats fed the glycine-deficient diet and rats fed the control diet. The data suggest that essential amino acid (EAA) deficiency inhibits global rates of liver protein synthesis via a block in translation initiation. Additionally, the translation of rp mRNAs is preferentially repressed in association with decreased S6K1 phosphorylation.

. Repression of protein synthesis and mTOR signaling in rat liver mediated by the AMPK activator aminoimidazole carboxamide ribonucleoside. Am J Physiol Endocrinol Metab 288: E980 -E988, 2005. First published December 21, 2004; doi:10.1152/ajpendo.00333.2004.-The studies described herein were designed to investigate the effects of 5-aminoimidazole-4-carboxamide-1-␤-D-ribonucleoside (AICAR), an activator of the AMP-activated protein kinase (AMPK), on the translational control of protein synthesis and signaling through the mammalian target of rapamycin (mTOR) in rat liver. Effects of AICAR observed in vivo were compared with those obtained in an in situ perfused liver preparation to investigate activation of AMPK in the absence of accompanying changes in hormones and nutrients. AMPK became hyperphosphorylated, as assessed by a gel-shift analysis, in response to AICAR both in vivo and in situ; however, increased relative phosphorylation at the Thr172 site on the kinase was observed only in perfused liver. Phosphorylation of AMPK either in vivo or in situ was associated with a repression of protein synthesis as well as decreased phosphorylation of a number of targets of mTOR signaling including ribosomal protein S6 kinase 1, eukaryotic initiation factor (eIF)4G, and eIF4E-binding protein (4E-BP)1. The phosphorylation changes in eIF4G and 4E-BP1 were accompanied by a reduction in the amount of eIF4E present in the active eIF4E ⅐ eIF4G complex and an increase in the amount present in the inactive eIF4E ⅐ 4E-BP1 complex. Reduced insulin signaling as well as differences in nutrient availability may have contributed to the effects observed in vivo as AICAR caused a fall in the serum insulin concentration. Overall, however, the results from both experimental models support a scenario in which AICAR directly represses protein synthesis and mTOR signaling in the liver through an AMPK-dependent mechanism. translation initiation; eukaryotic initiation factor 4E; eukaryotic initiation factor 4G; mammalian target of rapamycin THE AMP-ACTIVATED PROTEIN KINASE (AMPK) acts as an energy sensor that responds to changes in the intracellular ratio of AMP:ATP (13). Activation of AMPK results in the stimulation of a variety of cellular processes involved in ATP production, e.g., glucose uptake (36, 44, 54) and fatty acid oxidation (7,16,37,39), and a repression of energy-consuming processes, e.g., fatty acid (16) and protein (3,8,18) synthesis. The repression of protein synthesis by AMPK is associated with decreased signaling through the protein kinase referred to as the mammalian target of rapamycin (mTOR) (8,20,30,31). Decreased signaling through mTOR leads to downregulated phosphorylation of several proteins that play important roles in regulating the initiation phase of mRNA translation, including the eukaryotic initiation factor (eIF)4E-binding protein, 4E-BP1 (6), and the 70-kDa ribosomal protein (rp)S6 protein kinase, S6K1 (6). It also downregulates mRNA elongation through modulation of eukaryotic elongation factor (eEF)2 kinase...

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