Amino-acid limitation induces the GCN2 signaling pathway in myoblasts but not in myotubes.

There is a growing body of evidence that suggests that amino acids play an important role in controlling gene expression, but the cell specificity of the amino-acid-mediated regulation of gene expression in mammals remains unknown. Using a model of muscle cells (C2C12) at two stages of differentiation, i.e. myoblasts and myotubes, we employed transcriptional profiling to show that amino-acid deficiency does not regulate the same set of gene in differentiated and non-differentiated cells. Furthermore, in myotubes, the GCN2 pathway is not activated by amino-acid starvation due to an amino-acid supply from intracellular proteolysis associated with a low GCN2 expression.

Phospho-proteomic approach to identify new targets of leucine deprivation in muscle cells.

The aim of this study was to optimize a protocol that allows identifying changes at the phosphorylation level of specific proteins in response to cell stimulation by leucine starvation. To make possible the identification of differentially phosphorylated proteins by the combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we prepared fraction enriched in phosphoproteins. For that purpose, we adapted the immobilized metal affinity chromatography (IMAC) technique to make it compatible with 2D-PAGE. On the whole, this procedure allowed identifying regulated targets of leucine deprivation: molecular chaperones glucose-regulated protein 58 kDa (GRP58) and BiP (GRP78), RNA helicase DEAD box polypeptide 3, and eukaryotic translation initiation factor 4B (eIF4B).

Activation of mammalian target of rapamycin complex 1 and insulin resistance induced by palmitate in hepatocytes.

Excessive supply of fatty acids to the liver might be a contributing factor to hepatic insulin resistance associated with obesity and type 2 diabetes mellitus. The aim of this study was to investigate direct effects of palmitate on insulin signaling in hepatocytes. The ability of metformin to reverse changes induced by palmitate was also studied. Rat hepatocytes in primary culture exhibited a rightward shift of the insulin dose-response curve for PKB phosphorylation during culture with palmitate. The insulin-stimulated phosphorylation of GSK-3beta, a metabolic substrate of PKB, was diminished in palmitate hepatocytes. By contrast, the mTOR protein kinase was overstimulated in cells incubated with palmitate. Hepatocytes cultured with palmitate displayed hyperphosphorylation of IRS-1 at Ser residues 632/635, known to be phosphorylated by mTOR. Metformin treatment of the hepatocytes resulted in activation of the AMP-activated kinase, attenuation of the mTOR/S6K1 pathway, reduction of IRS-1 phosphorylation, and a leftward shift in the insulin dose-response curve for PKB activation. These data suggest a link between an oversupply of fatty acid to hepatocytes, a disproportionate stimulation of mTOR/S6K1, and resistance to insulin.

Insulin induction of glucokinase and fatty acid synthase in hepatocytes: analysis of the roles of sterol-regulatory-element-binding protein-1c and liver X receptor.

The transcription activator SREBP-1c (sterol-regulatory-element-binding protein-1c) is induced by insulin in the liver and is considered a master regulator of lipogenic genes such as FASN (fatty acid synthase). The question of whether SREBP-1c is also a mediator of insulin action on the regulatory enzyme of glucose metabolism GCK (glucokinase) is controversial. In the present paper, we induced SREBP-1c to various levels with insulin or the liver X receptor ligand T0901317 in primary hepatocytes and asked if these levels correlated with those of GCK or FASN mRNA expression, using the latter as positive control. Insulin and T0901317 triggered the accumulation of precursor and processed forms of SREBP-1c to similar levels and with comparable kinetics, and both effectors together caused synergistic increases in SREBP-1c protein levels. These effects were accompanied by commensurate elevation of FASN mRNA, notably by a synergistic response to both effectors. By contrast, GCK mRNA was unresponsive to T0901317 and was induced only by insulin. Treatment of hepatocytes with insulin and/or T0901317 resulted in the recruitment of SREBP-1c to the FASN promoter as shown by chromatin immunoprecipitation, whereas SREBP-1c did not bind to the GCK promoter. Lastly, we observed that the glycogen synthase kinase-3 inhibitor SB216763 produced a small increase in SREBP-1c protein level, which was further augmented in the presence of T0901317. The level of FASN mRNA varied in parallel with SREBP-1c, while GCK mRNA was unaffected. Collectively, these results showed that increases in SREBP-1c were neither necessary nor sufficient for GCK induction in hepatocytes, while at the same time they underscored the role of SREBP-1c as a key regulator of FASN.

Regulation of protein synthesis by leucine starvation involves distinct mechanisms in mouse C2C12 myoblasts and myotubes.

Leucine modulates protein translation in higher eukaryotes by affecting phosphorylation and the function of proteins that regulate the initiation and/or elongation steps. These include the initiation factor 4E binding protein 1 (4E-BP1), initiation factor 4E (eIF4E), initiation factor 2 (eIF2alpha), ribosomal S6 kinases (S6K1/2), and elongation factor 2 (eEF2). The alteration of protein translation by leucine starvation was studied during myogenic differentiation using the mouse C2C12 cell line as well as the role of rapamycin-sensitive mTOR (mammalian target of rapamycin) in the signaling of leucine in myotubes. A time course study showed that 1 h of leucine starvation decreased protein synthesis and S6K1 phosphorylation in myoblasts, whereas 3-5 h of starvation were necessary to induce such an alteration in myotubes. Although S6K1 phosphorylation was reduced in leucine-deprived myotubes, S6K2 and S6 phosphorylation were not affected. In contrast, rapamycin decreased the phosphorylation of S6K2 and S6 in myotubes. It is therefore likely that under the conditions present, the rapamycin-sensitive mTOR was not affected by leucine starvation. S6K1 dephosphorylation may thus be mTOR independent, and the functional mTOR/S6K2 pathway may maintain S6 phosphorylation. An increased phosphorylation of eEF2 in myoblasts and myotubes indicated that global protein synthesis was reduced via a decrease in translation elongation. An increased association between 4E-BP1 and eIF4E, and increased phosphorylation of eIF2alpha also contributed to decreasing protein synthesis in leucine-starved myoblasts. In contrast, in leucine-starved myotubes, there were no change in the 4E-BP1-eIF4E association or eIF2alpha phosphorylation, suggesting that these factors were not rate limiting for decreasing protein synthesis in leucine-deprived myotubes.

Amino acids as regulators of gene expression: molecular mechanisms.

Regulation of gene expression by nutrients in mammals is an important mechanism allowing them to adapt their physiological functions according to the supply of nutrient in the diet. It has been shown recently that amino acids are able to regulate by themselves the expression of numerous genes. CHOP, asparagine synthetase, and IGFBP-1 regulation following AA starvation will be described in this review with special interest in the molecular mechanisms involved.

Recent advances in the understanding of amino acid regulation of gene expression.

In mammals, the impact of nutrients on gene expression has become an important area of research. Because amino acids have multiple and important functions, their homeostasis has to be finely maintained. However, amino acidemia can be affected by certain nutritional conditions or various forms of stress. Consequently, mammals must adjust several of the physiological functions involved in the adaptation to amino acid availability by regulating expression of numerous genes. It has been shown that amino acids alone can modify the expression of target genes. However, understanding of amino acid-dependent control of gene expression has just started to emerge. This review focuses on recent advances in the understanding of mechanisms involved in the amino acid control of gene expression.

Leucine: a key amino acid in ageing-associated sarcopenia?

During ageing, a progressive loss of muscle mass has been well described in both man and rodents. This loss of proteins results from an imbalance between protein synthesis and degradation rates. Although some authors have shown a decrease of myofibrillar protein synthesis rates in human volunteers, this imbalance is not clearly apparent when basal rates of protein turnover are measured. A decrease in muscle protein synthesis stimulation was detected nevertheless in ageing rats during the postprandial period, suggesting that the 'meal signal' was altered during ageing. Many results now suggest that aged muscle is less sensitive to the stimulatory effect of amino acids at physiological concentrations but is still able to respond if the increase in aminoacidaemia is sufficiently large. Indeed amino acids play an important role in regulating muscle protein turnover both in vitro and in vivo. At the molecular level, amino acids modulate gene expression. Amino acid response elements have been characterised in the promoter of transcriptional factor CCAAT-enhancer binding protein homologous protein and asparagine synthetase genes. Among amino acids, leucine seems to play the major role in regulating the metabolic function. It inhibits proteolysis and stimulates muscle protein synthesis independently of insulin. Leucine has been shown to act as a real mediator by modulating specifically the activities of intracellular kinases linked to the translation of proteins such as phosphatidylinosinol 3' kinase and mammalian target of rapamycin-70 kDa ribosomal protein S6 (p70S6K) kinases. We recently demonstrated in vitro that protein synthesis of ageing rat muscles becomes resistant to the stimulatory effect of leucine in its physiological concentration range. However, when leucine concentration was increased greatly above its postprandial level, protein synthesis was stimulated normally. Moreover, we studied the effect of meal leucine supplementation on in vivo protein synthesis in adult and ageing rats. Leucine supplementation had no additional effect on muscle protein synthesis in adults but totally restored its stimulation in ageing rats. Whether chronic oral leucine supplementation would be beneficial for maintaining muscle protein mass in elderly men and women remains to be studied.