This study compared the effects of leucine and glutamine on the mTOR pathway, on protein synthesis and on muscle-specific gene expression in myogenic C(2)C(12) cells. Leucine increased the phosphorylation state of mTOR, on both Ser2448 and Ser2481, and its downstream effectors, p70(S6k), S6 and 4E-BP1. By contrast, glutamine decreased the phosphorylation state of mTOR on Ser2448, p70(S6k) and 4E-BP1, but did not modify the phosphorylation state of mTOR on Ser2481 and S6. Whilst the phosphorylation state of the mTOR pathway is usually related to protein synthesis, the incorporation of labelled methionine/cysteine was only transiently modified by leucine and was unaltered by glutamine. However, these two amino acids affected the mRNA levels of desmin, myogenin and myosin heavy chain in a time-dependant manner. In conclusion, leucine and glutamine have opposite effects on the mTOR pathway. Moreover, they induce modification of muscle-specific gene expression, unrelated to their effects on the mTOR/p70(S6k) pathway.

Like insulin, leucine stimulates the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) axis in various organs. Insulin proceeds via the canonical association of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent protein kinase-1 (PDK1), and protein kinase B (PKB/Akt). The signaling involved in leucine effect, although known to implicate a PI3K mechanism independent of PKB/Akt, is more poorly understood. In this study, we investigated whether PDK1 could also participate in the events leading to mTOR/p70S6K activation in response to leucine in the heart. In wild-type hearts, both leucine and insulin increased p70S6K activity whereas, in contrast to insulin, leucine was unable to activate PKB/Akt. The changes in p70S6K activity induced by insulin and leucine correlated with changes in phosphorylation of Thr389, the mTOR phosphorylation site on p70S6K, and of Ser2448 on mTOR, both related to mTOR activity. Leucine also triggered phosphorylation of the proline-rich Akt/PKB substrate of 40 kDa (PRAS40), a new pivotal mTOR regulator. In PDK1 knockout hearts, leucine, similarly to insulin, failed to induce the phosphorylation of mTOR and p70S6K, leading to the absence of p70S6K activation. The loss of leucine effect in absence of PDK1 correlated with the lack of PRAS40 phosphorylation. Moreover, the introduction in PDK1 of the L155E mutation, which is known to preserve the insulin-induced and PKB/Akt-dependent phosphorylation of mTOR/p70S6K, suppressed all leucine effects, including phosphorylation of mTOR, PRAS40, and p70S6K. We conclude that the leucine-induced stimulation of the cardiac PRAS40/mTOR/p70S6K pathway requires PDK1 in a way that differs from that of insulin.

Consuming diets rich in fatty acids leading to excessive weight gain has been identified as a major contributor to the pathogenesis of Type 2 Diabetes (T2D), a known risk factor for cardiovascular disease. Associated with diet-induced obesity (DIO) are elevated levels of plasma fatty acids (FA) and lipoprotein associated triglycerides (TG). Since the levels of plasma FA and lipoprotein associated TG are primarily controlled by the liver, it is likely that aberrant hepatic FA uptake and handling contributes to the dyslipidemia that precedes development of T2D. Indeed, we have shown that mRNA and protein expression of the FA transport protein, CD36, which is normally expressed at low levels in the mouse liver, is significantly elevated during DIO and is correlated with increased hepatic TG storage and TG secretion as very low-density lipoproteins (VLDL). In vivo gene delivery of a recombinant adenovirus harboring CD36 cDNA (Ad.CD36) into lean mice showed a significant increase in hepatic FA uptake in vivo and an accumulation in hepatic TG storage and VLDL-TG secretion compared to Ad.Null-injected mice, demonstrating that increased CD36 expression alone is sufficient to recapitulate the alterations in liver lipid storage and VLDL-TG secretion observed in DIO. Together these data show that increased hepatic CD36 expression may play a causative role in the dyslipidemia associated with DIO and further suggests that inhibition of hepatic CD36 may prove to be beneficial for the prevention of insulin resistance and the eventual development of T2D.