IRE1α and TRB3 do not contribute to the disruption of proximal insulin signaling caused by palmitate in C2C12 myotubes.

Endoplasmic reticulum (ER) stress is a central actor in the physiopathology of insulin resistance (IR) in various tissues. The subsequent unfolded protein response (UPR) interacts with insulin signaling through inositol-requiring 1α (IRE1α) activation and tribbles homolog 3 (TRB3) expressions. IRE1α impairs insulin actions through the activation of c-Jun N-terminal kinase (JNK), and TRB3 is a pseudokinase inhibiting Akt. In muscle cells, the link between ER stress and IR has only been demonstrated by using chemical ER stress inducers or overexpression techniques. However, the involvement of ER stress in lipid-induced muscle IR remains controversial. The aim of the study is to test whether palmitate-induced IRE1α signaling and TRB3 expression disturb insulin signaling in myogenic cells. C2C12 myotubes were exposed to palmitate and then stimulated with insulin. siRNA transfection was used to downregulate TRB3 and IRE1α. Palmitate increased TRB3 expression, activated IRE1α signaling, and reduced the insulin-dependent Akt phosphorylation. Knocking down TRB3 or IRE1α did not prevent the inhibitory effect of palmitate on Akt phosphorylation. Our results support the idea that ER stress is not responsible for lipid-induced IR in C2C12 myotubes.

Differential regulation of eEF2 and p70S6K by AMPKalpha2 in heart.

Eukaryotic elongation factor 2 (eEF-2) and mammalian target of rapamycin (mTOR)-p70 ribosomal protein S6 kinase (p70S6K) signaling pathways control protein synthesis and are inhibited during myocardial ischemia. Intracellular acidosis and AMP-activated protein kinase (AMPK) activation, both occurring during ischemia, have been proposed to participate in this inhibition. We evaluated the contribution of AMPKα2, the main cardiac AMPK catalytic subunit isoform, in eEF2 and mTOR-p70S6K regulation using AMPKα2 KO mice. Hearts were perfused ex vivo with or without insulin, and then submitted or not to ischemia. Insulin pre-incubation was necessary to activate mTOR-p70S6K and evaluate their subsequent inhibition by ischemia. Ischemia decreased insulin-induced mTOR-p70S6K phosphorylation in WT and AMPKα2 KO mice to a similar extent. This AMPKα2-independent p70S6K inhibition correlated well with the inhibition of PKB/Akt, located upstream of mTOR-p70S6K and can be mimicked in cardiomyocytes by decreasing pH. By contrast, ischemia-induced inhibitory phosphorylation of eEF-2 was drastically reduced in AMPKα2 KO mice. Interestingly, AMPKα2 also played a role under normoxia. Its deletion increased the insulin-induced p70S6K stimulation. This p70S6K over-stimulation was associated with a decrease in inhibitory phosphorylation of Raptor, an mTOR partner identified as an AMPK target. In conclusion, AMPKα2 controls cardiac p70S6K under normoxia and regulates eEF-2 but not the mTOR-p70S6K pathway during ischemia. This challenges the accepted notion that mTOR-p70S6K is inhibited by myocardial ischemia mainly via an AMPK-dependent mechanism.

Activation of the cardiac mTOR/p70(S6K) pathway by leucine requires PDK1 and correlates with PRAS40 phosphorylation.

Like insulin, leucine stimulates the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70(S6K)) 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/p70(S6K) activation in response to leucine in the heart. In wild-type hearts, both leucine and insulin increased p70(S6K) activity whereas, in contrast to insulin, leucine was unable to activate PKB/Akt. The changes in p70(S6K) activity induced by insulin and leucine correlated with changes in phosphorylation of Thr(389), the mTOR phosphorylation site on p70(S6K), and of Ser(2448) 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 p70(S6K), leading to the absence of p70(S6K) 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/p70(S6K), suppressed all leucine effects, including phosphorylation of mTOR, PRAS40, and p70(S6K). We conclude that the leucine-induced stimulation of the cardiac PRAS40/mTOR/p70(S6K) pathway requires PDK1 in a way that differs from that of insulin.

AMPK activation counteracts cardiac hypertrophy by reducing O-GlcNAcylation.

AMP-activated protein kinase (AMPK) has been shown to inhibit cardiac hypertrophy. Here, we show that submaximal AMPK activation blocks cardiomyocyte hypertrophy without affecting downstream targets previously suggested to be involved, such as p70 ribosomal S6 protein kinase, calcineurin/nuclear factor of activated T cells (NFAT) and extracellular signal-regulated kinases. Instead, cardiomyocyte hypertrophy is accompanied by increased protein O-GlcNAcylation, which is reversed by AMPK activation. Decreasing O-GlcNAcylation by inhibitors of the glutamine:fructose-6-phosphate aminotransferase (GFAT), blocks cardiomyocyte hypertrophy, mimicking AMPK activation. Conversely, O-GlcNAcylation-inducing agents counteract the anti-hypertrophic effect of AMPK. In vivo, AMPK activation prevents myocardial hypertrophy and the concomitant rise of O-GlcNAcylation in wild-type but not in AMPKα2-deficient mice. Treatment of wild-type mice with O-GlcNAcylation-inducing agents reverses AMPK action. Finally, we demonstrate that AMPK inhibits O-GlcNAcylation by mainly controlling GFAT phosphorylation, thereby reducing O-GlcNAcylation of proteins such as troponin T. We conclude that AMPK activation prevents cardiac hypertrophy predominantly by inhibiting O-GlcNAcylation.

Aging Reduces the Activation of the mTORC1 Pathway after Resistance Exercise and Protein Intake in Human Skeletal Muscle: Potential Role of REDD1 and Impaired Anabolic Sensitivity.

This study was designed to better understand the molecular mechanisms involved in the anabolic resistance observed in elderly people. Nine young (22 ± 0.1 years) and 10 older (69 ± 1.7 years) volunteers performed a one-leg extension exercise consisting of 10 × 10 repetitions at 70% of their 3-RM, immediately after which they ingested 30 g of whey protein. Muscle biopsies were taken from the vastus lateralis at rest in the fasted state and 30 min after protein ingestion in the non-exercised (Pro) and exercised (Pro+ex) legs. Plasma insulin levels were determined at the same time points. No age difference was measured in fasting insulin levels but the older subjects had a 50% higher concentration than the young subjects in the fed state (p < 0.05). While no difference was observed in the fasted state, in response to exercise and protein ingestion, the phosphorylation state of PKB (p < 0.05 in Pro and Pro+ex) and S6K1 (p = 0.059 in Pro; p = 0.066 in Pro+ex) was lower in the older subjects compared with the young subjects. After Pro+ex, REDD1 expression tended to be higher (p = 0.087) in the older group while AMPK phosphorylation was not modified by any condition. In conclusion, we show that the activation of the mTORC1 pathway is reduced in skeletal muscle of older subjects after resistance exercise and protein ingestion compared with young subjects, which could be partially due to an increased expression of REDD1 and an impaired anabolic sensitivity.