Role of AMPK in the regulation of protein synthesis and in the development of cardiac hypertrophy

The AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase acting as a cellular fuel gauge for the maintenance of energy homeostasis. The contractile activity of the heart being so “energy consuming”, the role of AMPK is central in this organ. It is even more critical under several pathological conditions including myocardial ischemia and cardiac hypertrophy. Among the different cellular processes regulated by AMPK, protein synthesis plays a key role. In this study, we first, evaluated the role of AMPK in the control of the signalling pathways involved in the regulation of protein synthesis, namely eEF2 and p70S6K, under normoxic and ischemic conditions using an AMPKα2 catalytic subunit knockout mouse model. Our results established the critical role of AMPK in the inhibition of the elongation factor eEF2 during ischemia but challenged the usual notion that the ischemia-mediated inhibition of the p70S6K pathway is controlled by AMPK. On the other hand, we showed that the absence of AMPKα2 promoted the stimulation of the p70S6K pathway under normoxic conditions. In the second part of our study, we evaluated the impact of AMPK activation on cardiac hypertrophy in a model of neonatal rat cardiomyocytes treated with the pro-hypertrophic agent phenylephrine. One of the particularities of this study was the use of a specific AMPK activator, the A-769662 compound. We demonstrated that A-769662 prevented the phenylephrine-induced stimulation of protein synthesis and of its regulators eEF2 and p70S6K. Moreover, the phenylephrine-dependent stimulation of two important gene transcription regulation pathways involving the MAP kinase ERK and the transcription factor NFAT was also counteracted by A-769662. Finally, by using a silencing RNA approach, we proved the implication of AMPK in the effect of the A-769662 compound on cardiac hypertrophy. In conclusion, our results provide evidence for the implication of AMPK in the control of various signalling elements involved in the regulation of protein synthesis and gene expression that could play an important role in various cardiac pathologies including myocardial ischemia and hypertrophy. AMPK activation might be a valuable tool for inhibiting or preventing adverse effect of such pathologies.(MED 3) -- UCL, 201

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

The type 1 insulin-like growth factor receptor (IGF-IR) pathway is mandatory for the follistatin-induced skeletal muscle hypertrophy

Myostatin inhibition by follistatin (FS) offers a new approach for muscle mass enhancement. The aim of the present study was to characterize the mediators responsible for the FS hypertrophic action on skeletal muscle in male mice. Because IGF-I and IGF-II, two crucial skeletal muscle growth factors, are induced by myostatin inhibition, we assessed their role in FS action. First, we tested whether type 1 IGF receptor (IGF-IR) is required for FS-induced hypertrophy. By using mice expressing a dominant-negative IGF-IR in skeletal muscle, we showed that IGF-IR inhibition blunted by 63% fiber hypertrophy caused by FS. Second, we showed that FS caused the same degree of fiber hypertrophy in wild-type and IGF-II knockout mice. We then tested the role of the signaling molecules stimulated by IGF-IR, in particular the Akt/mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (S6K) pathway. We investigated whether Akt phosphorylation is required for the FS action. By cotransfecting a dominant-negative form of Akt together with FS, we showed that Akt inhibition reduced by 65% fiber hypertrophy caused by FS. Second, we evaluated the role of mTOR in FS action. Fiber hypertrophy induced by FS was reduced by 36% in rapamycin-treated mice. Finally, because the activity of S6K is increased by FS, we tested its role in FS action. FS caused the same degree of fiber hypertrophy in wild-type and S6K1/2 knockout mice. In conclusion, the IGF-IR/Akt/mTOR pathway plays a critical role in FS-induced muscle hypertrophy. In contrast, induction of IGF-II expression and S6K activity by FS are not required for the hypertrophic action of FS

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

Sanchez Canedo C, Demeulder B, Ginion A, Bayascas JR, Balligand JL, Alessi DR, Vanoverschelde JL, Beauloye C, Hue L, Bertrand L. Activation of the cardiac mTOR/p70(S6K) pathway by leucine requires PDK1 and correlates with PRAS40 phosphorylation. Am J Physiol Endocrinol Metab 298: E761-E769, 2010. First published January 5, 2010; doi:10.1152/ajpendo.00421.2009.-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

AMPK activation inhibits cardiac hypertrophy. Here the authors show that this occurs independently of previously proposed mechanisms and that AMPK controls the phosphorylation of the aminotransferase GFAT, thereby preventing cardiac hypertrophy through the reduction of protein O-GlcNAcylation