The obtained images were analyzed using Image Master 6. 0.

Moreover, siRNA mediated PLD1 depletion or PLD1 inhibition de creased Akt phosphorylation levels, whereas PLD1 overexpression had the opposite effect. It is worth mentionning that PLD2 overexpression induced moderate, non significant, effects on S6K1 or Akt activa tion. buy AP24534 Together, these results suggest that, in L6 myotubes, PLD is involved in both mTORC1 and mTORC2 activation, mainly through its PLD1 isoform. We also observed that treating myotubes by dexametha sone or 1 butanol induced an inhibition of both S6K1 and Akt phosphorylation, therefore confirming that in atrophy promoting conditions mTOR signaling is inhibited. Furthermore, we verified that siRNA mediated depletion of Rictor decreased the phosphorylation of Akt, confirming that Akt is a substrate for mTORC2 in L6 myotubes.<br><br> Discussion Skeletal muscle displays a striking plasticity, mature muscle cells undergoing drastic changes in their size and specific protein content to adapt the tissue to different levels of mechanical stimulation or nutrient income, or to hypercatabolic pathological situations. mTOR signaling is known to play a central role in the AT7519 844442-38-2 mecha nisms that control muscle plasticity. The involve ment of PLD in muscle hypertrophy induced by mechanical loading has been hypothesized, due to the functional connection that exists between PLD activity and mTOR signaling. Mechanical stimuli have been shown to induce a PLD dependent mTORC1 activation in isolated muscles, however the participation of PLD in the hypertrophic response was not demonstrated.<br><br> Here we report that, in differentiated FDA approved Akt 阻害剤 myotubes, the suppression of PLD activity obtained by either addition of a primary alcohol or specific inhibitors, or by RNA interference, results in an atrophic effect, as evidenced by a size reduction and a decrease in the content in muscle proteins such as creatine kinase or MHC. Con versely, we observed that the overexpression of PLD is able to induce marked hypertrophic effects, showing that muscle cell size is positively regulated by PLD. In both the cases of PLD inhibition and overexpression, we ob served that trophic effects depend on PLD1, rather than PLD2. Although these two PLD isoforms display a strong sequence homology, and are both dependent on PIP2 for their activity, they exhibit quite different regula tory properties and subcellular localizations.<br><br> Whereas PLD1 has a low basal activity in vitro and is activated by small G proteins and protein kinase C, PLD2 has a high basal activity and does not respond to the PLD1 activators. Moreover, under steady state conditions, PLD1 has a predominently perinuclear loca tion, whereas PLD2 is found at the plasma membrane, which suggests that the isoforms have different bio logical functions. The respective participation of PLD1 and PLD2 in mTORC1 activation is still debated. Thus, PLD1 was shown to be indispensable for amino acid activation of mTORC1. Rheb, which is implicated in the activation of mTORC1, directly activates PLD1. However, PLD1 and PLD2 domin ant negative mutants have both been found to suppress mTORC1 and mTORC2 activity, and PLD2 over expression can activate mTORC1.