Mitochondria (two, five, 11). As observed for PC-TP-deficient mice, Them2-/-mice exhibit phenotypes that include reduced hepatic glucose production and resistance to diet-induced diabetes (12). The enzymatic activities of both purified recombinant THEM2 and PC-TP are improved by their interaction (ten, 11), but the biological relevance of this effect is unknown. To elucidate molecular mechanisms that may underlie improved glucose homeostasis in Pctp-/-mice, we examined the regulation of insulin signaling by PC-TP in cell culture systems and in vivo. Insulin binding to its receptor activates insulin receptor substrate (IRS) proteins, which market phosphoinositide 3-kinase (PI3K)- and 3-phosphoinositidedependent kinase (PDK1)-mediated phosphorylation of Akt at Thr308 (13, 14). Complete activation of Akt calls for further phosphorylation at Ser473, which can be mediated by mammalian target of rapamycin (mTOR) complex 2 (mTORC2) (15). Among the downstream targets of phosphorylated Akt are AMPK (16), GSK-3 (17) and tuberous sclerosis complex 2 (TSC2) (18). The TSC1-TSC2 complicated inhibits mTORC1 and its targets, including S6K1 (19). siRNA-mediated knockdown, genetic ablation or chemical inhibition of PC-TP enhanced insulin signaling by enhancing insulin-independent activation of IRS2, with knockdown of THEM2 yielding related effects. PC-TP and THEM2 also suppressed mTORC1 signaling upon binding TSC2. Chemical inhibition of PC-TP to stop phosphatidylcholine binding (7) disrupted the interactions of PC-TP with THEM2 and TSC2.Ondansetron Our findings reveal a phospholipid-dependent mechanism for suppressing insulin signaling: a PC-TP-THEM2 complicated reduces phosphorylation and stability of IRS2 and increases stability of the TSC1TSC2 complicated.Rifaximin NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal.PMID:23415682 Author manuscript; offered in PMC 2014 March 19.Ersoy et al.PageResultsInhibition on the activity of Akt and its crucial targets by PC-TP and THEMNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptIn serum-starved HEK 293E cells, knockdown of THEM2 was linked using a 50 reduction in PC-TP abundance, whereas knockdown of PC-TP tended to enhance THEM2 abundance (Fig. 1, A and B). We subsequent determined irrespective of whether PC-TP and THEM2 influenced the activity of Akt and its essential targets. Inside the absence of insulin, siRNA-mediated knockdown of either PC-TP or THEM2 resulted in 2- and 3-fold increases within the phosphorylation of Akt at Ser473, a 30 lower within the phosphorylation of AMPK for either situation, and 3- and 6-fold increases inside the phosphorylation of S6K1, respectively (Fig. 1, A and C). Phosphorylation of GSK-3 was increased 2-fold following PC-TP or THEM2 knockdown, while these effects did not achieve statistical significance. In response to insulin, the elevated phosphorylation of Akt at each Ser473 and Thr308 was additive towards the raise in basal activation of Akt (Fig. 1D). Due to the fact the 48 h time frame for PC-TP knockdown allowed for the possibility that effects on Akt activation were secondary to adaptive transcriptional or metabolic effects, we used compound A1, a tiny molecule inhibitor that rapidly inactivates PC-TP and increases the phosphorylation of Akt, S6K1, and GSK-3 in key human hepatocytes (7). Short-term (30 min) therapy with compound A1 (Fig. 1E) enhanced the phosphorylation of Akt in major hepatocyte cultures from Pctp+/+ mice but not in these from Pctp-/-mice. Chemical inhibition of PC-TP was ass.