A Role for Protein Phosphatase 2A–Like Activity, but Not Atypical Protein Kinase C, in the Inhibition of Protein Kinase B/Akt and Glycogen Synthesis by Palmitate Rosanna Cazzolli, Lee Carpenter, Trevor J. Biden, and Carsten Schmitz-Peiffer We have shown previously that palmitate treatment of C2C12 skeletal muscle myotubes causes inhibition of the protein kinase B (PKB) pathway and hence reduces insulin-stimulated glycogen synthesis through the ele- vation of intracellular ceramide levels. Ceramide is known to activate both atypical protein kinase C (aPKC)  and protein phosphatase (PP) 2A, and each of these effectors has been reported to inhibit PKB. In the present study, palmitate pretreatment was found to elevate PP2A-like activity in myotubes and to prevent its inhibition by insulin. Incubation with the phospha- tase inhibitor okadaic acid before insulin stimulation protected against the effect of the fatty acid on PKB phosphorylation. Palmitate was unable to inhibit PKB activity and glycogen synthesis in cells overexpressing the activated PKB mutant (T308D,S473D)-PKB, which is unaffected by phosphatase. In contrast, PKB activity and glycogen synthesis were still inhibited by palmitate in cells overexpressing a membrane-targeted and, hence, activated PKB mutant that retains sensitivity to phos- phatase. Although aPKC activity was also increased in palmitate-treated cells, overexpression of wild-type or kinase-dead aPKC did not alter the inhibitory effects of the lipid on either stimulation of PKB or glycogen synthesis by insulin. We conclude that palmitate dis- rupts insulin signaling in C2C12 myotubes by promoting PP2A-like activity and, therefore, the dephosphoryla- tion of PKB, which in turn reduces the stimulation of glycogen synthesis. Diabetes 50:2210 –2218, 2001 I nsulin resistance is a major characteristic of type 2 diabetes, and it can be defined as a failure of target tissues to increase glucose disposal in response to insulin. The insulin resistance seen in skeletal mus- cle is of particular importance because this is the major site of insulin-stimulated glucose uptake and glycogen synthesis (1). Many studies have revealed an association between increased lipid availability and insulin resistance (2– 4), suggesting that there is a causative link between the two, although the underlying mechanisms are not clear. The regulation of glycogen synthesis by insulin is thought to be mediated primarily through the protein kinase B (PKB) pathway, downstream of the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and the stimulation of phosphatidylinositol (PI) 3-kinase activity. PKB is transiently recruited to the plasma mem- brane by the increase in PI (3,4,5)-trisphosphate (PI[3,4,5]P 3 ) levels, where it becomes phosphorylated on two residues crucial to its activation (5). Phosphorylation of Thr308 is mediated by PI(3,4,5)P 3 -dependent kinase (PDK)-1, whereas the subsequent phosphorylation of Ser473 may be mediated by a hypothetical PDK-2 or by autophosphorylation (6). After activation in this manner, PKB is released from the membrane to phosphorylate substrates, including glycogen synthase kinase-3 (GSK-3), which is thus inactivated. GSK-3 is probably the major regulatory kinase of glycogen synthase. It phosphorylates the metabolic enzyme at three sites, causing marked inhibition, although phosphorylation by other kinases may also play a role. Therefore, inhibition of GSK-3, together with activation of protein phosphatase (PP) 1, may explain the increased synthesis of glycogen from glucose in re- sponse to insulin (7). We have recently shown that pretreatment of C2C12 skeletal muscle cells with the saturated free fatty acid palmitate leads to inhibition of PKB phosphorylation in response to insulin, without effects on upstream signaling events, such as IRS-1 tyrosine phosphorylation and PI 3-kinase activation, and thus it leads to inhibition of insulin-stimulated glycogen synthesis (8). The elevation of ceramide levels in the muscle cells, which occurs specifi- cally in response to palmitate but not to unsaturated free fatty acids, appears to be sufficient to account for these effects, because exogenously added ceramide was also able to inhibit PKB phosphorylation and glycogen synthe- sis (8). Ceramide has similar effects on PKB in a number of cell lines (9 –12), and in each case, these are observed in the absence of alterations in PI 3-kinase activity. Ceramide can be generated by de novo synthesis from palmitate (13), and also acutely upon the activation of sphingomylinases, such as in response to tumor necrosis From the Garvan Institute of Medical Research, Sydney, Australia. Address correspondence and reprint requests to Carsten Schmitz-Peiffer, Cell Signalling Group, Garvan Institute of Medical Research, 384, Victoria St., Darlinghurst, NSW 2010, Australia. E-mail: c.schmitz-peiffer@garvan.org.au. Received for publication 16 February 2001 and accepted in revised form 13 July 2001. aPKC, atypical protein kinase C; aPKCKD, kinase-dead aPKC; aPKCwt, wild-type aPKC; BSA, bovine serum albumin; CAPP, ceramide-activated protein phosphatase; EMEM, minimum essential medium with Earles’ salts; FCS, fetal calf serum; GFP, green fluorescence protein; GSK-3, glycogen synthase kinase-3; HA, hemagglutinin; IRS-1, insulin receptor substrate-1; m/p, myristoylated/palmitoylated; PBS, phosphate-buffered saline; PDK, phospha- tidylinositol (3,4,5)P 3 -dependent kinase; PI, phosphatidylinositol; PI(3,4,5)P 3 , PI (3,4,5)-trisphosphate; PKB, protein kinase B; PMSF, phenylmethyl sulfox- ide; PP, protein phosphatase; SF, serum-free. 2210 DIABETES, VOL. 50, OCTOBER 2001