As observed previously in L6 muscle cells (19), treatment of 3T3-L1 adipocytes with either 100 nmol/l insulin for 10 min or 100 mol/l ceramide for 2 h induced the phosphorylation of PKC (using a PKC/ antibody that recognizes the kinase phosphorylation sites Thr410/403 of both PKC and ) (Fig. we treated 3T3-L1 preadipocytes devoid of CEMs with ceramide and we saw a shift of the lipid-negative action on PKB/Akt to a PP2A-mediated mechanism. In fibroblasts with low CEM abundance, the ceramide-activated PP2A pathway dominated, but could be shifted to a ceramide-activated PKC pathway after caveolin-1 overexpression. == CONCLUSIONS == Our results show that ceramide can switch from a PKC-dependent mechanism to a PP2A pathway, acting negatively on PKB/Akt, and hence revealing a critical role of CEMs of the PM in this process. Insulin is a hormone essential for tissue development, growth, energy storage, and maintenance of glucose homeostasis. Defects in insulin secretion and action are key factors in the development of metabolic diseases such as diabetes, obesity, hypertension, atherosclerosis, and cardiovascular diseases (1). The mechanism Rabbit Polyclonal to NFAT5/TonEBP (phospho-Ser155) by which insulin resistance develops in peripheral tissue is not yet fully solved. Recent work has suggested that forcing cells to store fatty acids beyond their capacities could promote insulin resistance by inducing the accumulation of intracellular signaling molecules able to inhibit the action of insulin (2). Among these fatty acidderived lipids, ceramides are the most active to negatively regulate intermediates of the insulin-signaling pathway and to inhibit insulin-dependent pathways such as the uptake of glucose into muscle PR-619 and adipocytes (3,4). The process of insulin signal transduction is initiated by the activated insulin receptor kinase, which tyrosine phosphorylates intracellular target substrates, in particular the family of insulin receptor substrates (IRS 14 proteins) (5). Although numerous proteins can dock on activated IRS, it is generally accepted that phosphoinositide 3-kinase (PI 3-kinase) and signaling effectors that lie downstream from it, in particular protein kinase B (PKB, also known as Akt) and atypical protein kinase C / (aPKCs), play crucial roles in glucose homeostasis (6). PI 3-kinasegenerated membrane phosphatidylinositol-3,4,5-triphosphates (PIP3s) recruit to the plasma membrane (PM) and activate both aPKCs and PKB/Akt (7,8). Once recruited, PR-619 aPKCs are phosphorylated by a 3-phosphoinositidedependent protein kinase-1 (PDK1) on their Thr410/403 site (9). On the other hand, binding of PIP3s to the pleckstrin homology (PH) domain of PKB/Akt induces conformational changes in the kinase that expose two regulatory sites, Thr308 and Ser473 (for PKB/Akt1). Phosphorylation of Thr308 is mediated by PDK1 and Ser473 phosphorylation by TORC2 (mammalian target of rapamycin)-rictor (rapamycin-insensitive companion of mTOR) complex (10). The importance of the activation of aPKCs and PKB/Akt by insulin in mediating glucose metabolism is now well documented in insulin-sensitive tissues. Mice lacking PKB (Akt2) become insulin resistant and develop severe diabetes (11), and recently, Farese et al. (12) have demonstrated the importance of PKC in skeletal muscle PR-619 by selectively ablating this kinase in a mouse model. They showed that these mice developed insulin resistance, reduced glucose tolerance, and dyslipidemia, all common features of the metabolic syndrome. A consensus now exists that PKB/Akt is the primary target of ceramide. Indeed, defects in activation of this kinase induced by ceramide have been observed in cell types, such as white and brown adipocytes, skeletal and smooth PR-619 muscles, mammary cells, and nerve cells (13). In some cells, ceramide PR-619 acts on PKB/Akt through the direct activation of phosphatases such as the protein phosphatase-2A (PP2A) (14), a cytosolic serine/threonine phosphatase responsible for dephosphorylating PKB/Akt (15). Treatment of several cell types such as C2C12 muscle cells, PC12 nerve cells, and brown adipocytes with the PP2A inhibitor okadaic acid (OKA) (16) prevents the negative effects of ceramide on PKB/Akt (13). However, in L6 muscle cells and white adipose tissue, we and others have shown that ceramide inhibited insulin-stimulated glucose transport through a mechanism that does not involve a phosphatase (17,18). Ceramide activates PKC (19,20), which interacts and phosphorylates the PH domain of PKB/Akt on a Thr34 residue, preventing PKB/Akt to be recruited and activated at the PM in response to insulin (19). Thus, two mechanisms by which ceramide can inhibit PKB/Akt.