Plasma membrane subdomain compartmentalization contributes to distinct mechanisms of ceramide action on insulin signaling.

Objective: Ceramide is now recognized as a negative regulator of insulin signaling by impairing protein kinase B (PKB)/Akt activation. In different cells, two distinct mechanisms have been proposed to mediate ceramide inhibition of PKB/Akt: one involving atypical protein kinase C zeta (PKCzeta) and the other the protein phosphatase-2 (PP2A). We hypothesized that ceramide action through PKCzeta or PP2A might depend on plasma membrane (PM) structural organization and especially on caveolin-enriched domain (CEM) abundance.

Research Design And Methods: We have used different PKCzeta mutant constructs or the PP2A inhibitor, okadaic acid (OKA), to selectively inhibit PKCzeta- and PP2A-dependent pathways in cells expressing different caveolin-1 levels and evaluated the impact of insulin and ceramide on PKB/Akt activity in different PM subdomains.

Results: Although the PKCzeta-mediated negative effect of ceramide on insulin-stimulated PKB/Akt was dominant in adipocytes, a ceramide action through PP2A outside CEMs, prevented by OKA, was also unraveled. To test the importance of CEM to direct ceramide action through the PKCzeta pathway, 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 PKCzeta pathway after caveolin-1 overexpression.

Conclusions: Our results show that ceramide can switch from a PKCzeta-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.