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.


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.
Blouin
Cédric M
CM
Centre de Recherche des Cordeliers, INSERM, UMR-S 872, Paris, France.
Prado
Cécilia
C
Takane
Karen K
KK
Lasnier
Françoise
F
Garcia-Ocana
Adolfo
A
Ferré
Pascal
P
Dugail
Isabelle
I
Hajduch
Eric
E
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D022461:Caveolins / Q000378:metabolism; D002451:Cell Compartmentation / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology*; D002518:Ceramides / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007333:Insulin Resistance / Q000502:physiology; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology; D051379:Mice; D010168:Palmitates / Q000378:metabolism / Q000494:pharmacology; D010750:Phosphoproteins / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D054648:Protein Phosphatase 2 / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D000099221:Protein Kinase C zeta
D022461:Caveolins; D002518:Ceramides; D007328:Insulin; D008565:Membrane Proteins; D010168:Palmitates; D010750:Phosphoproteins; D011493:Protein Kinase C; D054648:Protein Phosphatase 2; D051057:Proto-Oncogene Proteins c-akt; D000099221:Protein Kinase C zeta; C083557:p112 protein, rat