Ceramide mediates insulin resistance by tumor necrosis factor-alpha in brown adipocytes by maintaining Akt in an inactive dephosphorylated state

Tumor necrosis factor (TNF)-alpha causes insulin resistance on glucose upta ke in fetal brown adipocytes. We explored the hypothesis that some effects of TNF-alpha could be mediated by the generation of ceramide, given that TN F-alpha treatment induced the production of ceramide in these primary cells . A short-chain ceramide analog, C2-ceramide, completely precluded insulin- stimulated glucose uptake and insulin-induced GLUT4 translocation to plasma membrane, as determined by Western blot or immunofluorescent localization of GLUT4. These effects were not produced in the presence of a biologically inactive ceramide analog, C2-dihydroceramide. Analysis of the phosphatidyl inositol (Pl) 3-kinase signaling pathway indicated that C2-ceramide preclud ed insulin stimulation of Akt kinase activity, but not of PI-3 kinase or pr otein kinase C-zeta activity. C2-ceramide completely abolished insulin-stim ulated Akt/protein kinase B phosphorylation on regulatory residues Thr 308 and Ser 473, as did TNF-alpha, and inhibited insulin-induced mobility shift in Akt1 and Akt2 separated in PAGE. Moreover, C2-ceramide seemed to activa te a protein phosphatase (PP) involved in dephosphorylating Akt because 1) PP2A activity was increased in C2-ceramide- and TNF-alpha -treated cells, 2 ) treatment with okadaic acid concomitantly with C2-ceramide completely res tored Akt phosphorylation by insulin, and 3) transient transfection of a co nstitutively active form of Akt did not restore Akt activity. Our results i ndicate that ceramide produced by TNF-alpha induces insulin resistance in b rown adipocytes by maintaining Akt in an inactive dephosphorylated state.