Akt mediates insulin rescue from apoptosis in brown adipocytes: effect of ceramide

We have recently shown that insulin can rescue serum deprived adipocytes fr om apoptosis in a PI 3 kinase and MAP kinase dependent manner. This study i nvestigated the contribution of Akt and p70S6-kinase in insulin rescue from two different apoptotic triggers, serum deprivation and ceramide treatment . Insulin rescued serum-deprived immortalized brown adipocytes from apoptos is through phosphatidylinositol (Pt) 3-kinase and Akt pathways, but indepen dently of p70S6-kinase, as demonstrated by the use of inhibitors such as LY 294002 or Rapamycin, and transfection experiments with dominant-negative co nstructs of Akt or p85 subunit of PI 3-kinase. A constitutively active Akt construct mimicked the insulin survival effect, decreasing the percentage o f hypodiploid cells, the percentage of apoptopic cells and precluding the f ormation of apoptotic nuclei. We propose that the insulin survival effect o n immortalized brown adipocytes is mediated through activation of Akt. Howe ver, insulin and EGF failed to rescue brown adipocytes from ceramide-induce d apoptosis, as determined by DNA laddering, hypodiploid cells and apoptoti c nuclei. Ceramide treatment blunted Akt activity but not Pi 3-kinase activ ity, and insulin and EGF were unable to activate Akt. Ceramide also caused apoptosis in cells transfected with a constitutively active Akt construct, since phosphorylation of Akt was impaired under these experimental conditio ns. This study suggests that activation of Akt may be an absolute requireme nt for the survival of brown adipocytes. (C) 2000 Harcourt Publishers Ltd.