Akt mediates insulin rescue from apoptosis in brown adipocytes: effect of ceramide.
We have recently shown that insulin can rescue serum deprived adipocytes from apoptosis in a PI 3 kinase and MAP kinase dependent manner. This study investigated 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 apoptosis through phosphatidylinositol (PI) 3-kinase and Akt pathways, but independently of p70S6-kinase, as demonstrated by the use of inhibitors such as LY294002 or Rapamycin, and transfection experiments with dominant-negative constructs of Akt or p85 subunit of PI 3-kinase. A constitutively active Akt construct mimicked the insulin survival effect, decreasing the percentage of hypodiploid cells, the percentage of apoptopic cells and precluding the formation of apoptotic nuclei. We propose that the insulin survival effect on immortalized brown adipocytes is mediated through activation of Akt. However, insulin and EGF failed to rescue brown adipocytes from ceramide-induced apoptosis, as determined by DNA laddering, hypodiploid cells and apoptotic nuclei. Ceramide treatment blunted Akt activity but not PI 3-kinase activity, 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 conditions. This study suggests that activation of Akt may be an absolute requirement for the survival of brown adipocytes.
Navarro
P
P
Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain.
Valverde
A M
AM
Rohn
J L
JL
Benito
M
M
Lorenzo
M
M
D017667:Adipocytes / Q000187:drug effects* / Q000502:physiology; D002001:Adipose Tissue, Brown / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002417:Cattle; D002461:Cell Line, Transformed; D002518:Ceramides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008979:Moloney murine leukemia virus; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D014162:Transfection
D002518:Ceramides; D004791:Enzyme Inhibitors; D007328:Insulin; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins; D019893:Ribosomal Protein S6 Kinases; D013110:Sphingosine; C064769:N-acetylsphingosine; C109343:dihydroceramide