title: Susceptibility to apoptosis in insulin-like growth factor-I receptor-deficient brown adipocytes.
abstract: Fetal brown adipocytes are insulin-like growth factor-I (IGF-I) target cells. To assess the importance of the IGF-I receptor (IGF-IR) in brown adipocytes during fetal life, we have generated immortalized brown adipocyte cell lines from the IGF-IR(-/-) mice. Using this experimental model, we demonstrate that the lack of IGF-IR in fetal brown adipocytes increased the susceptibility to apoptosis induced by serum withdrawal. Culture of cells in the absence of serum and growth factors produced rapid DNA fragmentation (4 h) in IGF-IR(-/-) brown adipocytes, compared with the wild type (16 h). Consequently, cell viability was decreased more rapidly in fetal brown adipocytes in the absence of IGF-IR. Furthermore, caspase-3 activity was induced much earlier in cells lacking IGF-IR. At the molecular level, IGF-IR deficiency in fetal brown adipocytes altered the balance of the expression of several proapoptotic (Bcl-xS and Bim) and antiapoptotic (Bcl-2 and Bcl-xL) members of the Bcl-2 family. This imbalance was irreversible even though in IGF-IR-reconstituted cells. Likewise, cytosolic cytochrome c levels increased rapidly in IGF-IR-deficient cells compared with the wild type. A rapid entry of Foxo1 into the nucleus accompanied by a rapid exit from the cytosol and an earlier activation of caspase-8 were observed in brown adipocytes lacking IGF-IR upon serum deprivation. Activation of caspase-8 was inhibited by 50% in both cell types by neutralizing anti-Fas-ligand antibody. Adenoviral infection of wild-type brown adipocytes with constitutively active Foxol (ADA) increased the expression of antiapoptotic genes, decreased Bcl-xL and induced caspase-8 and -3 activities, with the final outcome of DNA fragmentation. Up-regulation of uncoupling protein-1 (UCP-1) expression in IGF-IR-deficient cells by transduction with PGC-1alpha or UCP-1 ameliorated caspase-3 activation, thereby retarding apoptosis. Finally, insulin treatment prevented apoptosis in both cell types. However, the survival effect of insulin on IGF-IR(-/-) brown adipocytes was elicited even in the absence of phosphatidylinositol 3-kinase/Akt signaling. Thus, our results demonstrate for the first time the unique role of IGF-IR in maintaining the balance of death and survival in fetal brown adipocytes.
authors.affiliation: Departamento de Bioquímica y Biología Molecular II, Centro Mixto Consejo Superior de Investigaciones Cientificas, Universidad Complutense de Madrid, Facultad de Farmacia, Ciudad Universitaria, 28040-Madrid, Spain. valverde@farm.ucm.es
authors.forename: Angela M
authors.initials: AM
authors.lastname: Valverde
authors.forename: Cecilia
authors.initials: C
authors.lastname: Mur
authors.forename: Michael
authors.initials: M
authors.lastname: Brownlee
authors.forename: Manuel
authors.initials: M
authors.lastname: Benito
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D017667:Adipocytes / Q000378:metabolism; D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D016895:Culture Media, Serum-Free / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000648:ultrastructure; D053938:DNA Fragmentation; D053222:Fas Ligand Protein; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D007328:Insulin / Q000378:metabolism; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000235:genetics* / Q000502:physiology*; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D051020:bcl-X Protein; D019014:fas Receptor / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; C494812:Bcl2l1 protein, mouse; C000606776:Bcl2l11 protein, mouse; D002352:Carrier Proteins; D016895:Culture Media, Serum-Free; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D007328:Insulin; D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014157:Transcription Factors; D051020:bcl-X Protein; D019014:fas Receptor; D004247:DNA; D019869:Phosphatidylinositol 3-Kinases; D017526:Receptor, IGF Type 1; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases