title: Insulin-induced up-regulated uncoupling protein-1 expression is mediated by insulin receptor substrate 1 through the phosphatidylinositol 3-kinase/Akt signaling pathway in fetal brown adipocytes.
abstract: To investigate the role of insulin receptor substrate-1 (IRS-1) and its downstream signaling in insulin-induced thermogenic differentiation of brown adipocytes, we have reconstituted IRS-1-deficient fetal brown adipocytes (IRS-1(-/-)) with wild-type IRS-1 (IRS-1(wt)). The lack of IRS-1 resulted in the inability of insulin to induce IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity and Akt phosphorylation in IRS-1(-/-) brown adipocytes. In addition, these cells showed an impairment in activating alpha-Akt, beta-Akt, and gamma-Akt isoforms upon insulin stimulation. Reconstitution of IRS-1(-/-) brown adipocytes with IRS-1(wt) restored the IRS-1/PI 3-kinase/Akt signaling pathway. Treatment of wild-type brown adipocytes with insulin for 24 h up-regulated uncoupling protein-1 (UCP-1) expression and transactivated the UCP-1 promoter; this effect was abolished in the absence of IRS-1 or in the presence of an Akt inhibitor and further recovered after IRS-1(wt) reconstitution. Neither UCP-2 nor UCP-3 was up-regulated by insulin in wild-type and IRS-1-deficient brown adipocytes. Insulin stimulated the expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha) and its DNA binding activity in wild-type brown adipocytes but not in IRS-1(-/-) cells. However, insulin stimulation of both C/EBPalpha expression and binding activity was restored after IRS-1(wt) reconstitution of deficient cells. Retrovirus-mediated expression of C/EBPalpha and peroxisome proliferator-activated receptor gamma in IRS-1(-/-) brown adipocytes up-regulated UCP-1 protein content and transactivated UCP-1 promoter regardless of insulin stimulation. Both C/EBPalpha and peroxisome proliferator-activated receptor gamma reconstituted FAS mRNA expression, but only C/EBPalpha restored insulin sensitivity in the absence of IRS-1. Finally, reconstitution of IRS-1(-/-) brown adipocytes with the IRS-1 mutants IRS-1(Phe-895), which lacks IRS-1/growth factor receptor binding protein 2 binding but not IRS-1/p85-PI 3-kinase binding, or with IRS-1(Tyr-608/Tyr-628/Tyr-658), which only binds p85-PI 3-kinase, induced UCP-1 expression and transactivated the UCP-1 promoter. These data provide strong evidence for an essential role of IRS-1 through the PI 3-kinase/Akt signaling pathway inducing UCP-1 gene expression by insulin.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Centro Mixto Consejo Superior de Investigaciones Cientificas/Universidad Complutense de Madrid, Facultad de Farmacia, Universidad Complutense, Spain.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Valverde
authors.forename: Monica
authors.initials: M
authors.lastname: Arribas
authors.forename: Cecilia
authors.initials: C
authors.lastname: Mur
authors.forename: Paloma
authors.initials: P
authors.lastname: Navarro
authors.forename: Sebastián
authors.initials: S
authors.lastname: Pons
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Cassard-Doulcier
authors.forename: C Ronald
authors.initials: CR
authors.lastname: Kahn
authors.forename: Manuel
authors.initials: M
authors.lastname: Benito
mesh_terms: D017667:Adipocytes / Q000378:metabolism*; D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D007473:Ion Channels; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D024101:Mitochondrial Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation; D000071256:Uncoupling Protein 1; D015854:Up-Regulation
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D002352:Carrier Proteins; D004247:DNA; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D000071256:Uncoupling Protein 1; C526295:Irs1 protein, mouse; C000604372:Ucp1 protein, mouse