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Alterations in the Insulin Signaling Pathway Induced by Immortalization and H-ras Transformation of Brown Adipocytes¹

Department of Biochemistry and Molecular Biology, Institute of Biochemistry, and Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain

Address all correspondence and requests for reprints to: Dr. Manuel Benito, Department of Biochemistry and Molecular Biology, Institute of Biochemistry, and Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain.

In fetal brown adipocyte primary cultures, insulin rapidly (at 5 min) induced tyrosine phosphorylation of the insulin receptor ß-subunit; this effect was maximal at physiological concentrations (1 nM). Insulin also stimulated insulin receptor substrate-1 tyrosine phosphorylation and subsequently activated phosphatidylinositol 3-kinase. Moreover, a 3-fold increase in the Ras.GTP active form and a 6-fold increase in Raf-1 kinase activity were induced after insulin stimulation. An immortalized brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo cotransfection) showed a reduced maximal responsiveness to insulin in the same range of insulin concentrations studied (1–100 nM). Transformed brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo H-ras^lys12 cotransfection) developed insulin resistance upstream from Ras, showing an impairment in the insulin receptor autophosphorylation, and in insulin receptor substrate-1 tyrosine phosphorylation and its association with phosphatidylinositol 3-kinase upon treatment with 1 nM insulin, although insulin receptor number and affinity (K_d) remained unaltered. This lack of effect was ameliorated upon treatment with higher insulin concentrations, in a dose-dependent manner. However, downstream from Ras, events such as formation of the Ras.GTP active form, and Raf-1 kinase and 12-O-tetradecanoylphorbol-13-acetate response element-chloramphenicol transferase (transiently transfected) activities were overstimulated, compared with those in primary and immortalized cells, in an insulin-independent manner. Wheat-germ lectin-purified receptors from H-ras^lys12-transformed brown adipocytes showed a marked phosphorylation in the basal state, which was suppressed by serine-threonine phosphatase pretreatment. Moreover, alkaline phosphatase pretreatment restored the tyrosine kinase activity of the receptor in response to insulin. We conclude that the decreased tyrosine autophosphorylation rate of the insulin receptor from H-ras^lys12-transformed brown adipocytes is a consequence of its basal serine/threonine phosphorylation, resulting in severe insulin resistance.

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