Abstract
To characterize tissue-specific differences in insulin signaling, we compared the mechanisms of mitogen-activated protein (MAP) kinase activation by insulin in the mitogenically active 3T3-L1 fibroblasts with the metabolically active 3T3-L1 adipocytes. In both cell lines, insulin significantly increased p21(ras).GTP loading (1.5-2-fold) and MAP kinase activity (5-8-fold). Inhibition of Ras farnesylation with lovastatin blocked activation of p21(ras) and Raf-1 kinase in both 3T3-L1 fibroblasts and 3T3-L1 adipocytes. In 3T3-L1 fibroblasts, this was accompanied by an inhibition of the stimulatory effect of insulin on MAP kinase. In contrast, in 3T3-L1 adipocytes, despite an inhibition of activation of p21(ras) and Raf-1 by lovastatin, insulin continued to stimulate MAP kinase activity. Fractionation of the cell lysates on the FPLC Mono-Q column revealed that lovastatin inhibited insulin stimulation of ERK2 (and, to a lesser extent, ERK1) in 3T3-L1 fibroblasts and had no effect on the insulin-stimulated ERK2 in 3T3-L1 adipocytes. These results demonstrate an important distinction between the mechanism of insulin signaling in the metabolically and mitogenically active cells. Insulin activates MAP kinase by the Ras-dependent pathway in the 3T3-L1 fibroblasts and by the Ras-independent pathway in the 3T3-L1 adipocytes.
Highlights
From the Medical Research Service and Departments of Medicine and Pharmacology, Veterans Affairs Medical Center and the University of Colorado Health Sciences Center, Denver, Colorado 80220
To investigate the role of p21ras in mediating insulin activation of mitogen-activated protein (MAP) kinase, we initially employed the hydroxymethylglutaryl-CoA-reductase inhibitor lovastatin to block the activation of p21ras by insulin in 3T3-L1 fibroblasts and 3T3-L1 adipocytes
Insulin-stimulated MAP kinase activity was inhibited by lovastatin in the 3T3-L1 fibroblasts, but not in 3T3-L1 adipocytes (Fig. 2)
Summary
Vol 271, No 48, Issue of Nov 29, pp. 30625–30630, 1996 Printed in U.S.A. Insulin Stimulates Mitogen-activated Protein Kinase by a Ras-independent Pathway in 3T3-L1 Adipocytes*. To characterize tissue-specific differences in insulin signaling, we compared the mechanisms of mitogen-activated protein (MAP) kinase activation by insulin in the mitogenically active 3T3-L1 fibroblasts with the metabolically active 3T3-L1 adipocytes. Fractionation of the cell lysates on the FPLC Mono-Q column revealed that lovastatin inhibited insulin stimulation of ERK2 (and, to a lesser extent, ERK1) in 3T3-L1 fibroblasts and had no effect on the insulin-stimulated ERK2 in 3T3-L1 adipocytes These results demonstrate an important distinction between the mechanism of insulin signaling in the metabolically and mitogenically active cells. In 3T3-L1 adipocytes, PI 3-kinase and PKC exert a constitutively inhibitory influence on GTPase-activating protein (GAP), allowing insulin signaling to proceed through Sos and p21ras Removal of this inhibitory influence results in activation of GAP and inhibition of the p21ras1⁄7GTP loading. While in fibroblasts, insulin activates MAP kinase exclusively by a Rasdependent pathway, in adipocytes, insulin stimulates MAP kinase predominantly via a Ras-independent pathway
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