Expression of a dominant-negative Ras mutant does not affect stimulation of glucose uptake and glycogen synthesis by insulin |
| |
Authors: | J Dorrestijn D M Ouwens N Van den Berghe J L Bos Dr J A Maassen |
| |
Institution: | (1) Department of Medical Biochemistry, Section of Protein Synthesis and Hormone Regulation, Leiden University, Leiden, The Netherlands;(2) Department of Endocrinology and Metabolic Diseases, University Hospital, Leiden, The Netherlands;(3) Laboratory for Physiological Chemistry, Utrecht University, Utrecht, The Netherlands;(4) Department of Medical Biochemistry, Sylvius Laboratory, Wassenaarseweg 72, NL-2333 AL Leiden, The Netherlands |
| |
Abstract: | Summary It has previously been shown that insulin-induced stimulation of glucose uptake and glycogen synthesis requires activation of phosphatidylinositol-3-kinase (PI3kinase). Insulin also induces formation of RasGTP in cells and various studies have yielded inconsistent data with respect to the contribution of signalling pathways activated by RasGTP, to insulin-stimulated glucose uptake and glycogen synthesis. We have examined the requirement of RasGTP-mediated signalling for these insulin responses by expression of a dominant negative mutant of Ras (RasN17) in cells by vaccinia virus mediated gene transfer. This Ras-mutant abrogates the signalling pathways mediated by endogenous RasGTP. Subsequently, the ability of insulin to stimulate 2-deoxyglucose uptake and glycogen was examined. We observed that expression of RasN17 in 3T3L1 adipocytes did not affect the stimulation of hexose uptake by insulin. Similarly, expression of RasN17 in A14 cells, an NIH 3T3-derived cell line with high expression of insulin receptors, did not affect insulin-induced stimulation of glycogen synthesis. In both cell lines, insulin-induced phosphorylation of Mapkinase (Erk1,2) was abrogated after expression of RasN17, demonstrating the functional interference by RasN17 with signalling mediated by endogenous RasGTP. Wortmannin, an inhibitor of PBkinase, abolished dose-dependently the insulin-induced stimulation of hexose uptake and glycogen synthesis without an effect on RasGTP levels in both cell types. We conclude that stimulation of glucose transport and glycogen synthesis by insulin occurs independently of RasGTP-mediated signalling.Abbreviations DMEM
Dulbecco's modified Eagle's medium
- ECL
enhanced chemiluminescence
- MAPkinase
mitogen-activated protein kinase
- PI3kinase
phosphatidylinositol-3 kinase
- IRS
insulin receptor substrate
- BSA
bovine serum albumin |
| |
Keywords: | Glucose uptake glycogen synthesis Rasmediated signalling phosphatidylinositol-3 kinase |
本文献已被 SpringerLink 等数据库收录! |
|