Diabetologia

, Volume 39, Issue 5, pp 558–563 | Cite as

Expression of a dominant-negative Ras mutant does not affect stimulation of glucose uptake and glycogen synthesis by insulin

  • J. Dorrestijn
  • D. M. Ouwens
  • N. Van den Berghe
  • J. L. Bos
  • J. A. Maassen
Originals

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.

Keywords

Glucose uptake glycogen synthesis Rasmediated signalling phosphatidylinositol-3 kinase 

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

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Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • J. Dorrestijn
    • 1
  • D. M. Ouwens
    • 1
  • N. Van den Berghe
    • 2
  • J. L. Bos
    • 3
  • J. A. Maassen
    • 1
  1. 1.Department of Medical Biochemistry, Section of Protein Synthesis and Hormone RegulationLeiden UniversityLeidenThe Netherlands
  2. 2.Department of Endocrinology and Metabolic DiseasesUniversity HospitalLeidenThe Netherlands
  3. 3.Laboratory for Physiological ChemistryUtrecht UniversityUtrechtThe Netherlands

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