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Expression of a dominant-negative Ras mutant does not affect stimulation of glucose uptake and glycogen synthesis by
J Dorrestijn1, D M Ouwens, N Van den Berghe
1Department of Medical Biochemistry, Leiden University, The Netherlands.
Abstract:
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 PI3kinase, 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.
Insights
Insulin stimulates glucose uptake and glycogen synthesis independently of RasGTP signaling. This study used a dominant-negative Ras mutant (RasN17) to show that Ras-mediated pathways do not influence these key insulin responses.
Area of Science:
- Cellular signaling pathways
- Metabolic regulation
- Molecular biology
Background:
- Insulin-stimulated glucose uptake and glycogen synthesis are crucial metabolic processes.
- Phosphatidylinositol-3-kinase (PI3kinase) activation is essential for these insulin responses.
- The role of RasGTP-mediated signaling in insulin action has been debated due to inconsistent findings.
Purpose of the Study:
- To investigate the necessity of RasGTP-mediated signaling for insulin-stimulated glucose uptake and glycogen synthesis.
- To elucidate the specific contribution of Ras pathways to insulin's metabolic effects.
Main Methods:
- Utilized vaccinia virus-mediated gene transfer to express a dominant-negative Ras mutant (RasN17) in 3T3L1 adipocytes and A14 cells.
- Assessed insulin-induced 2-deoxyglucose uptake and glycogen synthesis.
- Monitored insulin-induced Mapkinase (Erk1,2) phosphorylation as an indicator of Ras pathway interference.
- Administered wortmannin, a PI3kinase inhibitor, to assess its effects on insulin signaling.
Main Results:
- Expression of RasN17 did not impede insulin-stimulated hexose uptake in 3T3L1 adipocytes.
- RasN17 expression did not affect insulin-induced glycogen synthesis in A14 cells.
- Insulin-induced Mapkinase (Erk1,2) phosphorylation was abolished by RasN17, confirming functional Ras pathway blockade.
- Wortmannin inhibited insulin-stimulated hexose uptake and glycogen synthesis dose-dependently, independent of RasGTP levels.
Conclusions:
- Insulin-stimulated glucose transport and glycogen synthesis are independent of RasGTP-mediated signaling.
- PI3kinase activation is the primary pathway mediating these insulin effects.
- Ras signaling pathways do not play a significant role in the regulation of glucose metabolism by insulin.