Insulin Induces Swelling-dependent Activation of the Epidermal Growth Factor Receptor in Rat Liver

Roland Reinehr,Annika Sommerfeld,Dieter Häussinger

doi:10.1074/jbc.m110.125781

Abstract

The aim of the study was to analyze whether the proliferative effects of insulin in rat liver involve cross-signaling toward the epidermal growth factor receptor (EGFR) and whether this is mediated by insulin-induced hepatocyte swelling. Studies were performed in the perfused rat liver and in primary rat hepatocytes. Insulin (35 nmol/liter) induced phosphorylation of the EGFR at position Tyr(845) and Tyr(1173), but not at Tyr(1045), suggesting that EGF is not involved in insulin-induced EGFR activation. Insulin-induced EGFR phosphorylation and subsequent ERK1/2 phosphorylation were sensitive to bumetanide, indicating an involvement of insulin-induced hepatocyte swelling. In line with this, hypoosmotic (225 mosmol/liter) hepatocyte swelling also induced EGFR and ERK1/2 activation. Insulin- and hypoosmolarity-induced EGFR activation were sensitive to inhibition by an integrin-antagonistic RGD peptide, an integrin beta1 subtype-blocking antibody, and the c-Src inhibitor PP-2, indicating the involvement of the recently described integrin-dependent osmosensing/signaling pathway (Schliess, F., Reissmann, R., Reinehr, R., vom Dahl, S., and Häussinger, D. (2004) J. Biol. Chem. 279, 21294-21301). As shown by immunoprecipitation studies, insulin and hypoosmolarity induced a rapid, RGD peptide-, integrin beta1-blocking antibody and PP-2-sensitive association of c-Src with the EGFR. As for control, insulin-induced insulin receptor substrate-1 phosphorylation remained unaffected by the RGD peptide, PP-2, or inhibition of the EGFR tyrosine kinase activity by AG1478. Both insulin and hypoosmolarity induced a significant increase in BrdU uptake in primary rat hepatocytes, which was sensitive to RGD peptide-, integrin beta1-blocking antibody, PP-2, AG1478, and PD098059. It is concluded that insulin- or hypoosmolarity-induced hepatocyte swelling triggers an integrin- and c-Src kinase-dependent EGFR activation, which may explain the proliferative effects of insulin.

Highlights

Insulin receptor substrate-1 (IRS-1)2 and activation of a variety of protein kinases such as mitogen-activated protein (MAP) kinases ERK1/2 and p38MAPK [3, 12]
Insulin (100 nmol/ liter) failed to induce epidermal growth factor receptor (EGFR) phosphorylation in 3-h cultured rat hepatocytes [19]. This experimental model may not pick up swelling-dependent components of insulin signaling [20], most likely due to not yet reorganized microtubules or impaired osmosensing. The latter is achieved by hepatocellular integrin/extracellular matrix (ECM) interactions, which require either the intact three-dimensional organ structure, e.g. the intact liver, or long term hepatocyte cultures that allow for endogenous ECM synthesis
Insulin (35 nmol/liter) induced within 5 min an activation of ERKs (Fig. 2A) and p38MAPK (Fig. 2B), which was recently shown to be mediated by insulin-induced hepatocyte swelling [15, 35]

Summary

Introduction

Insulin receptor substrate-1 (IRS-1) and activation of a variety of protein kinases such as mitogen-activated protein (MAP) kinases ERK1/2 and p38MAPK [3, 12]. Insulin (100 nmol/ liter) failed to induce EGFR phosphorylation in 3-h cultured rat hepatocytes [19] This experimental model may not pick up swelling-dependent components of insulin signaling [20], most likely due to not yet reorganized microtubules or impaired osmosensing. The latter is achieved by hepatocellular integrin/extracellular matrix (ECM) interactions, which require either the intact three-dimensional organ structure, e.g. the intact liver, or long term hepatocyte cultures that allow for endogenous ECM synthesis. As shown in the present study, insulin-induced cell swelling triggers activation of the EGFR through an integrin- and c-Src kinase-dependent osmosensing/signaling pathway that triggers insulin-induced hepatocyte proliferation

Objectives

Results

Conclusion