Regulation of intracellular signaling events that modulate insulin action

Abstract

Insulin regulates several mechanisms of fundamental importance to the body involving glucose, fat andprotein metabolism. Insulin resistance in skeletal muscle, liver and adipose tissue promotes an increaseddemand for insulin secretion. In individuals genetically predisposed to develop type 2 diabetes, thepancreatic â-cells, that under normal circumstances prevent hyperglycemia by increasing the insulinproduction, will eventually fail. Thus, type 2 diabetes results from the interaction of two defects: insulinresistance and â-cell dysfunction.The aim of this study was to examine intracellular signaling events that modulate insulin action andsecretion. Possible alternative signaling pathways, which may help to overcome insulin resistance, werealso investigated.The effects of insulin and different growth factors were compared. Epidermal growth factor (EGF) andtransforming growth factor (TGF) á, but not platelet derived growth factor (PDGF), increased glucosetransport to the same extent as insulin in human fat cells. Gene expression analyses, using real-time PCR,showed that EGF receptors (EGFR) were abundantly expressed in both human fat cells and skeletalmuscle while there was no evidence for either EGF or TGFá expression. The main tyrosinephosphorylatedbands induced by insulin were the insulin receptor (IR) and the insulin receptor substrates(IRS) proteins while EGF increased the tyrosine phosphorylation of EGFR, IRS-1 and IRS-2 and Grb2-associated binder 1 (Gab1) but not IR. Interestingly, EGF significantly increased the insulin-stimulateddownstream signaling (serine phosphorylation of PKB/Akt) in cells from insulin resistant (type 2diabetic) subjects but not control subjects.The effects of thiazolidinediones (PPAR agonists) and/or insulin on the expression of several genes andproteins involved in the insulin-signaling pathway were examined. The agonists tested did not increasethe gene or protein expression of IRS-1, PKB/Akt or GLUT4 in 3T3-L1 adipocytes. However, IRS-2mRNA and protein expression were clearly increased by the PPARã, but not PPARá, ligands. Similarly,IRS-2 mRNA expression was also increased by PPARã ligands in human adipose tissue.The effects of the phosphotyrosine phosphatase inhibitor, peroxovanadate (pV), and glucose on insulinsecretion and signaling were investigated in isolated rat islets. At a low glucose concentration (3.3 mM),pV stimulated insulin secretion 2- to 4 fold but this was not seen at a high (16.7 mM) glucose concentration.However, pV markedly enhanced the insulin secretion in depolarized cells at both low and high glucoseconcentrations. The effects of pV on the insulin signaling pathway were associated with an increasedtyrosine phosphorylation of IRS-1, IRS-2 and phosphorylation of the downstream proteins PKB/Aktand MAPK. Thus an enhanced intracellular protein tyrosine phosphorylation is, under appropriateconditions, associated with an increased insulin secretion.IRS-2 gene disruption is known to lead to insulin resistance, impaired insulin secretion and diabetes inanimals. We have shown for the first time that the insulin-sensitizers, the thiazolidinediones (TZD),increase the expression of IRS-2, thus linking the effects of TZD to a component of insulin s intracellularsignaling cascade. Furthermore, the possibility that additional signaling pathways may help to overcomeinsulin-resistant states is supported by this study. EGF and TGFá induced insulin-like effects in bothhuman adipocytes and human skeletal muscle. The ligand-specific activation of both insulin and EGFreceptor tyrosine kinases led to the tyrosine phosphorylation of IRS proteins while Gab1 was onlyphosphorylated by EGF. The ability of EGF to activate PI3-kinase pools additional to those of insulinprobably accounts for the effect of EGF to augment insulin s downstream signaling in insulin-resistantstates like type 2 diabetes.