Tyrosine kinase inhibitors inhibit glucose-stimulated insulin secretion.

Abstract

Protein phosphorylation by regulated protein kinases is an important regulatory mechanism in many secretory cell types, including pancreatic c CAMK) have been defined in some detail [reviewed in 11. However, there is little information on the involvement of tyrosine kinases (TKs) in stimulus-response coupling in 0-cells. TKs, which phosphorylate substrate proteins on tyrosine residues, have been implicated in a variety of cellular processes such as cell proliferation and cell transformation [2]. The recent availability of anti-phosphotyrosine antisera and the development of selective inhibitors of TKs have led to an increased understanding of the roles played by TKs in several secretory cell types, including platelets and neutrophils. We have now used an anti-phosphotyrosine antibody to determine whether tyrosine phosphorylated proteins are present in rat islets, and have used TK inhibitors to investigate the involvement of TKs in the insulin secretory responses to glucose, sulphonylureas and intracellular second messengers. All experiments were performed using collagenaseisolated rat islets of Langerhans. TKs were shown to be present in islets by the detection of tyrosine phosphorylated islet proteins using an antiphosphotyrosine monclonal antibody. At least ten substrates phosphorylated on tyrosine residues were present in islets, ranging in molecular weight from 16k2 to 130+6kDa. The role played by TKs in the secretory response to glucose, the major physiological secretagogue, was investigated using two structurally dissimilar TK inhibitors, genistein (GS) and tyrphostin A47 (TA47). Glucose-induced insulin secretion was inhibited in a dose-dependent manner by TA47 (12.5-200pM) and by GS (100-500pM), such that secretion was not significantly higher than basal at the highest concentration of inhibitor used (Figure 1). Inactive analogues of GS and TA47, daidzein and TAI, had no inhibitory effects on glucosestimulated insulin secretion, even at concentrations as high as 500pM, but TA1 exerted a small stimulatory effect (20mM glucose + 25pM TA1: 141 +17% glucose-stimulated secretion, P<O.Ol, n=9). These results may indicate that TKs are involved in the secretory response of islets to glucose, but since TK inhibitors have been shown to inhibit mitochondrial oxidative phosphorylation independently of their effects on TKs [3] it is possible that the inhibition of glucose-stimulated secretion is a result of impaired glucose handling. We therefore examined the effects of GS and TA47 on insulin secretion stimulated by the sulphonylurea tolbutamide, which exerts its effects by closing Rcell K+-ATP channels. Both GS and TA47 inhibited tolbutamideinduced insulin secretion (200pM tolbutamide, 629k 101 % basal; +500pM GS, 86*15% P<O.Ol; +200pM TA47, 271*57%, P<O.Ol, n=9), indicating that TK inhibitors exert effects distal to the transport and metabolism of glucose. The inhibitory effects of GS and TA47 were not confined to TKs since these agents also inhibited islet serine/threonine kinases, in vitro, to varying extents. Thus, TA47 (200pM) inhibited islet PKC and PKA activity by 60% and 54% respectively and markedly inhibited Ca’+/calrnodulin-dependent Figure 1. Effect of tyrosine kinase inhibitors on glucoseinduced insulin secretion. The insulin secretory response of isolated islets to 20mM glucose (open bar) was inhibited both by 200pM TA47 (P<O.001; hatched bar) and 500pM GS (P<O.001; cross-hatched bar), to levels not significantly higher than those obtained in the presence of 2mM glucose (filled bar). Data are expressed as a percentage of secretion obtained in the presence of 20mM glucose alone, and are the means (+SEM) of 9 observations.