Role of protein-tyrosine phospatases in insulin and glucagon secretion in pancreatic islets of healthy rats and spontaneously diabetic GK rats

Abstract: Both insulin and glucagon secretion from pancreatic beta- and alpha-cells, respectively, are regulated by nutrients such as glucose and amino acids. In addition, the mechanisms leading to exocytosis of hormones are influenced by kinases and phosphatases that phosphorylate and dephosphorylate regulatory proteins respectively. This study focuses on cellular mechanisms by which protein -tyrosine phosphatases, PTPases, may modify stimulus-secretion coupling for glucose in P-cells and stimulus-secretion coupling for arginine in a-cells, in pancreatic islets of normal Wistar and diabetic Goto-Kakizaki (GK) rats. For this purpose, peroxovanadate (pV), inhibitor of PTPases, was used as a study tool. In islets of healthy Wistar rats, pV exerted complex effects. At 3.3 mM glucose, 0.1-1 mM pV stimulated insulin secretion (p<0.05), while at 16.7 mM glucose 0.0 1 -0. 1 mM pV inhibited its secretion (p<0.05). These effects were not mediated by pV modulation of islet glucose metabolism, or by interference with Gi proteins. In addition, pV stimulated insulin release 3-fold in islets depolarised by 30 mM KG at both 3.3 and 16.7 mad glucose. PV markedly increased islet tyrosine phosphorylation of insulin signalling proteins such as IRS-1, IRS-2, PKB/Akt and MAPK. PV-stimulated tyrosine phosphorylation of IRS-2 was increased by high glucose level. In GK rat islets, pV dosedependently enhanced insulin release both at 3.3 and 16.7 mM glucose. Like in Wistar rat islets, these effects were not mediated by altered islet glucose metabolism. At 3.3 mM glucose, pV-stimulated insulin release was inhibited by wortmannin (p<0.05), an inhibitor of phosphatidylinositol 3-kinase (PI3K). Increased expression of PTPsigma (or PTP-NE3, LAR-PTP2) was demonstrated in pancreatic islets and the liver of GK rats, using in situ hybridisation and Western blots. Exposure of GK rat islets to PTPsigma antisense increased insulin response to glucose and, in parallel, decreased islet PTPsigma amounts. As to the pancreatic alpha-cells, at 3.3 mM glucose pV (0. 1 - 1 mM) dose-dependently increased glucagon release in both Wistar and GK rat islets. Arginine (10 mM) elicited significantly higher glucagon reponse in GK vs Wistar rat islets (p<0.01), and the arginine-induced glucagon release was inhibited by 0.01 mM pV. This inhibitory effect did not depend on islet paracrine effects or pertussis toxin sensitive Gi proteins, but was abolished by wortmannin. Also in glucagon-producing In-R1-G9 cells, arginine-induced glucagon release was suppressed by pV, paralleled by increased IRS- 1 and IRS-2 associated P13K activity. In conclusion, our findings show that intracellular tyrosine and serine phosphorylation are related to insulin secretion, and that the complex effects of pV on islet hormone release depend partly on prevailing glucose concentrations. At 16.7 mM glucose, in contrast to its inhibiting effect on insulin release in Wistar rat islets, pV stimulated insulin secretion in GK rat islets. Such a mechanism is in agreement with our findings that PTPsigma is over-expressed in GK rat islets and antisense to this PTPase improves insulin release in GK rat islets. Increased PTPase activity may also be implicated in arginine-induced glucagon secretion. The effect of pV on glucagon secretion seems to be mediated by activation of PI3K, and it may be relevant to pVinduced modulation of glucagon release both in GIC and Wistar rat islets.