Protein phosphorylation cascades regulating islet beta-cell insulin exocytosis through glucose sensing and drug signaling : with special focus on serine/threonine protein phosphates and hypoglycemic sulfonylureas

Abstract

In human type 2 diabetes, loss of glucose-sensitive insulin secretion from the pancreatic â-cell is an early pathogenetic event. Initially, this may be compensated for by stimulating insulin secretion by sulfonylurea drugs. The mechanisms involved in the regulation of insulin secretion, either by natural or pharmacological stimuli, are not entirely understood.

The beta-cell senses subtle changes in glucose concentration and it has been shown that ATP generated by glucose metabolism (and sulfonylureas) may close plasma membrane ATP-dependent K+ (KATP) channels resulting in depolarization and influx of Ca2+ through voltage-activated Ca2+ channels, an event setting in motion insulin exocytosis. However, glucose retains an excellent ability to secrete insulin even in the presence of maximally effective concentrations of K+ and diazoxide, an opener of K+ channels. Thus, signaling molecules other than ATP and Ca2+ must also be involved in glucose sensing in the beta-cell.

Rapidly reversible protein phosphorylation/dephosphorylation cycles are believed to play an essential role in integrating and coordinating incoming stimuli into an appropriate rate of insulin exocytosis. These cycles is now known to be dynamic processes, with cellular levels of protein phosphorylation being determined by the combined actions of numerous protein kinases and ser/thr protein phosphatases (PPases). Compared with protein kinases, relatively little attention have been paid to the role of PPases in the beta-cell.

We show that glycolytic and Krebs cycle intermediates, whose concentration increase upon glucose stimulation, not only inhibit the enzyme activities of divalent-cation independent PPases dose-dependently, but also directly promote insulin exocytosis from permeabilized beta-cells in a Ca2+-independent manner. They significantly enhance insulin exocytosis, by a mechanism non-additive to that of the specific PPase inhibitor okadaic acid, at nanomolar Ca2+ concentrations.

Specific glucose metabolites thus inhibit beta-cell divalent-cation independent PPase activities and directly stimulate Ca2+- independent insulin exocytosis, an effect that may in part explain the KATP-independent insulin exocytosis by glucose. A messenger role has been postulated for L-glutamate and inositol hexakisphosphate in linking glucose stimulation to sustained insulin exocytosis in the beta-cell, but the precise nature by which these metabolites controls insulin secretion remains elusive.

We show that glucose increases L-glutamate contents and promotes insulin secretion from INS-1E cells. L-glutamate and inositol hexakisphosphate, at physiological concentrations, also inhibit enzyme activities of ser/thr PPases in a dose-dependent fashion analogous to okadaic acid, whereas hypoglycemic sulfonylureas have no effect. Additionally, L-glutamate and okadaic acid directly and non-additively promote insulin exocytosis from permeabilized INS-1E cells in a Ca2+-independent manner, consistent with the view that the insulin-releasing effect of L-glutamate is at least in part mediated through its inactivation of PPases. Thus, as for certain other intermediary metabolites, an increase in phosphorylation state, through inhibition of protein dephosphorylation by L-glutamate and inositol hexakisphosphate, may be a novel regulatory mechanism linking glucose sensing to sustained insulin exocytosis in the beta-cell.

We also show that the sulfonylurea glibenclamide inhibits the beta-cell mitochondrial enzyme carnitine palmitoyltransferase 1 (CPT-1) activity, a key enzyme controlling fuel partitioning. This event diverts fatty acid metabolism from mitochondrial oxidation to diacylglycerol biosynthesis, which causes KATP-independent and protein kinase C-dependent exocytosis of insulin. Chronic CPT inhibition, through the progressive islet accumulation of glibenclamide, may thus explain the prolonged stimulation of insulin secretion in some diabetic patients that contributes to the sustained hypoglycemia of the sulfonylurea. Whether this mechanism also results in lipid overload in the beta-cell causing beta-cell lipoapoptosis, explaining the clinical phenomenon of secondary failure to sulfonylureas, remains to be elucidated