Leucine Culture Reveals That ATP Synthase Functions as a Fuel Sensor in Pancreatic β-Cells

Jichun Yang,Ryan K Wong,Xujing Wang,Jacob Moibi,Martin J Hessner,Scott Greene,Jianmei Wu,Siam Sukumvanich,Bryan A Wolf,Zhiyong Gao

doi:10.1074/jbc.m405309200

Abstract

Our goal was to investigate whether leucine culture affects beta-cell glucose sensing. One-day culture of rat islets with 10 mM leucine had no effect on glucose-induced insulin secretion. One-week leucine culture decreased the threshold for glucose-induced insulin secretion and increased maximal insulin secretion at 30 mM glucose. Glucose-induced cytosolic free Ca(2+) was increased at 1 week but not at 1 day of leucine culture. Without glucose, ATP content was not different with or without leucine culture for 1 week. With 20 mM glucose, ATP content was higher by 1.5-fold in islets cultured for 1 week with leucine than those without leucine. Microarray experiments indicated that culture of RINm5F cells with leucine increased expression of ATP synthase beta subunit 3.2-fold, which was confirmed by real time reverse transcription-PCR analysis (3.0- +/- 0.4-fold) in rat islets at 1 week but not after 1 day with leucine culture. Down-regulation of ATP synthase beta subunit by siRNA decreased INS1 cell ATP content and insulin secretion with 20 mM glucose. Overexpression of ATP synthase beta subunit in INS1 cell increased insulin secretion in the presence of 5 and 20 mM glucose. In conclusion, one-week leucine culture of rat islets up-regulated ATP synthase and increased ATP content, which resulted in elevated [Ca(2+)] levels and more insulin exocytosis by glucose. Depletion of ATP synthase beta subunit with siRNA produced opposite effects. These data reveal the fuel-sensing role of mitochondrial ATP synthase in the control of ATP production from glucose and the control of glucose-induced insulin secretion.

Highlights

Glucose is the main secretagogue of insulin secretion from pancreatic ␤-cells
Role of ␤-Cell Mitochondria in Fuel Sensing—Pancreatic ␤-cells secrete insulin in response to glucose through metabolism and production of ATP
Though glycolysis produces small amounts of ATP, the vast majority of ATP is produced through the mitochondrial oxidation of pyruvate, which is produced as the glycolytic product of glucose

Summary

Introduction

Glucose is the main secretagogue of insulin secretion from pancreatic ␤-cells. The mechanisms of glucose-induced insulin secretion have been studied extensively. Glucose increases pancreatic ␤-cell ATP/ADP ratio, which closes ATP-sensitive potassium (KATP) channels and depolarizes the cell membrane This results in an influx of extracellular Ca2ϩ and increase of free cytosolic [Ca2ϩ] that stimulates exocytosis of insulin granules [1,2,3,4,5]. Some recent publications indicate that superoxide produced by hyperglycemia activates uncouplingprotein-2 (UCP2) and destroys the proton gradient between inner and outer mitochondrial membranes This negatively affects the activity of ATP synthase, decreases ATP production, and impairs glucose-stimulated insulin secretion of pancreatic ␤-cells resulting in diabetes [8, 9]. The aim of this study was to investigate the role of ATP synthase in glucose-induced insulin secretion by attempting to change its expression level with leucine culture and siRNA

Objectives

Results

Conclusion