Essential Role of Ubiquitin-Proteasome System in Normal Regulation of Insulin Secretion

Miho Kawaguchi,Kohtaro Minami,Kazuaki Nagashima,Susumu Seino

doi:10.1074/jbc.m601228200

Abstract

Insulin secretion from pancreatic beta-cells occurs by sequential cellular processes, including glucose metabolism, electrical activity, Ca2+ entry, and regulated exocytosis. Abnormalities in any of these functions can impair insulin secretion. In the present study, we demonstrate that inhibition of proteasome activity severely reduces insulin secretion in the mouse pancreatic beta-cell line MIN6-m9. Although no significant effects on glucose metabolism including ATP production were found in the presence of proteasome inhibitors, both glucose- and KCl-induced Ca2+ entry were drastically reduced. As Ca2+-ionophore-induced insulin secretion was unaffected by proteasome inhibition, a defect in Ca2+ entry through voltage-dependent calcium channels (VDCCs) is the likely cause of the impaired insulin secretion. We found that the pore-forming alpha-subunit of VDCCs undergoes ubiquitination, which does not decrease but slightly increases expression of the alpha-subunit protein at the plasma membrane. However, electrophysiological analysis revealed that treatment with proteasome inhibitors results in a severe reduction in VDCC activity in MIN6-m9 cells, indicating that VDCC function is suppressed by proteasome inhibition. Furthermore, insulin secretion in isolated mouse pancreatic islets was also decreased by proteasome inhibition. These results demonstrate that the ubiquitin-proteasome system plays a critical role in insulin secretion by maintaining normal function of VDCCs.

Highlights

We previously established two different mouse pancreatic ␤-cell sublines from MIN6 cells, which facilitate study of the mechanisms of impaired insulin secretion [8]
The peak currents of voltage-dependent calcium channels (VDCCs) in MIN6-m9 cells exposed to proteasome inhibitors were significantly lower than in control cells (Fig. 6B). These data demonstrate that inhibition of proteasome activity in MIN6-m9 cells results in a loss of VDCC activity that results in impaired insulin secretion
It is well known that mutations in glucokinase, an enzyme having a critical role in glucose-sensing and metabolism in pancreatic ␤-cells, affects insulin secretion and causes a form of maturityonset diabetes of the young (MODY2) [27] and severe persistent hyperinsulinemic hypoglycemia [28]

Summary

Introduction

We previously established two different mouse pancreatic ␤-cell sublines from MIN6 cells, which facilitate study of the mechanisms of impaired insulin secretion [8]. E-64-d, a thiol protease inhibitor that does not suppress proteasome activity [15], showed no significant effects on insulin secretion in MIN6-m9 cells, even when an excessive concentration of the agent (100 ␮M) was applied (Fig. 1A). These data demonstrate that inhibition of proteasome activity causes impairment of glucose-induced insulin secretion in pancreatic ␤-cells.

Results

Conclusion