Preferential Release of Newly Synthesized Insulin Assessed by a Multi-Label Reporter System Using Pancreatic β-Cell Line MIN6

Ni Hou,Toshiyuki Takeuchi,Seiji Torii,Chisato Kubota-Murata,Meng Sun,Hideo Mogami,Ludger Johannes

doi:10.1371/journal.pone.0047921

Abstract

Newly synthesized hormones have been suggested to be preferentially secreted by various neuroendocrine cells. This observation indicates that there is a distinct population of secretory granules containing new and old hormones. Recent development of fluorescent timer proteins used in bovine adrenal chromaffin cells revealed that secretory vesicles segregate into distinct age-dependent populations. Here, we verify the preferential release of newly synthesized insulin in the pancreatic β-cell line, MIN6, using a combination of multi-labeling reporter systems with both fluorescent and biochemical procedures. This system allows hormones or granules of any age to be labeled, in contrast to the timer proteins, which require fluorescence shift time. Pulse-chase labeling with different color probes distinguishes insulin secretory granules by age, with younger granules having a predominantly intracellular localization rather than at the cell periphery.

Highlights

In pancreatic b-cells, insulin is initially synthesized as a precursor protein on the rough endoplasmic reticulum (ER), and is converted to proinsulin upon removal of a signal sequence during co-translational insertion into the ER lumen [1]
The resulting expression plasmid was transfected into the mouse pancreatic beta cell line MIN6, and the intracellular localization of the fusion protein was first analyzed by transient incubation with the cell membrane-permeable red fluorescent dye HT-TMR
TMR fluorescence of insulin-HT nearly entirely overlapped with green signals arising from insulin-enhanced green fluorescent protein (EGFP) fusion protein, whereas TMR signals were not detectable in untransfected cells, suggesting that the fluorescent signal on vesicles was specific

Summary

Introduction

In pancreatic b-cells, insulin is initially synthesized as a precursor protein (prepro-insulin) on the rough endoplasmic reticulum (ER), and is converted to proinsulin upon removal of a signal sequence during co-translational insertion into the ER lumen [1]. During maturation of ISGs to secretory granules (SGs), bioactive insulin is produced by the catalytic activities of PC1/3, PC2 and carboxypeptidase E [2]. Exocytosis, including SG targeting and membrane fusion, has been the focus of the studies on pancreatic b-cells that responded to glucose stimulation with pulsatile insulin secretion. Capacitance measurements of the cell membrane have been performed to monitor the exocytotic events in single b-cells, and individual SG exocytosis has been imaged directly using recently developed total internal reflection fluorescence (TIRF) microscopy [8]. Recent studies using simultaneous capacitance measurements and TIRF imaging suggested that insulin is stored in distinct SGs based on its age and younger insulin granules (up to 48 hours old) are secreted first from human pancreatic b-cells [9]

Methods

Results

Conclusion