Pancreatic β-Cells Secrete Insulin in Fast- and Slow-Release Forms

Abstract

Insulin vesicles contain a chemically rich mixture of cargo that includes ions, small molecules, and proteins. At present, it is unclear if all components of this cargo escape from the vesicle at the same rate or to the same extent during exocytosis. Here, we demonstrate through real-time imaging that individual rat and human pancreatic ␤-cells secrete insulin in heterogeneous forms that disperse either rapidly or slowly. In healthy pancreatic ␤-cells maintained in culture, most vesicles discharge insulin in its fastrelease form, a form that leaves individual vesicles in a few hundred milliseconds. The fast-release form of insulin leaves vesicles as rapidly as C-peptide leaves vesicles. Healthy ␤-cells also secrete a slow-release form of insulin that leaves vesicles more slowly than C-peptide, over times ranging from seconds to minutes. Individual ␤-cells make vesicles with both forms of insulin, though not all vesicles contain both forms of insulin. In addition, we confirm that insulin vesicles store their cargo in two functionally distinct compartments: an acidic solution, or halo, and a condensed core. Thus, our results suggest two important features of the condensed core: 1) It exists in different states among the vesicles undergoing exocytosis and 2) its dissolution determines the availability of insulin during exocytosis. Diabetes 55:600 -607, 2006 I nsufficient insulin secretion in the face of insulin resistance leads to the disease type 2 diabetes (1). The mechanism responsible for insufficient insulin secretion remains unclear. Before it is possible to understand why insulin secretion fails to compensate for insulin resistance during the progression of type 2 diabetes (2), it is essential to understand insulin secretion at all levels of biological complexity, ranging from the whole pancreas within a living animal to the pancreatic ␤-cell and its fundamental unit of secretion, the insulin secretory vesicle.Insulin vesicles contain a chemically complex mixture of ions, small molecules, and proteins (3,4). Among the ions, there are large amounts of two divalent cations: zinc and calcium (5). Both of these ions are believed to play important roles in the regulated secretion of insulin (6). Among the vesicle proteins, insulin-related peptides comprise ϳ75% of the mass (7). The remaining 25% of the protein mass includes more than a hundred different membrane and cargo proteins. Although our understanding of regulated secretion of insulin has progressed greatly in recent years, the mechanisms that control the trafficking, processing, and secretion of insulin vesicle cargo are still matters of debate and extensive research (8).Previously, we (9) and others (10 -13) have studied secretion from single insulin vesicles by imaging fluorescently tagged proteins consisting of a vesicle cargo protein linked to a fluorescent protein. The fluorescently tagged vesicle cargo proteins, hereafter referred to as fluorescent cargo proteins, have provided a valuable and powerful tool for studies of single vesicle exocytosis. H...

Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR)

“…We demonstrate here that ZIMIR can be used to image the exocytotic activity of a variety of insulin-secreting cell preparations, ranging from dispersed cells to the intact islet, reporting changes across the entire cell surface in each case (in contrast to TIRF microscopy) (10,16). Each approach revealed interesting features of the organization and timing of GSIS at both the cellular and subcellular levels.…”

“…However, because FluoZin-3 or other Zn 2+ sensors are applied to the extracellular bath, the sensitivity of detecting local Zn 2+ release near the plasma membrane is compromised by the background fluorescence from the bulk solution. Consequently, total internal reflection of fluorescence (TIRF) microscopy has been applied to FluoZin-3 or RhodZin-3 imaging to reject bulk fluorescence signal and to study secretion at the interface between a cell and the underlying glass coverslip (10,16). More sensitive imaging probes that are compatible with wide-field epifluorescence detection or confocal laser scanning microscopy (CLSM) would be extremely valuable for following the pattern or the site of insulin release in 3D in cell populations over time.…”

“…Pancreatic β cells contain high concentrations of Zn 2+ in the secretory granules, much of it coordinated with insulin (9), which is coreleased with the hormone on stimulation (10). This phenomenon has been exploited to develop experiments using the fluorescent Zn 2+ sensors Zinquin (11), FluoZin-3 (12), RhodZin-3 or Newport Green DCF (10), and ZnAF-2 (13) as a surrogate for measuring insulin release (14)(15)(16).…”

Orchestration of Glucose Homeostasis

“…When stimulated by rapid changes in plasma glucose, insulin release from the ␤-cells is biphasic. This biphasic pattern, originally described by Grodsky and colleagues (65,66), has been much studied and reflects two pools of releasable insulin within the ␤-cells, which have recently been imaged directly (67). It is interesting to contemplate the possible significance of biphasic release with regards to overall glucose regulation.…”

Human Insulin Vesicle Dynamics During Pulsatile Secretion

“…We focused in particular on those cells that responded to glucose stimulation with pulsatile insulin secretion, using total internal reflection fluorescence (TIRF) microscopy (13) to image directly individual insulin vesicles that were made fluorescent (14,15). Because a TIRF microscope confines light to a thin optical slice at the base of a cell, it is especially well suited for monitoring the dynamics of membrane-proximal insulin vesicles.…”