Mechanisms of biphasic insulin-granule exocytosis - roles of the cytoskeleton, small GTPases and SNARE proteins

Abstract

The release of insulin from pancreatic islets requires negative regulation to ensure low levels of insulin release under resting conditions, as well as positive regulation to facilitate robust responsiveness to conditions of elevated fuel or glucose. The first phase of release involves the plasma-membrane fusion of a small pool of granules, termed the readily releasable pool; these granules are already at the membrane under basal conditions, and discharge their cargo in response to nutrient and also non-nutrient secretagogues. By contrast, second-phase secretion is evoked exclusively by nutrients, and involves the mobilization of intracellular granules to t-SNARE sites at the plasma membrane to enable the distal docking and fusion steps of insulin exocytosis. Nearly 40 years ago, the actin cytoskeleton was first recognized as a key mediator of biphasic insulin release, and was originally presumed to act as a barrier to block granule docking at the cell periphery. More recently, however, the discovery of cycling GTPases that are involved in F-actin reorganization in the islet beta-cell, combined with the availability of reagents that are more specific and tools with which to study the mechanisms that underlie granule movement, have contributed greatly to our understanding of the role of the cytoskeleton in regulating biphasic insulin secretion. Herein, we provide historical perspective and review recent progress that has been made towards integrating cytoskeletal reorganization and cycling of small Rho-, Rab- and Ras-family GTPases into our current models of stimulus-secretion coupling and second-phase insulin release.

Fig. 1.

The stimulus-secretion coupling pathway of glucose-dependent insulin exocytosis. Glucose enters the cells via the GLUT2 transporter (1) and undergoes glycolytic and mitochondrial metabolism (2), which ultimately has the effect of increasing the ATP:ADP ratio (3). An increased ATP:ADP ratio leads to the closure of ATP-sensitive K_ATP channels (4) and to membrane depolarization (5), which triggers the opening of voltage-dependent Ca²⁺ channels (VDCCs) (6). The resulting influx of Ca²⁺ (7) induces the fusion of insulin-containing granules with the plasma membrane and insulin release from the cell (8). PM, plasma membrane.

Fig. 2.

F-actin reorganization, granule mobilization and glucose-stimulated insulin secretion. Under basal conditions (left panel), F-actin not only functions as a barrier to block SNARE-complex formation, but also supplies transportation tracks for insulin-containing granules. Glucose stimulation (right panel) triggers transient F-actin reorganization to allow the granules access to the plasma membrane (PM) for subsequent docking, fusion (mediated by interactions between VAMP2 and syntaxins) and insulin release.

Fig. 3.

Granule recruitment supports the second phase of insulin secretion. When present at elevated levels, extracellular glucose enters the islet β-cell through the GLUT2 transporter and rapidly undergoes intracellular metabolism. Through the classic stimulus-secretion coupling pathway, the resulting increase in [Ca²⁺]_i triggers the exocytosis of pre-docked granules in the RRP to give rise to the first phase of insulin secretion (1). Concurrently, the metabolic signal also induces F-actin reorganization (2) and recruits granules to the plasma membrane (PM) to support the sustained second phase of insulin secretion (3).

Fig. 4.

Roles of small GTPases and their cycling factors in glucose-stimulated insulin secretion. The small Rho-family GTPases Cdc42 and Rac1 are implicated in a common pathway that regulates glucose-induced actin remodeling. Cdc42 has also been implicated in directing granule targeting to t-SNARE sites through its ability to associate directly with VAMP2. The Rab GTPases Rab3A and Rab27A, as well as the Ras-family GTPases RalA and Rap1, have been proposed to function in insulin-granule docking and/or priming. PM, plasma membrane.