Second-phase insulin secretion gets cool.

STIMULUS-SECRETION COUPLING of insulin is a highly orchestrated process initiated by the uptake of glucose by pancreatic -cells culminating with the fusion of insulin vesicles with the plasma membrane and release of insulin into the extracellular space. A hallmark feature of stimulus-secretion coupling is the biphasic nature of insulin secretion following an elevation in plasma glucose levels (9). The primary mechanisms involved in glucose-stimulated insulin secretion have been established (9). Briefly, glucose uptake by membrane glucose transporters (GLUT1, humans; GLUT2, rodents) leads to enhanced glucose metabolism and subsequent elevation in the ratio of ATP to MgADP. Under basal glucose conditions, ATP-sensitive K channels open, thereby maintaining the membrane potential around 70 mV. The increased ratio of intracellular ATP to MgADP induces closure of ATP-sensitive K channels, resulting in cell membrane depolarization and the subsequent activation of voltagegated L-type Ca channels. The influx of extracellular Ca through the L-type Ca channels induces oscillatory elevations in intracellular Ca levels, which promotes the fusion of insulin-containing vesicles with the cell membrane through the interaction with SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. The oscillatory electrical activity and intracellular Ca are regulated by the opening of Ca -activated K channels. This entire process is suppressed or terminated by the inactivation of the L-type Ca channels and the opening of the voltage-gated and Ca -activated K channels (9). High-resolution temporal measurements of insulin exocytosis have been achieved by single-cell capacitance analysis and total internal reflection fluorescence (TIRF) microscopy. Separate pools of insulin vesicles, readily releasable and reserve pools, are present in the -cells (9). Fusion-competent granules docked and primed in close apposition with the plasma membrane are responsible for the first phase of insulin release (9). TIRF microscopy has shown that first-phase exocytotic events are localized to the plasma membrane, where the SNARE protein syntaxin 1A clusters (8). The second phase results from the mobilization of insulin granules (newcomers) in the reserve pool and occurs externally to syntaxin 1A clusters. Our basic understanding of the processes involved in stimulus-secretion coupling has been established. However, precise molecular and cellular mechanisms underlying the temporal and spatial regulation of insulin secretion are less understood. Moreover, cellular pathways and proteins have been shown to differentially regulate firstand second-phase insulin release. Small G proteins and their regulatory proteins [guanine nucleotide-exchange factors (GEF) and guanine nucleotide dissociation inhibitors (GDI)] play a vital role in regulating the docking and priming of insulin vesicles, insulin vesicular fusion, and cytoskeletal remodeling in -cells. These small G proteins cycle between a GDP-bound inactive state and an active GTP-bound configuration. GEF facilitate the conversion of the GDP-bound small G proteins into the GTP-bound active form, while GDI prevent GDP dissociation (2). There is a distinct temporal activation of the small Rho GTPases, Cdc42, and Rac1, in -cells. A rapid but transient activation of Cdc42 occurs within 3 min of glucose stimulation (7). Loss of Cdc42 reduces second-phase insulin secretion (11). Activated Cdc42 leads to the activation of PAK1 (p21-activated kinase), leading to the subsequent phosphorylation of the GDI RhoGDI and the dissociation of Rho-GDI from GDP-bound Rac1. As anticipated, -cell Rac1 activation occurs much later (15–20 min) than Cdc42 following glucose stimulation due to the multiple steps in the activation pathway (10). Both activated Cdc42 and Rac1 stimulate the translocation of insulinsecretory vesicles to the plasma membrane through their effects on cytoskeletal remodeling (4, 7). In addition to RhoGDI, caveolin-1 is another GDI of Cdc42 and Rac1 (3, 11). Caveolin-1 is associated primarily as a scaffolding protein localized in cholesterol-rich membrane domains (caveolae). However, caveolin-1 was found to interact with Cdc42 that were bound to VAMP2 on insulin granules present near the plasma membrane (5). Upon glucose stimulation, caveolin-1 dissociates from Cdc42, allowing for Cdc42 activation. The preferential interaction of Rho-GDI with Cdc42 and Rac1 is governed by tyrosine and serine phosphorylation (11). Dissociation of Rho-GDI with Cdc42 is dependent on its phosphorylation at Tyr, whereas phosphorylation at this tyrosine residue as well as at Ser and Ser are required for dissociating Rac1 from Rho-GDI. Loss of Rho-GDI enhances second-phase insulin release while having no effect on basal or first-phase insulin secretion. In contrast, mutation of the RhoGDI phosphorylation sites specifically inhibits second-phase insulin release (10). These findings support the concept that Rho GTPases and their regulation by GDIs have a fundamental importance in regulating the second phase of insulin secretion. In this issue of this Journal, Kepner et al. (1) have now identified expression of the GEF, Cool-1 (clone out of library-1)/ PIX ( PAK-interacting exchange factor), in pancreatic -cells. PIX has been shown to regulate the activity of PAK, Cdc42, and Rac. Using a candidate PCR screen approach, the authors identified eight putative GEFs known to interact with Cdc42. Of those eight, only PIX expression was confirmed by Western blotting in islets and MIN6 cells. Moreover, human and mouse islets and MIN6