The small Rho family GTPases Cdc42 and Rac1 have each been shown to function in insulin exocytosis and are presumed to function in actin remodeling and insulin granule mobilization. However, whether either GTPase is required for the mobilization phase of insulin release (second phase) and are linked in a common signaling pathway has remained unknown. Here we demonstrate that small interfering RNA-mediated depletion of Cdc42 from isolated islets results in the selective loss of secondphase insulin release. Consistent with a role in this nutrient-dependent phase, Cdc42 activation was detected exclusively in response to d-glucose and was unresponsive to KCl or non-metabolizable glucose analogs in MIN6 β-cells. Cdc42 activation occurred early in secretion (3 min), whereas Rac1 activation required ∼15–20 min, suggesting Cdc42 as proximal and Rac1 as distal regulators of second-phase secretion. Importantly, Rac1 activation and function was linked in a common pathway downstream of Cdc42; Cdc42 depletion ablated glucose-induced Rac1 activation, and expression of constitutively active Rac1 in Cdc42-depleted cells functionally restored glucose-stimulated insulin secretion. Occurring at a time midway between Cdc42 and Rac1 activations was the phosphorylation of p21-activated-kinase 1 (Pak1), and this phosphorylation event required Cdc42. Moreover, small interfering RNA-mediated Pak1 depletion abolished Rac1 activation and glucose-stimulated insulin release, suggesting that Pak1 may mediate the link between Cdc42 and Rac1 in this pathway. Taken together, these data substantiate the existence of a novel signaling pathway in the islet β-cell whereby Cdc42 functions as a key proximal transmitter of the glucose signal early in stimulus-secretion coupling to support the later stage of insulin release.

The p21-activated kinase PAK1 is implicated in tumorigenesis, and efforts to inhibit PAK1 signaling as a means to induce tumor cell apoptosis are underway. However, PAK1 has also been implicated as a positive effector of mechanisms in clonal pancreatic beta cells and skeletal myotubes that would be crucial to maintaining glucose homeostasis in vivo. Of relevance, human islets of Type 2 diabetic donors contained ~80% less PAK1 protein compared with non-diabetics, implicating PAK1 in islet signaling/scaffolding functions. Mimicking this, islets from PAK1(-/-) knock-out mice exhibited profound defects in the second/sustained-phase of insulin secretion. Reiteration of this specific defect by human islets treated with the PAK1 signaling inhibitor IPA3 revealed PAK1 signaling to be of primary functional importance. Analyses of human and mouse islet beta cell signaling revealed PAK1 activation to be 1) dependent upon Cdc42 abundance, 2) crucial for signaling downstream to activate ERK1/2, but 3) dispensable for cofilin phosphorylation. Importantly, the PAK1(-/-) knock-out mice were found to exhibit whole body glucose intolerance in vivo. Exacerbating this, the PAK1(-/-) knock-out mice also exhibited peripheral insulin resistance. Insulin resistance was coupled to ablation of insulin-stimulated GLUT4 translocation in skeletal muscle from PAK1(-/-) knock-out mice, and in sharp contrast to islet beta cells, skeletal muscle PAK1 loss was underscored by defective cofilin phosphorylation but normal ERK1/2 activation. Taken together, these data provide the first human islet and mammalian in vivo data unveiling the key and crucial roles for differential PAK1 signaling in the multi-tissue regulation of whole body glucose homeostasis.

Type 2 diabetes has been coined “a two-hit disease,” as it involves specific defects of glucose-stimulated insulin secretion from the pancreatic beta cells in addition to defects in peripheral tissue insulin action required for glucose uptake. Both of these processes, insulin secretion and glucose uptake, are mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein core complexes composed of syntaxin, SNAP-23/25, and VAMP proteins. The SNARE core complex is regulated by the Sec1/Munc18 (SM) family of proteins, which selectively bind to their cognate syntaxin isoforms with high affinity. The process of insulin secretion uses multiple Munc18-syntaxin isoform pairs, whereas insulin action in the peripheral tissues appears to use only the Munc18c-syntaxin 4 pair. Importantly, recent reports have linked obesity and Type 2 diabetes in humans with changes in protein levels and single nucleotide polymorphisms (SNPs) of Munc18 and syntaxin isoforms relevant to these exocytotic processes, although the molecular mechanisms underlying the observed phenotypes remain incomplete (5, 104, 144). Given the conservation of these proteins in two seemingly disparate processes and the need to design and implement novel and more effective clinical interventions, it will be vitally important to delineate the mechanisms governing these conserved SNARE-mediated exocytosis events. Thus, we provide here an up-to-date historical review of advancements in defining the roles and molecular mechanisms of Munc18-syntaxin complexes in the pathophysiology of Type 2 diabetes.

Glucose-induced insulin exocytosis is coupled to associations between F-actin and SNARE proteins, although the nature and function of these interactions remains unknown. Toward this end we show here that both Syntaxin 1A and Syntaxin 4 associated with F-actin in MIN6 cells and that each interaction was rapidly and transiently diminished by stimulation of cells with d-glucose. Of the two isoforms, only Syntaxin 4 was capable of interacting directly with F-actin in an in vitro sedimentation assay, conferred by the N-terminal 39-112 residues of Syntaxin 4. The 39-112 fragment was capable of selective competitive inhibitory action, disrupting endogenous F-actin-Syntaxin 4 binding in MIN6 cells. Disruption of F-actin-Syntaxin 4 binding correlated with enhanced glucose-stimulated insulin secretion, mediated by increased granule accumulation at the plasma membrane and increased Syntaxin 4 accessibility under basal conditions. However, no increase in basal level Syntaxin 4-VAMP2 association occurred with either latrunculin treatment or expression of the 39-112 fragment. Taken together, these data disclose a new underlying mechanism by which F-actin negatively regulates exocytosis via binding and blocking Syntaxin 4 accessibility, but they also reveal the existence of additional signals and/or steps required to trigger the subsequent docking and fusion steps of exocytosis.

Type 2 diabetes (T2D) is a heterogenous disease, and conventionally, peripheral insulin resistance (IR) was thought to precede islet β-cell dysfunction, promoting progression from prediabetes to T2D. New evidence suggests that T2D-lean individuals experience early β-cell dysfunction without significant IR. Regardless of the primary event (i.e., IR vs. β-cell dysfunction) that contributes to dysglycemia, significant early-onset oxidative damage and mitochondrial dysfunction in multiple metabolic tissues may be a driver of T2D onset and progression. Oxidative stress, defined as the generation of reactive oxygen species (ROS), is mediated by hyperglycemia alone or in combination with lipids. Physiological oxidative stress promotes inter-tissue communication, while pathological oxidative stress promotes inter-tissue mis-communication, and new evidence suggests that this is mediated via extracellular vesicles (EVs), including mitochondria containing EVs. Under metabolic-related stress conditions, EV-mediated cross-talk between β-cells and skeletal muscle likely trigger mitochondrial anomalies leading to prediabetes and T2D. This article reviews the underlying molecular mechanisms in ROS-related pathogenesis of prediabetes, including mitophagy and mitochondrial dynamics due to oxidative stress. Further, this review will describe the potential of various therapeutic avenues for attenuating oxidative damage, reversing prediabetes and preventing progression to T2D.

The disruption of Munc18c binding to syntaxin 4 impairs insulin-stimulated GLUT4 vesicle translocation in 3T3L1 adipocytes. To investigate the physiological function and requirement for Munc18c in the regulation of GLUT4 translocation and glucose homeostasis in vivo, we used homologous recombination to generate Munc18c-knockout (KO) mice. Homozygotic disruption of the Munc18c gene resulted in early embryonic lethality, whereas heterozygous KO mice (Munc18c^−/+) had normal viability. Munc18c^−/+ mice displayed significantly decreased insulin sensitivity in an insulin tolerance test and a >50% reduction in skeletal muscle insulin-stimulated GLUT4 translocation when compared with wild-type (WT) mice. Furthermore, glucose-stimulated insulin secretion was significantly reduced in islets isolated from Munc18c^−/+ mice compared with those from WT mice. Despite the defects in insulin action and secretion, Munc18c^−/+ mice demonstrated the ability to clear glucose to the same level as WT mice in a glucose tolerance test when fed a normal diet. However, after consuming a high-fat diet for only 5 weeks, the Munc18c^−/+ mice manifested severely impaired glucose tolerance compared with high-fat−fed WT mice. Taken together, these data suggest that the reduction of Munc18c protein in the Munc18c^−/+ mice results in impaired insulin sensitivity with a latent increased susceptibility for developing severe glucose intolerance in response to environmental perturbations such as intake of a high-calorie diet rich in fat and carbohydrate.