Debbie C. Thurmond

CAP defines a second signalling pathway required for insulin-stimulated glucose transport

Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl protooncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP–Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP–Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl–CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.

Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10

The stimulation of glucose uptake by insulin in muscle and adipose tissue requires translocation of the GLUT4 glucose transporter protein from intracellular storage sites to the cell surface. Although the cellular dynamics of GLUT4 vesicle trafficking are well described, the signalling pathways that link the insulin receptor to GLUT4 translocation remain poorly understood. Activation of phosphatidylinositol-3-OH kinase (PI(3)K) is required for this trafficking event, but it is not sufficient to produce GLUT4 translocation. We previously described a pathway involving the insulin-stimulated tyrosine phosphorylation of Cbl, which is recruited to the insulin receptor by the adapter protein CAP. On phosphorylation, Cbl is translocated to lipid rafts. Blocking this step completely inhibits the stimulation of GLUT4 translocation by insulin. Here we show that phosphorylated Cbl recruits the CrkII–C3G complex to lipid rafts, where C3G specifically activates the small GTP-binding protein TC10. This process is independent of PI(3)K, but requires the translocation of Cbl, Crk and C3G to the lipid raft. The activation of TC10 is essential for insulin-stimulated glucose uptake and GLUT4 translocation. The TC10 pathway functions in parallel with PI(3)K to stimulate fully GLUT4 translocation in response to insulin.

Molecular Basis of Insulin-stimulated GLUT4 Vesicle Trafficking LOCATION! LOCATION! LOCATION!

Among all the diverse actions of insulin, one of the most critical and intensively studied is its regulation of glucose homeostasis. In the postabsorptive state, insulin action in muscle and adipose tissue results in increased glucose uptake from the circulation, thereby maintaining plasma euglycemia and preventing hyperglycemia (1–3). Defects in this pathway result in insulin resistance, a condition in which excessive concentrations of insulin are required to reduce blood glucose levels and often precede the development of frank Type II diabetes (3, 4). Although it has been appreciated for almost two decades that this major action of insulin results from a redistribution of glucose transporter proteins from intracellular storage sites to the plasma membrane (5, 6), the cellular mechanisms responsible for these trafficking events and the defects associated with insulin resistance have remained largely enigmatic.

Munc18c function is required for insulin-stimulated plasma membrane fusion of GLUT4 and insulin-responsive amino peptidase storage vesicles.

ABSTRACT To examine the functional role of the interaction between Munc18c and syntaxin 4 in the regulation of GLUT4 translocation in 3T3L1 adipocytes, we assessed the effects of introducing three different peptide fragments (20 to 24 amino acids) of Munc18c from evolutionarily conserved regions of the Sec1 protein family predicted to be solvent exposed. One peptide, termed 18c/pep3, inhibited the binding of full-length Munc18c to syntaxin 4, whereas expression of the other two peptides had no effect. In parallel, microinjection of 18c/pep3 but not a control peptide inhibited the insulin-stimulated translocation of endogenous GLUT4 and insulin-responsive amino peptidase (IRAP) to the plasma membrane. In addition, expression of 18c/pep3 prevented the insulin-stimulated fusion of endogenous and enhanced green fluorescent protein epitope-tagged GLUT4- and IRAP-containing vesicles into the plasma membrane, as assessed by intact cell immunofluorescence. However, unlike the pattern of inhibition seen with full-length Munc18c expression, cells expressing 18c/pep3 displayed discrete clusters of GLUT4 abd IRAP storage vesicles at the cell surface which were not contiguous with the plasma membrane. Together, these data suggest that the interaction between Munc18c and syntaxin 4 is required for the integration of GLUT4 and IRAP storage vesicles into the plasma membrane but is not necessary for the insulin-stimulated trafficking to and association with the cell surface.

Syntaxin 4 heterozygous knockout mice develop muscle insulin resistance

To investigate the physiological function of syntaxin 4 in the regulation of GLUT4 vesicle trafficking, we used homologous recombination to generate syntaxin 4-knockout mice. Homozygotic disruption of the syntaxin 4 gene results in early embryonic lethality, whereas heterozygous knockout mice, Syn4(+/-), had normal viability with no significant impairment in growth, development, or reproduction. However, the Syn4(+/-) mice manifested impaired glucose tolerance with a 50% reduction in whole-body glucose uptake. This defect was attributed to a 50% reduction in skeletal muscle glucose transport determined by 2-deoxyglucose uptake during hyperinsulinemic-euglycemic clamp procedures. In parallel, insulin-stimulated GLUT4 translocation in skeletal muscle was also significantly reduced in these mice. In contrast, Syn4(+/-) mice displayed normal insulin-stimulated glucose uptake and metabolism in adipose tissue and liver. Together, these data demonstrate that syntaxin 4 plays a critical physiological role in insulin-stimulated glucose uptake in skeletal muscle. Furthermore, reduction in syntaxin 4 protein levels in this tissue can account for the impairment in whole-body insulin-stimulated glucose metabolism in this animal model.

Filamentous Actin Regulates Insulin Exocytosis through Direct Interaction with Syntaxin 4

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.

Inhibition or Ablation of p21-activated Kinase (PAK1) Disrupts Glucose Homeostatic Mechanisms in Vivo

Background: P21-activated kinase (PAK1) is a downstream effector of the GTPase Cdc42. Results: Inhibition of Cdc42-PAK1 signaling in human islets inhibited insulin secretion. PAK1 knock-out mice showed defects in insulin release and skeletal muscle insulin action, underlying impaired whole body glucose homeostasis. Conclusion: Attenuated PAK1 abundance/activation may contribute to type 2 diabetes susceptibility. Significance: Cdc42-PAK1 signaling is crucial for regulating glucose homeostasis in vivo. 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.

Caveolin-1 Functions as a Novel Cdc42 Guanine Nucleotide Dissociation Inhibitor in Pancreatic β-Cells

The cycling of the small Rho family GTPase Cdc42 is required for insulin granule exocytosis, although the regulatory proteins involved in Cdc42 cycling in pancreatic β-cells are unknown. Here we demonstrate that the caveolar protein caveolin-1 (Cav-1) is a Cdc42-binding protein in β-cells. Cav-1 associated with Cdc42-VAMP2-bound granules present near the plasma membrane under basal conditions. However, stimulation with glucose induced the dissociation of Cav-1 from Cdc42-VAMP2 complexes, coordinate with the timing of Cdc42 activation. Analyses of the Cav-1 scaffolding domain revealed a motif conserved in guanine nucleotide dissociation inhibitors (GDIs), which suggested a novel role for Cav-1 as a Cdc42 GDI in β-cells. The novel role was further supported by: 1) in vitro binding analyses that demonstrated a direct interaction between Cav-1 and Cdc42; 2) GST-Cdc42 interaction assays showing preferential Cav-1 binding to GDP-Cdc42 over that of GTP-Cdc42; 3) Cav-1 depletion studies resulting in an inappropriate 40% induction of activated Cdc42 in the absence of stimuli and also a 40% increase in basal insulin release from both MIN6 cells and islets. Expression of wild-type Cav-1 in Cav-1-depleted cells restored basal level secretion to normal, whereas expression of a scaffolding domain mutant of Cav-1 failed to normalize secretion. Taken together, these data suggest that Cav-1 functions as a Cdc42 GDI in β-cells, maintaining Cdc42 in an inactive state and regulating basal secretion in the absence of stimuli. Through its interaction with the Cdc42-VAMP2-bound insulin granule complex, Cav-1 may contribute to the specific targeting of granules to “active sites” of exocytosis organized by caveolae.

Molecular machinery involved in the insulin-regulated fusion of GLUT4-containing vesicles with the plasma membrane

The GLUT4 facilitative glucose transporter protein is primarily expressed in muscle and adipose tissue and accounts for the majority of post-prandial glucose uptake. In the basal or non-stimulated state, GLUT4 is localized to intracellular membrane compartments sequestered away from circulating glucose. However, in response to agonist stimulation, there is a marked redistribution of the GLUT4 protein to the cell surface membrane providing a transport route for the uptake of glucose. This GLUT4 translocation can be divided into four general steps: (i) GLUT4 vesicle trafficking outofthe storage pool, (ii) docking just below the cell surface, (iii) priming via the interactions of the SNARE proteins present on the vesicular and plasma membranes, and (iv) fusion of the GLUT4 vesicle with the plasma membrane. This review focuses on recent advances made in identification and characterization of the molecular events and protein interactions involved in these steps of insulin-stimulated GLUT4 translocation.The GLUT4 facilitative glucose transporter protein is primarily expressed in muscle and adipose tissue and accounts for the majority of post-prandial glucose uptake. In the basal or non-stimulated state, GLUT4 is localized to intracellular membrane compartments sequestered away from circulating glucose. However, in response to agonist stimulation, there is a marked redistribution of the GLUT4 protein to the cell surface membrane providing a transport route for the uptake of glucose. This GLUT4 translocation can be divided into four general steps: (i) GLUT4 vesicle trafficking out of the storage pool, (ii) docking just below the cell surface, (iii) priming via the interactions of the SNARE proteins present on the vesicular and plasma membranes, and (iv) fusion of the GLUT4 vesicle with the plasma membrane. This review focuses on recent advances made in identification and characterization of the molecular events and protein interactions involved in these steps of insulin-stimulated GLUT4 translocation.

Signaling mechanisms of glucose-induced F-actin remodeling in pancreatic islet β cells.

The maintenance of whole-body glucose homeostasis is critical for survival, and is controlled by the coordination of multiple organs and endocrine systems. Pancreatic islet β cells secrete insulin in response to nutrient stimuli, and insulin then travels through the circulation promoting glucose uptake into insulin-responsive tissues such as liver, skeletal muscle and adipose. Many of the genes identified in human genome-wide association studies of diabetic individuals are directly associated with β cell survival and function, giving credence to the idea that β-cell dysfunction is central to the development of type 2 diabetes. As such, investigations into the mechanisms by which β cells sense glucose and secrete insulin in a regulated manner are a major focus of current diabetes research. In particular, recent discoveries of the detailed role and requirements for reorganization/remodeling of filamentous actin (F-actin) in the regulation of insulin release from the β cell have appeared at the forefront of islet function research, having lapsed in prior years due to technical limitations. Recent advances in live-cell imaging and specialized reagents have revealed localized F-actin remodeling to be a requisite for the normal biphasic pattern of nutrient-stimulated insulin secretion. This review will provide an historical look at the emergent focus on the role of the actin cytoskeleton and its regulation of insulin secretion, leading up to the cutting-edge research in progress in the field today.