James G. Burchfield

Identification of a distal GLUT4 trafficking event controlled by actin polymerization.

The insulin-stimulated trafficking of GLUT4 to the plasma membrane in muscle and fat tissue constitutes a central process in blood glucose homeostasis. The tethering, docking, and fusion of GLUT4 vesicles with the plasma membrane (PM) represent the most distal steps in this pathway and have been recently shown to be key targets of insulin action. However, it remains unclear how insulin influences these processes to promote the insertion of the glucose transporter into the PM. In this study we have identified a previously uncharacterized role for cortical actin in the distal trafficking of GLUT4. Using high-frequency total internal reflection fluorescence microscopy (TIRFM) imaging, we show that insulin increases actin polymerization near the PM and that disruption of this process inhibited GLUT4 exocytosis. Using TIRFM in combination with probes that could distinguish between vesicle transport and fusion, we found that defective actin remodeling was accompanied by normal insulin-regulated accumulation of GLUT4 vesicles close to the PM, but the final exocytotic fusion step was impaired. These data clearly resolve multiple steps of the final stages of GLUT4 trafficking, demonstrating a crucial role for actin in the final stage of this process.

Variations in the requirement for v-SNAREs in GLUT4 trafficking in adipocytes

Vesicle transport in eukaryotic cells is regulated by SNARE proteins, which play an intimate role in regulating the specificity of vesicle fusion between discrete intracellular organelles. In the present study we investigated the function and plasticity of v-SNAREs in insulin-regulated GLUT4 trafficking in adipocytes. Using a combination of knockout mice, v-SNARE cleavage by clostridial toxins and total internal reflection fluorescence microscopy, we interrogated the function of VAMPs 2, 3 and 8 in this process. Our studies reveal that the simultaneous disruption of VAMPs 2, 3 and 8 completely inhibited insulin-stimulated GLUT4 insertion into the plasma membrane, due to a block in vesicle docking at the plasma membrane. These defects could be rescued by re-expression of VAMP2, VAMP3 or VAMP8 alone, but not VAMP7. These data indicate a plasticity in the requirement for v-SNAREs in GLUT4 trafficking to the plasma membrane and further define an important role for the v-SNARE proteins in pre-fusion docking of vesicles.

Exocytotic vesicle behaviour assessed by total internal reflection fluorescence microscopy.

The regulated trafficking or exocytosis of cargo‐containing vesicles to the cell surface is fundamental to all cells. By coupling the technology of fluorescently tagged fusion proteins with total internal reflection fluorescence microscopy (TIRFM), it is possible to achieve the high spatio‐temporal resolution required to study the dynamics of sub‐plasma membrane vesicle trafficking and exocytosis. TIRFM has been used in a number of cell types to visualize and dissect the various steps of exocytosis revealing how molecules identified via genetic and/or biochemical approaches are involved in the regulation of this process. Here, we summarize the contribution of TIRFM to our understanding of the mechanism of exocytosis and discuss the novel methods of analysis that are required to exploit the large volumes of data that can be produced using this technique.

The Rab GTPase-Activating Protein TBC1D4/AS160 Contains an Atypical Phosphotyrosine-Binding Domain That Interacts with Plasma Membrane Phospholipids To Facilitate GLUT4 Trafficking in Adipocytes

ABSTRACT The Rab GTPase-activating protein TBC1D4/AS160 regulates GLUT4 trafficking in adipocytes. Nonphosphorylated AS160 binds to GLUT4 vesicles and inhibits GLUT4 translocation, and AS160 phosphorylation overcomes this inhibitory effect. In the present study we detected several new functional features of AS160. The second phosphotyrosine-binding domain in AS160 encodes a phospholipid-binding domain that facilitates plasma membrane (PM) targeting of AS160, and this function is conserved in other related RabGAP/Tre-2/Bub2/Cdc16 (TBC) proteins and an AS160 ortholog in Drosophila. This region also contains a nonoverlapping intracellular GLUT4-containing storage vesicle (GSV) cargo-binding site. The interaction of AS160 with GSVs and not with the PM confers the inhibitory effect of AS160 on insulin-dependent GLUT4 translocation. Constitutive targeting of AS160 to the PM increased the surface GLUT4 levels, and this was attributed to both enhanced AS160 phosphorylation and 14-3-3 binding and inhibition of AS160 GAP activity. We propose a model wherein AS160 acts as a regulatory switch in the docking and/or fusion of GSVs with the PM.

Diverse roles for protein kinase C δ and protein kinase C ε in the generation of high-fat-diet-induced glucose intolerance in mice: regulation of lipogenesis by protein kinase C δ

Aims/hypothesisThis study aimed to determine whether protein kinase C (PKC) δ plays a role in the glucose intolerance caused by a high-fat diet, and whether it could compensate for loss of PKCε in the generation of insulin resistance in skeletal muscle.MethodsPrkcd−/−, Prkce−/− and wild-type mice were fed high-fat diets and subjected to glucose tolerance tests. Blood glucose levels and insulin responses were determined during the tests. Insulin signalling in liver and muscle was assessed after acute in vivo insulin stimulation by immunoblotting with phospho-specific antibodies. Activation of PKC isoforms in muscle from Prkce−/− mice was assessed by determining intracellular distribution. Tissues and plasma were assayed for triacylglycerol accumulation, and hepatic production of lipogenic enzymes was determined by immunoblotting.ResultsBoth Prkcd−/− and Prkce−/− mice were protected against high-fat-diet-induced glucose intolerance. In Prkce−/− mice this was mediated through enhanced insulin availability, while in Prkcd−/− mice the reversal occurred in the absence of elevated insulin. Neither the high-fat diet nor Prkcd deletion affected maximal insulin signalling. The activation of PKCδ in muscle from fat-fed mice was enhanced by Prkce deletion. PKCδ-deficient mice exhibited reduced liver triacylglycerol accumulation and diminished production of lipogenic enzymes.Conclusions/interpretationDeletion of genes encoding isoforms of PKC can improve glucose intolerance, either by enhancing insulin availability in the case of Prkce, or by reducing lipid accumulation in the case of Prkcd. The absence of PKCε in muscle may be compensated by increased activation of PKCδ in fat-fed mice, suggesting that an additional role for PKCε in this tissue is masked.

Cluster analysis of insulin action in adipocytes reveals a key role for Akt at the plasma membrane.

The phosphatidylinositol 3-kinase/Akt pathway regulates many biological processes, including insulin-regulated GLUT4 insertion into the plasma membrane. However, Akt operates well below its capacity to facilitate maximal GLUT4 translocation. Thus, reconciling modest changes in Akt expression or activity as a cause of metabolic dysfunction is complex. To resolve this, we examined insulin regulation of components within the signaling cascade in a quantitative kinetic and dose-response study combined with hierarchical cluster analysis. This revealed a strong relationship between phosphorylation of Akt substrates and GLUT4 translocation but not whole cell Akt phosphorylation. In contrast, Akt activity at the plasma membrane strongly correlated with GLUT4 translocation and Akt substrate phosphorylation. Additionally, two of the phosphorylated sites in the Akt substrate AS160 clustered separately, with Thr(P)-642 grouped with other Akt substrates. Further experiments suggested that atypical protein kinase Cζ phosphorylates AS160 at Ser-588 and that these two sites are mutually exclusive. These data indicate the utility of hierarchical cluster analysis for identifying functionally related biological nodes and highlight the importance of subcellular partitioning of key signaling components for biological specificity.

Deletion of PKCε Selectively Enhances the Amplifying Pathways of Glucose-Stimulated Insulin Secretion via Increased Lipolysis in Mouse β-Cells

OBJECTIVE Insufficient insulin secretion is a hallmark of type 2 diabetes, and exposure of β-cells to elevated lipid levels (lipotoxicity) contributes to secretory dysfunction. Functional ablation of protein kinase C ε (PKCε) has been shown to improve glucose homeostasis in models of type 2 diabetes and, in particular, to enhance glucose-stimulated insulin secretion (GSIS) after lipid exposure. Therefore, we investigated the lipid-dependent mechanisms responsible for the enhanced GSIS after inactivation of PKCε. RESEARCH DESIGN AND METHODS We cultured islets isolated from PKCε knockout (PKCεKO) mice in palmitate prior to measuring GSIS, Ca2+ responses, palmitate esterification products, lipolysis, lipase activity, and gene expression. RESULTS The enhanced GSIS could not be explained by increased expression of another PKC isoform or by alterations in glucose-stimulated Ca2+ influx. Instead, an upregulation of the amplifying pathways of GSIS in lipid-cultured PKCεKO β-cells was revealed under conditions in which functional ATP-sensitive K+ channels were bypassed. Furthermore, we showed increased esterification of palmitate into triglyceride pools and an enhanced rate of lipolysis and triglyceride lipase activity in PKCεKO islets. Acute treatment with the lipase inhibitor orlistat blocked the enhancement of GSIS in lipid-cultured PKCεKO islets, suggesting that a lipolytic product mediates the enhancement of glucose-amplified insulin secretion after PKCε deletion. CONCLUSIONS Our findings demonstrate a mechanistic link between lipolysis and the amplifying pathways of GSIS in murine β-cells, and they suggest an interaction between PKCε and lipolysis. These results further highlight the therapeutic potential of PKCε inhibition to enhance GSIS from the β-cell under conditions of lipid excess.

Quantitative proteomic analysis of the adipocyte plasma membrane

The adipocyte is a key regulator of mammalian metabolism. To advance our understanding of this important cell, we have used quantitative proteomics to define the protein composition of the adipocyte plasma membrane (PM) in the presence and absence of insulin. Using this approach, we have identified a high confidence list of 486 PM proteins, 52 of which potentially represent novel cell surface proteins, including a member of the adiponectin receptor family and an unusually high number of hydrolases with no known function. Several novel insulin-responsive proteins including the sodium/hydrogen exchanger, NHE6 and the collagens III and VI were also identified, and we provide evidence of PM-ER association suggestive of a unique functional association between these two organelles in the adipocyte. Together these studies provide a wealth of potential therapeutic targets for the manipulation of adipocyte function and a valuable resource for metabolic research and PM biology.

The diverse roles of protein kinase C in pancreatic β-cell function

Members of the serine/threonine PKC (protein kinase C) family perform diverse functions in multiple cell types. All members of the family are activated in signalling cascades triggered by occupation of cell surface receptors, but the cPKC (conventional PKC) and nPKC (novel PKC) isoforms are also responsive to fatty acid metabolites. PKC isoforms are involved in various aspects of pancreatic beta-cell function, including cell proliferation, differentiation and death, as well as regulation of secretion in response to glucose and muscarinic receptor agonists. Recently, the nPKC isoform, PKCepsilon, has also been implicated in the loss of insulin secretory responsiveness that underpins the development of Type 2 diabetes.

Novel Systems for Dynamically Assessing Insulin Action in Live Cells Reveals Heterogeneity in the Insulin Response

Regulated GLUT4 trafficking is a key action of insulin. Quantitative stepwise analysis of this process provides a powerful tool for pinpointing regulatory nodes that contribute to insulin regulation and insulin resistance. We describe a novel GLUT4 construct and workflow for the streamlined dissection of GLUT4 trafficking; from simple high throughput screens to high resolution analyses of individual vesicles. We reveal single cell heterogeneity in insulin action highlighting the utility of this approach – each cell displayed a unique and highly reproducible insulin response, implying that each cell is hard‐wired to produce a specific output in response to a given stimulus. These data highlight that the response of a cell population to insulin is underpinned by extensive heterogeneity at the single cell level. This heterogeneity is pre‐programmed within each cell and is not the result of intracellular stochastic events.