Insulin-regulated Trafficking Research Articles

Pleiotropic effects of Syntaxin16 identified by gene editing in cultured adipocytes.

Adipocytes play multiple roles in the regulation of glucose metabolism which rely on the regulation of membrane traffic. These include secretion of adipokines and serving as an energy store. Central to their energy storing function is the ability to increase glucose uptake in response to insulin, mediated through translocation of the facilitative glucose transporter GLUT4 to the cell surface. The trans-Golgi reticulum localized SNARE protein syntaxin 16 (Sx16) has been identified as a key component of the secretory pathway required for insulin-regulated trafficking of GLUT4. We used CRISPR/Cas9 technology to generate 3T3-L1 adipocytes lacking Sx16 to understand the role of the secretory pathway on adipocyte function. GLUT4 mRNA and protein levels were reduced in Sx16 knockout adipocytes and insulin stimulated GLUT4 translocation to the cell surface was reduced. Strikingly, neither basal nor insulin-stimulated glucose transport were affected. By contrast, GLUT1 levels were upregulated in Sx16 knockout cells. Levels of sortilin and insulin regulated aminopeptidase were also increased in Sx16 knockout adipocytes which may indicate an upregulation of an alternative GLUT4 sorting pathway as a compensatory mechanism for the loss of Sx16. In response to chronic insulin stimulation, Sx16 knockout adipocytes exhibit elevated insulin-independent glucose transport and significant alterations in lactate metabolism. We further show that the adipokine secretory pathways are impaired in Sx16 knockout cells. Together this demonstrates a role for Sx16 in the control of glucose transport, the response to elevated insulin, cellular metabolic profiles and adipocytokine secretion.

Disruption of Adipose Rab10-Dependent Insulin Signaling Causes Hepatic Insulin Resistance.

Insulin controls glucose uptake into adipose and muscle cells by regulating the amount of GLUT4 in the plasma membrane. The effect of insulin is to promote the translocation of intracellular GLUT4 to the plasma membrane. The small Rab GTPase, Rab10, is required for insulin-stimulated GLUT4 translocation in cultured 3T3-L1 adipocytes. Here we demonstrate that both insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane are reduced by about half in adipocytes from adipose-specific Rab10 knockout (KO) mice. These data demonstrate that the full effect of insulin on adipose glucose uptake is the integrated effect of Rab10-dependent and Rab10-independent pathways, establishing a divergence in insulin signal transduction to the regulation of GLUT4 trafficking. In adipose-specific Rab10 KO female mice, the partial inhibition of stimulated glucose uptake in adipocytes induces insulin resistance independent of diet challenge. During euglycemic-hyperinsulinemic clamp, there is no suppression of hepatic glucose production despite normal insulin suppression of plasma free fatty acids. The impact of incomplete disruption of stimulated adipocyte GLUT4 translocation on whole-body glucose homeostasis is driven by a near complete failure of insulin to suppress hepatic glucose production rather than a significant inhibition in muscle glucose uptake. These data underscore the physiological significance of the precise control of insulin-regulated trafficking in adipocytes.

Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures

Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4) vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV) or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1–2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies.

Diacylglycerol promotes GLUT4 translocation to the cell surface in a PKCε-dependent and PKCλ/ι and -ζ-independent manner

AimEmerging evidence has pointed to the participation of protein kinase C (PKC) in insulin-regulated trafficking of the glucose transporter GLUT4. The present study investigated the effect of the PKC activator diacylglycerol (DAG) on GLUT4 trafficking and glucose uptake. Main methods3T3L1-GLUT4myc fibroblast cells expressing GLUT4myc were differentiated into adipocytes. Western blotting, glucose assay, and real-time RT-PCR were carried out in 3T3L1-GLUT4myc adipocytes. PKCλ/ι, -ζ, -ε, and -γ were knocked-down by transfecting each siRNA. Activity of PKC isozymes was assayed under the cell-free conditions. Key findingsInsulin increased cell surface localization of GLUT4 in 3T3L1-GLUT4myc adipocytes, and a similar effect was obtained with 1,2-dioleoyl-sn-glycerol (DO-DAG), 1-oleoyl-2-acetyl-sn-glycerol (OA-DAG), or 1,2-dipalmitoyl-sn-glycerol (DP-DAG). Like insulin, DO-DAG stimulated glucose uptake into adipocytes, but no significant synergistic increase in the glucose uptake was found with co-treatment with insulin and DO-DAG. Insulin activated Akt in adipocytes, but no Akt activation was induced by any investigated DAG. In the cell-free PKC assay, DAGs examined here activated PKCα, -βI, -βII, -γ, -δ, and -ε, but the atypical PKC isozymes PKCλ/ι and -ζ were not activated. Insulin-induced GLUT4 translocation to the cell surface was inhibited by knocking-down PKCλ/ι and -ζ, but not PKCγ or -ε. In contrast, DO-DAG-induced GLUT4 translocation to the cell surface was clearly prevented by knocking-down PKCε. SignificanceThe results of the present study indicate that DAG stimulates GLUT4 translocation to the cell surface by activating PKCε, regardless of PKCλ/-ι and -ζ.

Posttranslational modifications of GLUT4 affect its subcellular localization and translocation.

The facilitative glucose transporter type 4 (GLUT4) is expressed in adipose and muscle and plays a vital role in whole body glucose homeostasis. In the absence of insulin, only ~1% of cellular GLUT4 is present at the plasma membrane, with the vast majority localizing to intracellular organelles. GLUT4 is retained intracellularly by continuous trafficking through two inter-related cycles. GLUT4 passes through recycling endosomes, the trans Golgi network and an insulin-sensitive intracellular compartment, termed GLUT4-storage vesicles or GSVs. It is from GSVs that GLUT4 is mobilized to the cell surface in response to insulin, where it increases the rate of glucose uptake into the cell. As with many physiological responses to external stimuli, this regulated trafficking event involves multiple posttranslational modifications. This review outlines the roles of posttranslational modifications of GLUT4 on its function and insulin-regulated trafficking.

Endosomal sorting of GLUT4 and Gap1 is conserved between yeast and insulin-sensitive cells

The insulin-regulated trafficking of the facilitative glucose transporter GLUT4 in human fat and muscle cells and the nitrogen-regulated trafficking of the general amino acid permease Gap1 in the yeast Saccharomyces cerevisiae share several common features: Both Gap1 and GLUT4 are nutrient transporters that are mobilised to the cell surface from an intracellular store in response to an environmental cue; both are polytopic membrane proteins harbouring amino acid targeting motifs in their C-terminal tails that are required for their regulated trafficking; ubiquitylation of both Gap1 and GLUT4 plays an important role in their regulated trafficking, as do the ubiquitin-binding GGA (Golgi-localised, γ-ear-containing, ARF-binding) adaptor proteins. Here, we find that when expressed heterologously in yeast, human GLUT4 is subject to nitrogen-regulated trafficking in an ubiquitin-dependent manner similar to Gap1. In addition, by expressing a GLUT4/Gap1 chimeric protein in adipocytes we show that the carboxy-tail of Gap1 directs intracellular sequestration and insulin-regulated trafficking in adipocytes. These findings demonstrate that the trafficking signals and their cognate molecular regulatory machinery that mediate regulated exocytosis of membrane proteins are conserved across evolution.

Insulin-Regulated Trafficking of GLUT4 Requires Ubiquitination

A major consequence of insulin binding its receptor on fat and muscle cells is translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the cell surface where it serves to clear glucose from the bloodstream. Sorting of GLUT4 into its insulin-sensitive store requires the GGA [Golgi-localized, γ-ear-containing, ADP ribosylation factor (ARF)-binding] adaptor proteins, but the signal on GLUT4 to direct this sorting step is unknown. Here, we have identified a role for ubiquitination of GLUT4 in this process. We demonstrate that GLUT4 is ubiquitinated in 3T3-L1 adipocytes, and that a ubiquitin-resistant version fails to translocate to the cell surface of these cells in response to insulin. Our data support a model in which ubiquitination acts as a signal for the trafficking of GLUT4 from the endosomal/trans-Golgi network (TGN) system into its intracellular storage compartment, from where it is mobilized to the cell surface in response to insulin.

Snapin Interacts with the Exo70 Subunit of the Exocyst and Modulates GLUT4 Trafficking

The exocyst is a multisubunit complex that has been implicated in the transport of vesicles from the Golgi complex to the plasma membrane, possibly acting as a vesicle tether and contributing to the specificity of membrane fusion. Here we characterize a novel interaction between the Exo70 subunit of the exocyst and Snapin, a ubiquitous protein known to associate with at least two t-SNAREs, SNAP23 and SNAP25. The interaction between Exo70 and Snapin is mediated via an N-terminal coil-coil domain in Exo70 and a C-terminal helical region in Snapin. Exo70 competes with SNAP23 for Snapin binding, suggesting that Snapin does not provide a direct link between the exocyst and the SNARE complex but, rather, mediates cross-talk between the two complexes by sequential interactions. The insulin-regulated trafficking of GLUT4 to the plasma membrane serves to facilitate glucose uptake in adipocytes, and both SNAP23 and the exocyst have been implicated in this process. In this study, depletion of Snapin in adipocytes using RNA interference inhibits insulin-stimulated glucose uptake. Thus, Snapin interacts with the exocyst and plays a modulatory role in GLUT4 vesicle trafficking.

Golgin-160 Is Required for the Golgi Membrane Sorting of the Insulin-responsive Glucose Transporter GLUT4 in Adipocytes

The peripheral Golgi protein golgin-160 is induced during 3T3L1 adipogenesis and is primarily localized to the Golgi cisternae distinct from the trans-Golgi network (TGN) in a general distribution similar to p115. Small interfering RNA (siRNA)-mediated reduction in golgin-160 protein resulted in an increase accumulation of the insulin-responsive amino peptidase (IRAP) and the insulin-regulated glucose transporter (GLUT4) at the plasma membrane concomitant with enhanced glucose uptake in the basal state. The redistribution of GLUT4 was rescued by expression of a siRNA-resistant golgin-160 cDNA. The basal state accumulation of plasma membrane GLUT4 occurred due to an increased rate of exocytosis without any significant effect on the rate of endocytosis. This GLUT4 trafficking to the plasma membrane in the absence of golgin-160 was independent of TGN/Golgi sorting, because it was no longer inhibited by the expression of a dominant-interfering Golgi-localized, gamma-ear-containing ARF-binding protein mutant and displayed reduced binding to the lectin wheat germ agglutinin. Moreover, expression of the amino terminal head domain (amino acids 1-393) had no significant effect on the distribution or insulin-regulated trafficking of GLUT4 or IRAP. In contrast, expression of carboxyl alpha helical region (393-1498) inhibited insulin-stimulated GLUT4 and IRAP translocation, but it had no effect on the sorting of constitutive membrane trafficking proteins, the transferrin receptor, or vesicular stomatitis virus G protein. Together, these data demonstrate that golgin-160 plays an important role in directing insulin-regulated trafficking proteins toward the insulin-responsive compartment in adipocytes.

GLUT4 retention in adipocytes requires two intracellular insulin-regulated transport steps.

Insulin regulates glucose uptake into fat and muscle by modulating the distribution of the GLUT4 glucose transporter between the surface and interior of cells. The GLUT4 trafficking pathway overlaps with the general endocytic recycling pathway, but the degree and functional significance of the overlap are not known. In this study of intact adipocytes, we demonstrate, by using a compartment-specific fluorescence-quenching assay, that GLUT4 is equally distributed between two intracellular pools: the transferrin receptor-containing endosomes and a specialized compartment that excludes the transferrin receptor. These pools of GLUT4 are in dynamic communication with one another and with the cell surface. Insulin-induced redistribution of GLUT4 to the surface requires mobilization of both pools. These data establish a role for the general endosomal system in the specialized, insulin-regulated trafficking of GLUT4. Trafficking through the general endosomal system is regulated by rab11. Herein, we show that rab11 is required for the transport of GLUT4 from endosomes to the specialized compartment and for the insulin-induced translocation to the cell surface, emphasizing the importance of the general endosomal pathway in the specialized trafficking of GLUT4. Based on these findings we propose a two-step model for GLUT4 trafficking in which the general endosomal recycling compartment plays a specialized role in the insulin-regulated traffic of GLUT4. This compartment-based model provides the framework for understanding insulin-regulated trafficking at a molecular level.

Insulin-Regulated Trafficking of Dual-Labeled Glucose Transporter 4 in Primary Rat Adipose Cells

In isolated rat adipose cells, physiologically relevant insulin target cells, glucose transporter 4 (GLUT4) subcellular trafficking can be assessed by transfection of exofacially HA-tagged GLUT4. To simultaneously visualize the transfected GLUT4, we fused GFP with HA-GLUT4. With the resulting chimeras, GFP-HA-GLUT4 and HA-GLUT4-GFP, we were able to visualize for the first time the cell-surface localization, total expression, and intracellular distribution of GLUT4 in a single cell. Confocal microscopy reveals that the intracellular proportions of both GFP-HA-GLUT4 and HA-GLUT4-GFP are properly targeted to the insulin-responsive aminopeptidase-positive vesicles. Dynamic studies demonstrate close similarities in the trafficking kinetics between the two constructs and with native GLUT4. However, while the basal subcellular distribution of HA-GLUT4-GFP and the response to insulin are indistinguishable from those of HA-GLUT4 and endogenous GLUT4, most of the GFP-HA-GLUT4 is targeted to the plasma membrane with little further insulin response. Thus, HA-GLUT4-GFP will be useful to study GLUT4 trafficking in vivo while GFP on the N-terminus interferes with intracellular retention.

Insulin promotes rapid delivery of N-methyl-D- aspartate receptors to the cell surface by exocytosis.

Insulin potentiates N-methyl-d-aspartate receptors (NMDARs) in neurons and Xenopus oocytes expressing recombinant NMDARs. The present study shows that insulin induced (i) an increase in channel number times open probability (nP(o)) in outside-out patches excised from Xenopus oocytes, with no change in mean open time, unitary conductance, or reversal potential, indicating an increase in n and/or P(o); (ii) an increase in charge transfer during block of NMDA-elicited currents by the open channel blocker MK-801, indicating increased number of functional NMDARs in the cell membrane with no change in P(o); and (iii) increased NR1 surface expression, as indicated by Western blot analysis of surface proteins. Botulinum neurotoxin A greatly reduced insulin potentiation, indicating that insertion of new receptors occurs via SNARE-dependent exocytosis. Thus, insulin potentiation occurs via delivery of new channels to the plasma membrane. NMDARs assembled from mutant subunits lacking all known sites of tyrosine and serine/threonine phosphorylation in their carboxyl-terminal tails exhibited robust insulin potentiation, suggesting that insulin potentiation does not require direct phosphorylation of NMDAR subunits. Because insulin and insulin receptors are localized to glutamatergic synapses in the hippocampus, insulin-regulated trafficking of NMDARs may play a role in synaptic transmission and plasticity, including long-term potentiation.

Demonstration of insulin-responsive trafficking of GLUT4 and vpTR in fibroblasts.

Insulin-responsive trafficking of the GLUT4 glucose transporter and the insulin-regulated aminopeptidase (IRAP) in adipose and muscle cells is well established. Insulin regulation of GLUT4 trafficking in these cells underlies the role that adipose tissue and muscle play in the maintenance of whole body glucose homeostasis. GLUT4 is expressed in a very limited number of tissues, most highly in adipose and muscle, while IRAP is expressed in many tissues. IRAP's physiological role in any of the tissues in which it is expressed, however, is unknown. The fact that IRAP, which traffics by the same insulin-regulated pathway as GLUT4, is expressed in 'non-insulin responsive' tissues raises the question of whether these other cell types also have a specialized insulin-regulated trafficking pathway. The existence of an insulin-responsive pathway in other cell types would allow regulation of IRAP activity at the plasma membrane as a potentially important physiological function of insulin. To address this question we use reporter molecules for both GLUT4 and IRAP trafficking to measure insulin-stimulated translocation in undifferentiated cells by quantitative fluorescence microscopy. One reporter (vpTR), a chimera between the intracellular domain of IRAP and the extracellular and transmembrane domains of the transferrin receptor, has been previously characterized. The other is a GLUT4 construct with an exofacial HA epitope and a C-terminal GFP. By comparing these reporters to the transferrin receptor, a marker for general endocytic trafficking, we demonstrate the existence of a specialized, insulin-regulated trafficking pathway in two undifferentiated cell types, neither of which normally express GLUT4. The magnitude of translocation in these undifferentiated cells (approximately threefold) is similar to that reported for the translocation of GLUT4 in muscle cells. Thus, undifferentiated cells have the necessary retention and translocation machinery for an insulin response that is large enough to be physiologically important.

Vesicle-associated Membrane Protein 2 Plays a Specific Role in the Insulin-dependent Trafficking of the Facilitative Glucose Transporter GLUT4 in 3T3-L1 Adipocytes

Vesicle-associated membrane protein 2 (VAMP2) has been implicated in the insulin-regulated trafficking of GLUT4 in adipocytes. It has been proposed that VAMP2 co-localizes with GLUT4 in a postendocytic storage compartment (Martin, S., Tellam, J., Livingstone, C., Slot, J. W., Gould, G. W., and James, D. E. (1996) J. Cell Biol. 134, 625-635), suggesting that it may play a role distinct from endosomal v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) such as cellubrevin that are also expressed in adipocytes. The present study examines the effects of recombinant glutathione S-transferase (GST) fusion proteins encompassing the entire cytoplasmic tails of VAMP1, VAMP2, and cellubrevin on insulin-stimulated GLUT4 translocation in streptolysin O permeabilized 3T3-L1 adipocytes. GST-VAMP2 inhibited insulin-stimulated GLUT4 translocation by approximately 35%, whereas GST-VAMP1 and GST-cellubrevin were without effect. A synthetic peptide corresponding to the unique N terminus of VAMP2 also inhibited insulin-stimulated GLUT4 translocation in a dose-dependent manner. This peptide had no effect on either guanosine 5'-3-O-(thio)triphosphate-stimulated GLUT4 translocation or on insulin-stimulated GLUT1 translocation. These results imply that GLUT4 and GLUT1 may undergo insulin-stimulated translocation to the cell surface from separate intracellular compartments. To confirm this, adipocytes were incubated with a transferrin-horseradish peroxidase conjugate to fill the itinerant endocytic system after which cells were incubated with H2O2 and diaminobenzidine. This treatment completely blocked insulin-stimulated movement of GLUT1, whereas in the case of GLUT4, movement to the surface was delayed but still reached similar levels to that observed in insulin-stimulated control cells after 30 min. These results suggest that the N terminus of VAMP2 plays a unique role in the insulin-dependent recruitment of GLUT4 from its intracellular storage compartment to the cell surface.

Trafficking of glucose transporters--signals and mechanisms.

The uptake of glucose into mammalian cells, catalysed by members of the GLUT family of glucose transporters, is regulated by a variety of hormones, growth factors and other agents. In adipocytes, skeletal muscle and heart the principal regulator is the hormone insulin, which rapidly stimulates glucose uptake by bringing about the translocation of the GLUT4 glucose transporter isoform from an intracellular vesicular compartment to the cell surface. Recent studies have implicated the C-terminal hydrophilic region of this protein as being primarily responsible for its insulin-regulated trafficking. In an attempt to identify the protein machinery involved in this trafficking, we have used glutathione S-transferase fusion proteins bearing hydrophilic domains of various GLUT transporters in affinity purification experiments on detergent-solubilized extracts of 3T3-L1 adipocyte intracellular membranes. The C-terminal region of GLUT4 was found specifically to bind a number of polypeptides in these extracts, which are therefore candidates for components of the trafficking machinery. Although these proteins did not bind to the corresponding region of the more widely-distributed GLUT1 glucose transporter isoform, regulation of this transporter also appears to be of physiological importance in some cell types. To study such regulation we have used as a model system the interleukin-3 (IL-3)-dependent haemopoietic cell line IC.DP. These cells express a temperature sensitive mutane of the v-abl tyrosine kinase, whose activation at the permissive temperature permits cell survival in the absence of IL-3 by suppression of apoptosis, although the growth factor is still required for proliferation. Both IL-3 and activation of the kinase were found to stimulate glucose transport by promoting the translocation of GLUT1 to the cell surface. Moreover, inhibition of glucose uptake by addition of transport inhibitors markedly increased the rate of apoptosis, an effect which could be reversed by the provision of alternative energy sources. These observations suggest that the trafficking of GLUT1, regulated by growth factors or oncogenes, may play an important role in the suppression of apoptosis in haemopoietic cells.

Insulin resistance, diabetes, and the insulin-regulated trafficking of GLUT-4.

AB~T~S affects ~,5% of people in the United States of America and the incidence is much higher in sporadic populations such as Pima Indians in North America and Australian Aborigines. There are two major forms of diabetes, referred to as insulin-dependent diabetes (IDD) ~ and non-insulin-dependent diabetes (NIDD). The etiology of these diseases is quite different despite similarities in their pathophysiologies. IDD often arises in early life and is due to autoimmune destruction of pancreatic/3 cells resulting in partial or complete loss of/3 cell function. NIDD is more common in later life (>40 yr) and ,x,85 % of all diabetics have this form. The increased prevalence of NIDD in developed countries together with its association with heart disease and stroke makes this one of the most devastating diseases in the western world. NIDD appears to be due to a defect in glucose disposal (DeFronzo et al., 1992). Both glucose per se and insulin, play important roles in facilitating glucose uptake into target organs (for review see DeFronzo et ai., 1992). Following ingestion of a carbohydrate meal, glucose uptake into many organs is increased simply due to the elevated blood glucose. A more pronounced increase which is evoked by insulin, is observed specifically in muscle and adipose tissue. The net result is that in the postprandial state, glucose is rapidly and efficiently cleared from the blood and stored primarily in muscle and adipose. Each of these processes is defective in NIDD making it extremely difficult to identify the primary lesion. Studies in first degree relatives of NIDDs indicate that one of the earliest detectable defects is impaired insulin action in muscle and adipose tissue or insulin resistance (Martin et al., 1992). Thus, identifying the physiological and molecular biological processes that control insulin-stimulated glucose utilization in these organs is of major significance.