Targeting motifs in GLUT4

The trafficking of the insulin-sensitive glucose transporter, GLUT4, is the paradigm of how cells control the movement of membrane proteins through intricate pathways of transport in response to external stimuli, and how, by doing so, regulate their function. The GLUT4 intracellularly sequestered in resting adipocytes and muscle cells becomes exposed on their surface in response to an increase in insulin levels and muscle contraction, where it facilitates glucose uptake. Ceasing of the stimuli is followed by endocytosis of the GLUT4 molecules exposed on the plasma membrane and their recycling to the original stores, where they are retained. This review discusses current understanding of the organelles that host GLUT4 and the motifs that mediate its trafficking.     

Insulin action on glucose transporters through molecular switches, tracks and tethers

Glucose entry into muscle cells is precisely regulated by insulin, through recruitment of GLUT4 (glucose transporter-4) to the membrane of muscle and fat cells. Work done over more than two decades has contributed to mapping the insulin signalling and GLUT4 vesicle trafficking events underpinning this response. In spite of this intensive scientific research, there are outstanding questions that continue to challenge us today. The present review summarizes the knowledge in the field, with emphasis on the latest breakthroughs in insulin signalling at the level of AS160 (Akt substrate of 160 kDa), TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) and their target Rab proteins; in vesicle trafficking at the level of vesicle mobilization, tethering, docking and fusion with the membrane; and in the participation of the cytoskeleton to achieve optimal temporal and spatial location of insulin-derived signals and GLUT4 vesicles.     

Insulin stimulation of GLUT4 exocytosis, but not its inhibition of endocytosis, is dependent on RabGAP AS160

Insulin maintains whole body blood glucose homeostasis, in part, by regulating the amount of the GLUT4 glucose transporter on the cell surface of fat and muscle cells. Insulin induces the redistribution of GLUT4 from intracellular compartments to the plasma membrane, by stimulating a large increase in exocytosis and a smaller inhibition of endocytosis. A considerable amount is known about the molecular events of insulin signaling and the complex itinerary of GLUT4 trafficking, but less is known about how insulin signaling is transmitted to GLUT4 trafficking. Here, we show that the AS160 RabGAP, a substrate of Akt, is required for insulin stimulation of GLUT4 exocytosis. A dominant-inhibitory mutant of AS160 blocks insulin stimulation of exocytosis at a step before the fusion of GLUT4-containing vesicles with the plasma membrane. This mutant, however, does not block insulin-induced inhibition of GLUT4 endocytosis. These data support a model in which insulin signaling to the exocytosis machinery (AS160 dependent) is distinct from its signaling to the internalization machinery (AS160 independent).     

GARNL1, a major RalGAP α subunit in skeletal muscle,regulates insulin-stimulated RalA activation and GLUT4 trafficking via interaction with 14-3-3 proteins

Insulin and muscle contraction each stimulate translocation of the glucose transporter GLUT4 to the plasma membrane in skeletal muscle, an important process regulating whole-body glucose homeostasis. RalA mediates insulin-stimulated GLUT4 translocation; however, it is unclear how this small GTPase is regulated in skeletal muscle in response to insulin. Here, we identified GARNL1/RalGAPα1, a major α subunit of the Ral-GTPase activating protein in skeletal muscle, as a protein whose phosphorylation and binding to the regulatory 14-3-3 proteins is stimulated by insulin and also by muscle contraction. The insulin-stimulated interaction with 14-3-3 involved PKB/Akt-mediated phosphorylation of Thr⁷³⁵ on GARNL1/RalGAPα1. Knockdown of GARNL1/RalGAPα1 increased, while overexpression of GARNL1/RalGAPα1^Thr735Ala mutant protein decreased, the RalA activation and the RalA-dependent GLUT4 translocation in response to insulin in muscle cells. These findings show that GARNL1/RalGAPα1 is the missing link that connects the insulin-PKB/Akt signaling pathway with the activation of the RalA small GTPase in muscle cells. GARNL1/RalGAPα1 and its phosphorylation and/or binding to 14-3-3s are critical for GLUT4 trafficking through RalA in muscle cells.     

How many signals impinge on GLUT4 activation by insulin?

GLUT4 is the main glucose transporter activated by insulin in skeletal muscle cells and adipocytes. GLUT4 storage vesicles (GSVs) traffic in endocytic and exocytic compartments. In the basal state, GLUT4 compartments are preferentially sequestered in perinuclear deposits wherein stimuli including insulin and non-insulin factors can increase GLUT4 vesicle formation, its exocytosis, and fusion to plasma membrane. In addition to well-established effectors of insulin signaling pathway, such as PKCzeta and Akt, the cytoskeletal network is implicated in GLUT4 translocation. This review will discuss the mechanisms and activation of GLUT4 trafficking and incorporating to PM from three aspects: known molecules of the insulin signaling pathway; Rho and Rab family proteins and cytoskeletal molecules.     

Exploring the whereabouts of GLUT4 in skeletal muscle (review)

The glucose transporter GLUT4 is expressed in muscle, fat cells, brain and kidney. In contrast to other glucose transporters, GLUT4 in unstimulated cells is mostly intracellular. Stimuli such as insulin and muscle contractions then cause the translocation of GLUT4 to the cell surface. Questions related to GLUT4 storage compartments, trafficking to the surface membrane, and nature of the intracellular pools, have kept many groups busy for the past 20 years. Yet, one of the main questions in the field remains the universality of GLUT4 features. Can one extrapolate work done on fat cells to muscle or brain? Or vice-versa? Can one use cultures to predict GLUT4 behaviour in fully differentiated tissues? This review summarizes the authors' knowledge of GLUT4 biology in skeletal muscle, which is the predominant tissue for glucose homeostasis. The results are compared to those obtained with the fat cell system, and an attempt is made to assess the universality principle.     

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.     

Book reviews

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.     

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

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.     

Effect of plasma membrane cholesterol depletion on glucose transport regulation in leukemia cells

GLUT1 is the predominant glucose transporter in leukemia cells, and the modulation of glucose transport activity by cytokines, oncogenes or metabolic stresses is essential for their survival and proliferation. However, the molecular mechanisms allowing to control GLUT1 trafficking and degradation are still under debate. In this study we investigated whether plasma membrane cholesterol depletion plays a role in glucose transport activity in M07e cells, a human megakaryocytic leukemia line. To this purpose, the effect of cholesterol depletion by methyl-β-cyclodextrin (MBCD) on both GLUT1 activity and trafficking was compared to that of the cytokine Stem Cell Factor (SCF). Results show that, like SCF, MBCD led to an increased glucose transport rate and caused a subcellular redistribution of GLUT1, recruiting intracellular transporter molecules to the plasma membrane. Due to the role of caveolae/lipid rafts in GLUT1 stimulation in response to many stimuli, we have also investigated the GLUT1 distribution along the fractions obtained after non ionic detergent treatment and density gradient centrifugation, which was only slightly changed upon MBCD treatment. The data suggest that MBCD exerts its action via a cholesterol-dependent mechanism that ultimately results in augmented GLUT1 translocation. Moreover, cholesterol depletion triggers GLUT1 translocation without the involvement of c-kit signalling pathway, in fact MBCD effect does not involve Akt and PLCγ phosphorylation. These data, together with the observation that the combined MBCD/SCF cell treatment caused an additive effect on glucose uptake, suggest that the action of SCF and MBCD may proceed through two distinct mechanisms, the former following a signalling pathway, and the latter possibly involving a novel cholesterol dependent mechanism.     

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.     

Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking

Glucose transporter 4 (GLUT4) is the major insulin-regulated glucose transporter expressed mainly in muscle and adipose tissue. GLUT4 is stored in a poorly characterized intracellular vesicular compartment and translocates to the cell surface in response to insulin stimulation resulting in an increased glucose uptake. This process is essential for the maintenance of normal glucose homeostasis and involves a complex interplay of trafficking events and intracellular signaling cascades. Recent studies have identified sortilin as an essential element for the formation of GLUT4 storage vesicles during adipogenesis and Golgi-localized gamma-ear-containing Arf-binding protein (GGA) as a key coat adaptor for the entry of newly synthesized GLUT4 into the specialized compartment. Insulin-stimulated GLUT4 translocation from this compartment to the plasma membrane appears to require the Akt/protein kinase B substrate termed AS160 (Akt substrate of 160kDa). In addition, the VPS9 domain-containing protein Gapex-5 in complex with CIP4 appears to function as a Rab31 guanylnucleotide exchange factor that is necessary for insulin-stimulated GLUT4 translocation. Here, we attempt to summarize recent advances in GLUT4 vesicle biogenesis, intracellular trafficking and membrane fusion.     

Insulin Stimulates Syntaxin4 SNARE Complex Assembly via a Novel Regulatory Mechanism

Insulin stimulates glucose transport into fat and muscle cells by increasing the exocytic trafficking rate of the GLUT4 facilitative glucose transporter from intracellular stores to the plasma membrane. Delivery of GLUT4 to the plasma membrane is mediated by formation of functional SNARE complexes containing syntaxin4, SNAP23, and VAMP2. Here we have used an in situ proximity ligation assay to integrate these two observations by demonstrating for the first time that insulin stimulation causes an increase in syntaxin4-containing SNARE complex formation in adipocytes. Furthermore, we demonstrate that insulin brings about this increase in SNARE complex formation by mobilizing a pool of syntaxin4 held in an inactive state under basal conditions. Finally, we have identified phosphorylation of the regulatory protein Munc18c, a direct target of the insulin receptor, as a molecular switch to coordinate this process. Hence, this report provides molecular detail of how the cell alters membrane traffic in response to an external stimulus, in this case, insulin.     

Insulin stimulates the entry of GLUT4 into the endosomal recycling pathway by a quantal mechanism

The insulin-sensitive glucose transporter GLUT4 mediates the uptake of glucose into adipocytes and muscle cells. In this study we have used a novel 96-well plate fluorescence assay to study the kinetics of GLUT4 trafficking in 3T3-L1 adipocytes. We have found evidence for a graded release mechanism whereby GLUT4 is released into the plasma membrane recycling system in a nonkinetic manner as follows: the kinetics of appearance of GLUT4 at the plasma membrane is independent of the insulin concentration; a large proportion of GLUT4 molecules do not participate in plasma membrane recycling in the absence of insulin; and with increasing insulin there is an incremental increase in the total number of GLUT4 molecules participating in the recycling pathway rather than simply an increased rate of recycling. We propose a model whereby GLUT4 is stored in a compartment that is disengaged from the plasma membrane recycling system in the basal state. In response to insulin, GLUT4 is quantally released from this compartment in a pulsatile manner, leaving some sequestered from the recycling pathway even in conditions of excess insulin. Once disengaged from this location we suggest that in the continuous presence of insulin this quanta of GLUT4 continuously recycles to the plasma membrane, possibly via non-endosomal carriers that are formed at the perinuclear region.     

脂肪细胞中Munc18c的缺失不影响胰岛素诱导的GLUT4转运(已撤稿2016-01-13)

动物脂肪和肌肉组织中葡萄糖的摄取是通过受胰岛素调控的GLUT4储存囊泡的运输实现的.Sec1p的同源物Munc18c被认为是通过控制SNARE复合物的装配来使GLUT4囊泡锚定到质膜上的重要物质.我们发现Munc18c的缺失没有影响GLUT4的转运上膜,也没有影响Syntaxin4在细胞膜上的定位.在缺少Munc18c和功能性Syntaxin2的时候,GLUT4的转运可能和Munc18b有关.在3T3-L1脂肪细胞中与Syntaxin4具有强烈相互作用的是Munc18c而不是Munc18a和Munc18b.然而,当缺少Munc18c时,Munc18a和Munc18b与Syntaxin4体现出较弱的相互作用.因此,Syntaxin4可能在胰岛素刺激GLUT4转运过程中起到重要的作用,且与SM蛋白的相互作用是有代偿性的.     

Bridging the GAP between insulin signaling and GLUT4 translocation

Upon binding and activating its cell-surface receptor, insulin triggers signaling cascades that regulate many cellular processes. Regarding glucose homeostasis, insulin suppresses hepatic glucose production and increases glucose transport into muscle and adipose tissues. At the cellular level, glucose uptake results from the insulin-stimulated translocation of the glucose transporter 4 (GLUT4) from intracellular storage sites to the plasma membrane. Although the signaling molecules that function proximal to the activated insulin receptor have been well characterized, it is not known how the distal insulin-signaling cascade interfaces with and mobilizes GLUT4-containing compartments. Recently, several candidate signaling molecules, including AS160, PIKfyve and synip, have been identified that might provide functional links between the insulin signaling cascade and GLUT4 compartments. Future work will focus on delineating the precise GLUT4 trafficking steps regulated by these molecules.     

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body¹. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes^2,3.The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.     

Insulin stimulated GLUT4 translocation – Size is not everything!

Insulin-regulated trafficking of the facilitative glucose transporter GLUT4 has been studied in many cell types. The translocation of GLUT4 from intracellular membranes to the cell surface is often described as a highly specialised form of membrane traffic restricted to certain cell types such as fat and muscle, which are the major storage depots for insulin-stimulated glucose uptake. Here, we discuss evidence that favours the argument that rather than being restricted to specialised cell types, the machinery through which insulin regulates GLUT4 traffic is present in all cell types. This is an important point as it provides confidence in the use of experimentally tractable model systems to interrogate the trafficking itinerary of GLUT4.     

Endothelin-1 impairs glucose transporter trafficking via a membrane-based mechanism

Endothelin-1 (ET-1) disrupts insulin-regulated glucose transporter GLUT4 trafficking. Since the negative consequence of chronic ET-1 exposure appears to be independent of signal disturbance along the insulin receptor substrate-1/phosphatidylinositol (PI) 3-kinase (PI3K)/Akt-2 pathway of insulin action, we tested if ET-1 altered GLUT4 regulation engaged by osmotic shock, a PI3K-independent stimulus that mimics insulin action. Regulation of GLUT4 by hyperosmotic stress was impaired by ET-1. Because of the mutual disruption of both insulin- and hyperosmolarity-stimulated GLUT4 translocation, we tested whether shared signaling and/or key phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated cytoskeletal events of GLUT4 trafficking were targets of ET-1. Both insulin and hyperosmotic stress signaling to Cbl were impaired by ET-1. Also, plasma membrane PIP2 and cortical actin levels were reduced in cells exposed to ET-1. Exogenous PIP2, but not PI 3,4,5-bisphosphate, restored actin structure, Cbl activation, and GLUT4 translocation. These data show that ET-1-induced PIP2/actin disruption impairs GLUT4 trafficking elicited by insulin and hyperosmolarity. In addition to showing for the first time the important role of PIP2-regulated cytoskeletal events in GLUT4 regulation by stimuli other than insulin, these studies reveal a novel function of PIP2/actin structure in signal transduction.     

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.