Investigations into insulin-regulated trafficking of the facilitative glucose transporter GLUT4 in adipocytes: novel insights from in situ studies

Trafficking of the facilitative glucose transporter GLUT4 is regulated by insulin in fat and muscle cells. Under basal conditions, GLUT4 is retained intracellularly by continually cycling through the endosomal system, but translocates to the plasma membrane in response to insulin stimulation. Intracellular GLUT4-containing vesicles fall into two categories: cellugyrin-positive (sortilin-free) and sortilin-positive (cellugyrin-negative). The former are the source of GLUT4 that cycles through the plasma membrane under basal conditions while the latter are the source of GLUT4 that translocates to the cell surface upon insulin stimulation. Fusion of GLUT4-containing vesicles with the plasma membrane is mediated by formation of SNARE complexes including the plasma membrane localized t-SNAREs Syntaxin 4 and SNAP23, and the v-SNARE VAMP2 present in the GLUT4-containing vesicles. The Sec1/Munc18 (SM) protein Munc18c also plays a key role in insulin-stimulated GLUT4 translocation, although its precise role remains controversial. Munc18c binds directly to both Syntaxin 4 and VAMP2 as well as to the assembled SNARE complex through a series of different binding modes. It has been suggested that SM/Syntaxin interactions facilitate SNARE complex formation by bringing about a conformational switch to release an inhibitory effect of syntaxins’ Habc domain. In this study I have used in situ Proximity Ligation Assay (PLA) to visualize the effects of insulin stimulation on interactions between Syntaxin 4, SNAP23, VAMP2 and Munc18c in 3T3-L1 adipocytes and fibroblasts. I find that insulin treatment results in an increase of the formation of assembled Syntaxin 4/SNAP23/VAMP2 SNARE complexes as well as recruitment of Munc18c to these complexes. These studies also reveal the existence of two pools of Syntaxin 4 under basal conditions: one in complex with SNAP23 (lacking VAMP2 and Munc18c); the other in complex with Munc18c and VAMP2 (lacking SNAP23). Additionally I have used in vitro binding studies to demonstrate that Syntaxin 4 binds directly to VAMP2 in a SNARE motif related manner and that this interaction is inhibitory to the rate of Syntaxin 4/SNAP23/VAMP2 SNARE complex assembly. Syntaxin 4 also binds directly to SNAP23, an interaction that enhances SNARE complex formation. Munc18c is phosphorylated on Tyr-521 in response to insulin-stimulation of 3T3-L1 adipocytes. I report here, that wild-type Munc18c inhibits SNARE complex formation, whereas a phosphomimetic version facilitates this process. Finally PLA studies reveal that the Syntaxin 4 pool in complex with VAMP2 and Munc18c associates with sortilin-positive vesicles, and that it is this pool which facilitates fusion of GLUT4 carrying vesicles upon insulin-stimulation. These studies also demonstrate the other Syntaxin 4 pool, that in complex with SNAP23, associates with cellugyrin-positive vesicles, and likely regulates the basal cycling of GLUT4 through the plasma membrane. I have used the data presented in this thesis to formulate a model whereby the two pools of Syntaxin 4 described are functionally distinct, and differ in their ability to mediate delivery of GLUT4 to the plasma membrane in response to insulin through the function of Munc18c

Alternate routes to the cell surface underpin insulin-regulated membrane trafficking of GLUT4

Insulin-stimulated delivery of glucose transporters (GLUT4) from specialized intracellular GLUT4 storage vesicles (GSVs) to the surface of fat and muscle cells is central to whole-body glucose. This translocation and subsequent internalization of GLUT4 back into intracellular stores transits numerous small membrane-bound compartments (internal GLUT4-containing vesicles; IGVs) including GSVs, but the function of these different compartments is not clear. Cellugyrin and sortilin define distinct populations of IGV; sortilin-positive IGVs represent GSVs, but the function of cellugyrin-containing IGVs is unknown. Here we demonstrate a role for cellugyrin in intracellular sequestration of GLUT4 in HeLa cells and have used a proximity ligation assay to follow changes in pairwise associations between cellugyrin, sortilin, GLUT4 and membrane trafficking machinery following insulin-stimulation of 3T3-L1 adipoctyes. Our data suggest that insulin stimulates traffic from cellugyrin- to sortilin- membranes, and that cellugyrin-IGVs provide an insulin-sensitive reservoir to replenish GSVs following insulin-stimulated exocytosis of GLUT4. Furthermore, our data support the existence of a pathway from cellugyrin-membranes to the surface of 3T3-L1 adipocytes that bypasses GSVs under basal conditions, and that insulin diverts traffic away from this into GSVs

Large scale, single-cell FRET-based glucose uptake measurements within heterogeneous populations

Fluorescent biosensors are powerful tools allowing the concentration of metabolites and small molecules, and other properties such as pH and molecular crowding to be measured inside live single cells. The technology has been hampered by lack of simple software to identify cells and quantify biosensor signals in single cells. We have developed a new software package, FRETzel, to address this gap and demonstrate its use by measuring insulin-stimulated glucose uptake in individual fat cells of varying sizes for the first time. Our results support the long-standing hypothesis that larger fat cells are less sensitive to insulin than smaller ones, a finding that has important implications for the battle against type 2 diabetes. FRETzel has been optimized using the messy and crowded environment of cultured adipocytes, demonstrating its utility for quantification of FRET biosensors in a wide range of other cell types, including fibroblasts and yeast via a simple user-friendly quantitative interface

Phosphorylation of Syntaxin 4 by the insulin receptor drives exocytic SNARE complex formation to deliver GLUT4 to the cell surface

A major consequence of insulin binding its receptor on fat and muscle cells is the stimulation of glucose transport into these tissues. This is achieved through an increase in the exocytic trafficking rate of the facilitative glucose transporter GLUT4 from intracellular stores to the cell surface. Delivery of GLUT4 to the cell surface requires the formation of functional SNARE complexes containing Syntaxin 4, SNAP23, and VAMP2. Insulin stimulates the formation of these complexes and concomitantly causes phosphorylation of Syntaxin 4. Here, we use a combination of biochemistry and cell biological approaches to provide a mechanistic link between these observations. We present data to support the hypothesis that Tyr-115 and Tyr-251 of Syntaxin 4 are direct substrates of activated insulin receptors, and that these residues modulate the protein’s conformation and thus regulate the rate at which Syntaxin 4 forms SNARE complexes that deliver GLUT4 to the cell surface. This report provides molecular details on how the cell regulates SNARE-mediated membrane traffic in response to an external stimulus

The deubiquitinating enzyme USP25 binds tankyrase and regulates trafficking of the facilitative glucose transporter GLUT4 in adipocytes

Key to whole body glucose homeostasis is the ability of fat and muscle cells to sequester the facilitative glucose transporter GLUT4 in an intracellular compartment from where it can be mobilized in response to insulin. We have previously demonstrated that this process requires ubiquitination of GLUT4 while numerous other studies have identified several molecules that are also required, including the insulin-responsive aminopeptidase IRAP and its binding partner, the scaffolding protein tankyrase. In addition to binding IRAP, Tankyrase has also been shown to bind the deubiquinating enzyme USP25. Here we demonstrate that USP25 and Tankyrase interact, and colocalise with GLUT4 in insulin-sensitive cells. Furthermore depletion of USP25 from adipocytes reduces cellular levels of GLUT4 and concomitantly blunts the ability of insulin to stimulate glucose transport. Collectively, these data support our model that sorting of GLUT4 into its insulin-sensitive store involves a cycle of ubiquitination and subsequent deubiquitinatio

Knockout of Syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion

Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport that is underpinned by the insulin-stimulated delivery of glucose transporter type 4 (SLC2A4, also known and hereafter referred to as GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, and increase cell surface GLUT4 levels. Adipocytokines, such as adiponectin, are secreted via a similar mechanism. We used genome editing to knock out syntaxin-4, a protein reported to mediate fusion between GLUT4-containing vesicles and the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of haemagglutinin (HA)-tagged GLUT4 conjugated to GFP showed that syntaxin-4-knockout cells retain significant GLUT4 translocation capacity, demonstrating that syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells

CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

Post-prandial blood glucose is cleared by Glucose Transporter 4 (GLUT4) released from an intracellular GLUT4 storage compartment (GSC) to the surface of muscle and adipose tissue in response to insulin. Here we map the biosynthetic pathway for human GSC formation, which involves the clathrin isoform CHC22. We observe that GLUT4 transits more slowly through the early secretory pathway than the constitutively-secreted GLUT1 transporter, and show CHC22 colocalizes with p115 in the endoplasmic-reticulum-to-Golgi-intermediate compartment (ERGIC). We find CHC22 functions in membrane traffic from the early secretory pathway during formation of the replication vacuole of Legionella pneumophila, which also acquires components of the GLUT4 pathway. We show that p115 but not GM130 is required for GSC formation, indicating GSC biogenesis from the ERGIC bypasses the Golgi. This GSC biogenesis pathway is attenuated in mice, which lack CHC22, and rely mainly on recapture of surface GLUT4 to populate their GSC. GLUT4 traffic to the GSC is enhanced by CHC22 function at the human ERGIC, which has implications for pathways to insulin resistance

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

Proximity ligation assay to study the GLUT4 membrane trafficking machinery

In this chapter a detailed protocol of proximity ligation assay (PLA) is described thoroughly. PLA is a technique that allows detection of protein associations in situ, providing a sensitive and selective approach for protein-protein interaction studies. We demonstrate the technique by applying it for trafficking studies of the facilitative glucose transporter GLUT4. Trafficking of GLUT4 from perinuclear depots to the plasma membrane is regulated by insulin in adipocytes and muscle cells, and mediated by formation of functional SNARE complexes containing Syntaxin4, SNAP23, and VAMP2. The Sec1/Munc18 (SM) protein Munc18c also plays a key role in insulin-stimulated GLUT4 translocation via a series of different interactions with the SNARE complex and/or with the SNARE proteins individually. Studying the interactions that occur between SNARE proteins themselves and also with Munc18c in insulin-responsive cells is critical to further understand SNARE protein function and GLUT4 trafficking mechanism in general

OTS1 and OTS2 SUMO proteases link plant development and survival under salt stress

High salinity is an important factor limiting agriculture as major crops are salt sensitive. Understanding salt stress signalling is key to producing salt tolerant crops. The small ubiquitin-like modifier (SUMO) is a crucial regulator of signalling proteins in eukaryotes. Attachment of SUMO onto substrates is reversible and SUMO-proteases which specifically cleave the SUMO-substrate linkages play a vital regulatory role during SUMOylation. We have identified two SUMO proteases OTS1 and OTS2 that act redundantly to regulate salt stress responses in Arabidopsis. ots1 ots2 double mutants show extreme sensitivity to salt. However during non-salt conditions, ots1 ots2 double mutants are phenotypically similar to wild-type plants in terms of growth and development. Overexpressing SUMO1 in the ots1 ots2 double mutants severally diminishes plant size as quantified by rosette diameter even under non-stressed conditions. This reduction in plant growth is reminiscent of ots1 ots2 double mutants under salt stress. Our data indicates that overSUMOylation of target proteins can have severe effects on plant growth and that SUMO proteases like OTS1/2 are key to maintaining cellular balance of SUMOylation. We propose that upon environmental stress the hyperSUMOylation of key target proteins act to retard growth to survive stress periods