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.

Diabetes is accompanied by changes in the levels of proteins involved in endosomal GLUT4 trafficking in obese human skeletal muscle.

INTRODUCTION: The regulated delivery of the glucose transporter GLUT4 from intracellular stores to the plasma membrane underpins insulin-stimulated glucose transport. Insulin-stimulated glucose transport is impaired in skeletal muscle of patients with type-2 diabetes, and this may arise because of impaired intracellular trafficking of GLUT4. However, molecular details of any such impairment have not been described. We hypothesized that GLUT4 and/or levels of proteins involved in intracellular GLUT4 trafficking may be impaired in skeletal muscle in type-2 diabetes and tested this in obese individuals without and without type-2 diabetes. METHODS: We recruited 12 participants with type-2 diabetes and 12 control participants. All were overweight or obese with BMI of 25-45 kg/m2 . Insulin sensitivity was measured using an insulin suppression test (IST), and vastus lateralis biopsies were taken in the fasted state. Cell extracts were immunoblotted to quantify levels of a range of proteins known to be involved in intracellular GLUT4 trafficking. RESULTS: Obese participants with type-2 diabetes exhibited elevated fasting blood glucose and increased steady state glucose infusion rates in the IST compared with controls. Consistent with this, skeletal muscle from those with type-2 diabetes expressed lower levels of GLUT4 (30%, p = .014). Levels of Syntaxin4, a key protein involved in GLUT4 vesicle fusion with the plasma membrane, were similar between groups. By contrast, we observed reductions in levels of Syntaxin16 (33.7%, p = 0.05), Sortilin (44%, p = .006) and Sorting Nexin-1 (21.5%, p = .039) and -27 (60%, p = .001), key proteins involved in the intracellular sorting of GLUT4, in participants with type-2 diabetes. CONCLUSIONS: We report significant reductions of proteins involved in the endosomal trafficking of GLUT4 in skeletal muscle in obese people with type 2 diabetes compared with age- and weight-matched controls. These abnormalities of intracellular GLUT4 trafficking may contribute to reduced whole body insulin sensitivity.

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. This article has an associated First Person interview with Hannah L. Black and Rachel Livingstone, joint first authors of the paper.

Cardiovascular benefits of GLP-1 agonists in type 2 diabetes: a comparative review.

Type 2 diabetes (T2D) carries risks of both cardiovascular (CV) (myocardial infarction, stroke, and peripheral vascular disease) and microvascular (retinopathy/nephropathy/neuropathy) complications. Glucose-lowering is an effective strategy for preventing microvascular complications, but the extent to which it can reduce CV complications is less certain. Glucagon-like peptide-1 (GLP-1) agonists are potent glucose-lowering agents but also have potentially beneficial effects on other traditional (body weight, blood pressure (BP), and LDL cholesterol) and non-traditional risk factors (low grade inflammation and endothelial dysfunction). The results of four large CV outcome trials with GLP-1 agonists are now available. These have compared lixisenatide (ELIXA), liraglutide (LEADER), semaglutide (SUSTAIN-6), and long-acting exenatide (EXSCEL) with placebo and standard of care over 2-4 years; four others (including with dulaglutide and albiglutide) are ongoing. LEADER and SUSTAIN-6 have demonstrated reductions in rates of major adverse CV events with active GLP-1 treatment but ELIXA and EXSCEL have not. In this review, we discuss the mechanisms by which GLP-1 receptor agonists act on the CV system and the design and conduct of these trials. Contrary to the assertions that (a) all GLP-1 agonists reduce CV disease in T2D but to different extents or (b) the magnitude of CV protection is predominantly related to glucose-lowering, we argue that CV benefit is specific to agents that provide longer acting agonism at the GLP-1 receptor. The mechanisms involve reduction in body weight and BP, and lowering of LDL-cholesterol and glucose, but pleiotropic effects-including suppression of low grade inflammation, vasodilation, and natriuresis-are also likely relevant.

SNARE phosphorylation: a control mechanism for insulin-stimulated glucose transport and other regulated exocytic events.

Trafficking within eukaryotic cells is a complex and highly regulated process; events such as recycling of plasma membrane receptors, formation of multivesicular bodies, regulated release of hormones and delivery of proteins to membranes all require directionality and specificity. The underpinning processes, including cargo selection, membrane fusion, trafficking flow and timing, are controlled by a variety of molecular mechanisms and engage multiple families of lipids and proteins. Here, we will focus on control of trafficking processes via the action of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of proteins, in particular their regulation by phosphorylation. We will describe how these proteins are controlled in a range of regulated trafficking events, with particular emphasis on the insulin-stimulated delivery of glucose transporters to the surface of adipose and muscle cells. Here, we focus on a few examples of SNARE phosphorylation which exemplify distinct ways in which SNARE machinery phosphorylation may regulate membrane fusion.

A new perspective on metformin therapy in type 1 diabetes.

Metformin is quite frequently used off-label in type 1 diabetes to limit insulin dose requirement. Guidelines recommend that it can improve glucose control in those who are overweight and obese but evidence in support of this is limited. Recently-published findings from the REducing with MetfOrmin Vascular Adverse Lesions (REMOVAL) trial suggest that metformin therapy in type 1 diabetes can reduce atherosclerosis progression, weight and LDL-cholesterol levels. This provides a new perspective on metformin therapy in type 1 diabetes and suggests a potential role for reducing the long-term risk of cardiovascular disease.

Improving the Quality of Assessment and Management of Hypoglycaemia in Hospitalised Patients with Diabetes Mellitus by Introducing 'Hypo Boxes' to General Medical Wards with a Specialist Interest in Diabetes.

Diabetes is becoming more prevalent in the UK and this is represented in the in-patient cohort, such that 1 in 6 hospital patients have diabetes (1). The UK National Diabetes In-Patient Audit in 2012 estimated that 30% of patients experience one episode of hypoglycaemia during admission. Hypoglycaemia is associated with increased morbidity and mortality, and longer length of hospital stay. It is therefore important that hypoglycaemia is managed promptly and effectively to reduce associated morbidity. The Joint British Diabetes Society recommends that all wards should have access to 'Hypo Boxes' (2). We assessed all episodes of hypoglycaemia (<4.0 mmol/l) in the diabetes wards in over a 4 week period. 'Hypo Boxes' were installed to the wards and the appropriateness of treatment and time to correction of hypoglycaemia was re-assessed. Assessment of hypoglycaemia pre-intervention revealed 45 episodes of hypoglycaemia in 14 patients, and 42% (n=19) of episodes were deemed to have been treated appropriately. Only 17.8% of episodes were corrected within 30 minutes, and 33.3% were corrected within 60 minutes. A third of patients (35%) did not have a further blood glucose checked. Following intervention, there was a marked improvement in management. The proportion of appropriately managed episodes increased to 82% (n=35) and management of episodes of severe hypoglycaemia (<3.0 mmol/l) increased to 94%. The time to correction increased with 40% of episodes corrected to >4.0 mmol/l within 30 minutes, and a further 54% between 30-60 minutes. In conclusion, the introduction of 'Hypo Boxes' improved the assessment and management of hypoglycaemia.