GLUT4 translocation by insulin in intact muscle cells: detection by a fast and quantitative assay

AbstractWe report a rapid and sensitive colorimetric approach to quantitate the amount of glucose transporters exposed at the surface of intact cells, using L6 muscle cells expressing GLUT4 containing an exofacial myc epitope. Unstimulated cells exposed to the surface 5 fmol GLUT4myc per mg protein. This value increased to 10 fmol/mg protein in response to insulin as 2-deoxyglucose (10 μM) uptake doubled. The results are substantiated by immunofluorescent detection of GLUT4myc in unpermeabilized cells and by subcellular fractionation. We further show that wortmannin and the cytoskeleton disruptors cytochalasin D and latrunculin B completely blocked these insulin effects. The rapid quantitative assay described here could be of high value to study insulin signals and to screen for potential anti-diabetic drugs

How booking platforms will influence patient journeys

How booking platforms will influence patient journey

Multiple mechanisms of regulating glucose transporters and glucose transport in skeletal muscle cells

grantor: University of TorontoGlucose is a universal energy substrate of mammalian cells. The Thesis examines the two main pathways of regulating glucose transport in muscle cells--insulin and the metabolic demand pathway. Insulin and the mitochondrial uncoupler dinitrophenol (DNP) were used to stimulate these two pathways in L6 muscle cells in order to dissect differences involving 3 major cellular regulatory pathways: (1) the actin cytoskeleton and temporal spatial regulation of signals and glucose transporters; (2) signalling pathways activated by these stimuli; and (3) cellular mechanisms controlling chronic glucose transporter expression. The results demonstrate that insulin provokes a rapid and marked aggregation of filamentous actin into structures that provide the coordinates for the insulin-derived signals to meet glucose transporter organelles, and direct their insertion into membrane ruffles. The results also reveal that, in contrast to insulin, DNP-stimulated glucose uptake largely depends on cytosolic Ca2+ and Ca2+-sensitive PKCs, but likely does not engage the fuel sensing enzyme AMPK. Finally, insulin and DNP were shown to elicit different long-term effects on glucose transporter expression: whereas prolonged exposure to insulin increases glucose transporter biosynthesis, chronic exposure to DNP increases the half-life of the glucose transporters in an isoform-specific manner. By uncovering the different cellular factors accessed by insulin and DNP to control glucose transport, the Thesis enhances our understanding of the diverse means employed by the muscle cell to control glucose homeostasis, and may have implications for the therapeutic treatment of diseases associated with impaired glucose utilization, such as Type 2 diabetes.Ph.D

Application of personalized medicine to chronic disease: a feasibility assessment

Abstract Personalized Medicine has the potential to improve health outcomes and reduce the cost of care; however its adoption has been slow in Canada. Bridgepoint Health is a complex continuous care provider striving to reduce the burden of polypharmacy in chronic patients. The main goal of the study was to explore the feasibility of utilizing personalized medicine in the treatment of chronic complex patients as a preliminary institutional health technology assessment. We analyzed stroke treatment optimization as a clinical indication that could serve as a “proof of concept” for the widespread implementation of pharmacogenetics. The objectives of the study were three-fold: 1. Review current practice in medication administration for stroke treatment at Bridgepoint Health 2. Critically analyze evidence that pharmacogenetic testing could (or could not) enhance drug selection and treatment efficacy for stroke patients; 3. Assess the cost-benefit potential of a pharmacogenetic intervention for stroke. Review current practice in medication administration for stroke treatment at Bridgepoint Health Critically analyze evidence that pharmacogenetic testing could (or could not) enhance drug selection and treatment efficacy for stroke patients; Assess the cost-benefit potential of a pharmacogenetic intervention for stroke. We conducted a review of stroke treatment practices at Bridgepoint Health, scanned the literature for drug-gene and drug-outcome interactions, and evaluated the potential consequences of pharmacogenetic testing using the ACCE model. There is a substantial body of evidence suggesting that pharmacogenetic stratification of stroke treatment can improve patient outcomes in the long-term, and provide substantial efficiencies for the healthcare system in the short-term. Specifically, pharmacogenetic stratification of antiplatelet and anticoagulant therapies for stroke patients may have a major impact on the risk of disease recurrence, and thus should be explored further for clinical application. Bridgepoint Health, and other healthcare institutions taking this path, should consider launching pilot projects to assess the practical impact of pharmacogenetics to optimize treatment for chronic continuous care

VAMP2, but Not VAMP3/Cellubrevin, Mediates Insulin-dependent Incorporation of GLUT4 into the Plasma Membrane of L6 Myoblasts

Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin