Halothane increases cytosolic Ca2 and inhibits Na/H exchange in L6 muscle cells

ABSTRAC

Exercise modulates the insulin-induced translocation of glucose transporters in rat skeletal muscle

AbstractInsulin and acute exercise (45 min of treadmill run) increased glucose uptake into perfused rat hindlimbs 5-fold and 3.2-fold, respectively. Following exercise, insulin treatment resulted in a further increase in glucose uptake. The subcellular distribution of the muscle glucose transporters GLUT-1 and GLUT-4 was determined in plasma membranes and intracellular membranes. Neither exercise nor exercise→ insulin treatment altered the distribution of GLUT-1 transporters in these medmbrane fractions. In contrast, exercise, insulin and exercise→ insulin treatment caused comparable increases in GLUT-4 transporters in the plasma membrane. The results suggest that exercise might limit insulin-induced GLUT-4 recruitment and that following exercise, insulin may alter the intrinsic activity of plasma membrane glucose transporters

Phosphatidylinositol 4-kinase, but not phosphatidylinositol 3-kinase, is present in GLUT4-containing vesicles isolated from rat skeletal muscle

Insulin stimulates the rate of glucose transport into muscle and adipose cells by translocation of glucose transporter (GLUT4)-containing vesicles from an intracellular storage pool to the surface membrane. This event is mediated through the insulin receptor substrates (IRSs), which in turn activate phosphatidylinositol (PI) 3-kinase isoforms. It has been suggested that insulin causes attachment of PI 3-kinases to the intracellular GLUT4-containing vesicles in rat adipose cells. Furthermore, it has also been shown that GLUT4-containing vesicles in adipose cells contain a PI 4-kinase. In the present study we investigate whether GLUT4-containing vesicles isolated from rat skeletal muscle display PI 3-kinase and/or PI 4-kinase activities. Insulin stimulation caused a rapid increase (5-15-fold increase compared with control) in the intracellular cytosolic IRS-1-associated PI-3 kinase activity. This PI 3-kinase activity was also present in a membrane preparation containing the insulin-regulatable pool of GLUT4 transporters. However, when GLUT4-containing vesicles were isolated by immunoprecipitation from basal and insulin-stimulated (3 min) skeletal muscle, the vesicles displayed PI 4-kinase, but not PI 3-kinase, activity. Insulin did not regulate the PI 4-kinase activity in the GLUT4-containing vesicles. In conclusion, GLUT4-containing vesicles from rat skeletal muscle contain a PI 4-kinase, but not a PI 3-kinase. It is suggested that, in skeletal muscle, insulin causes activation of the IRS/PI 3-kinase complex in an intracellular membrane compartment associated closely with the GLUT4-containing vesicles, but not in the GLUT4-containing vesicles themselves

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