GLUT-4myc ectopic expression in L6 myoblasts generates a GLUT-4-specific pool conferring insulin sensitivity

Abstract

Insulin stimulates glucose uptake into muscle and fat cells via recruitment of the glucose transporter 4 (GLUT-4) from intracellular store(s) to the cell surface. Robust stimulation of glucose uptake by insulin coincides with the expression of GLUT-4 during differentiation of muscle and fat cells, but it is not known if GLUT-4 expression suffices to confer insulin sensitivity to glucose uptake. We have therefore examined the effect of expression of a myc epitope-tagged GLUT-4 (GLUT-4myc) into L6 myoblasts, which do not express endogenous GLUT-4 until differentiated into myotubes. Ectopic expression of GLUT-4myc markedly improved insulin sensitivity of glucose uptake in L6 myoblasts. The GLUT-4myc protein distributed equally to the cell surface and intracellular compartments in myoblasts, and the intracellular fraction of GLUT-4myc further increased in myotubes. In myoblasts, the intracellular GLUT-4myc compartment contained the majority of the insulin-regulatable amino peptidase (IRAP) but less than half of the GLUT-1, suggesting segregation of GLUT-4myc and IRAP to a specific cellular locus. Insulin stimulation of phosphatidylinositol 3-kinase and protein kinase B-α activities was similar for L6-GLUT-4myc myoblasts and myotubes. At both stages, GLUT-4myc responded to insulin by translocating to the cell surface. These results suggest that GLUT-4myc segregates into a specific compartment in L6 myoblasts and confers insulin sensitivity to these cells. L6-GLUT-4myc myoblasts, which are easily transfectable with various constructs, are a useful resource to study insulin action.

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

“…To this end, we used L6 myoblasts that stably express an exofacially myctagged GLUT4 protein, allowing for the measurement of cell surface GLUT4myc incorporation in single cells. Previous work from our laboratory had shown that GLUT4myc is recruited to the myoblast surface in response to insulin (Ueyama et al, 1999). The extent and characteristics of this translocation are virtually identical to those of the endogenous GLUT4 that is expressed only in mature L6 myotubes (Ueyama et al, 1999).…”

“…Previous work from our laboratory had shown that GLUT4myc is recruited to the myoblast surface in response to insulin (Ueyama et al, 1999). The extent and characteristics of this translocation are virtually identical to those of the endogenous GLUT4 that is expressed only in mature L6 myotubes (Ueyama et al, 1999). In both parental L6 myotubes and L6-GLUT4myc myoblasts, GLUT4 is concentrated in an intracellular membrane compartment that includes all of the IRAP and largely excludes GLUT1 (Ueyama et al, 1999).…”

“…The extent and characteristics of this translocation are virtually identical to those of the endogenous GLUT4 that is expressed only in mature L6 myotubes (Ueyama et al, 1999). In both parental L6 myotubes and L6-GLUT4myc myoblasts, GLUT4 is concentrated in an intracellular membrane compartment that includes all of the IRAP and largely excludes GLUT1 (Ueyama et al, 1999). Given the amenability of myoblasts to transient transfection by various cDNA constructs, this experimental model is highly suitable to explore the translocation of GLUT4 vesi- V.K.…”

“…In L6-GLUT4myc myoblasts, the GLUT4myc protein segregates intracellularly along with the insulin-regulated aminopeptidase (IRAP) and away from GLUT1 into an insulinsensitive compartment (Ueyama et al, 1999). In response to insulin, GLUT4myc is readily recruited to the plasma membrane, resulting in a twofold gain in surface-exposed GLUT4myc (Ueyama et al, 1999;Wang et al, 1999).…”

“…In response to insulin, GLUT4myc is readily recruited to the plasma membrane, resulting in a twofold gain in surface-exposed GLUT4myc (Ueyama et al, 1999;Wang et al, 1999). The insulin effect is illustrated by immunofluorescence detection of the myc epitope in intact L6-GLUT4myc myoblasts (Figure 7A).…”