Abstract 17501: Insulin-Mediated GLUT4 Translocation Requires Intracellular and Extracellular Calcium and is Impaired by Short-Term High Fat Feeding
The analysis of mechanisms that govern GLUT4 translocation in cardiomyocytes has been limited by lack of suitable reagents with which to quantify GLUT4 vesicle translocation and insertion into sarcolemma and T-Tubules. We therefore generated transgenic mice with doxycycline-inducible expression of a myc-epitope tagged GLUT4, and used this model to evaluate the mechanisms responsible for impaired glucose uptake following 2-weeks of high-fat feeding (HFD) and the contribution of intracellular and extracellular calcium to insulin-mediated GLUT4 translocation. Using immunofluorescence in non-permeabilized cardiomyocytes, insulin stimulated a 2.5 fold increase in surface-exposed GLUT4myc in sarcolemma, and a similar fold translocation to T-Tubules as determined by GLUT4myc and Ca2+V L-type channel co-localization (p<0.05). 2-weeks of HFD impairs basal and insulin-mediated glucose uptake despite normal activation of Akt. This corresponds with a 40% reduction in GLUT4myc exposure under basal conditions a 50% reduction in sarcolemmal GLUT4myc and an even greater reduction in T-tubule GLUT4 translocation following insulin treatment. In cardiomyocytes isolated from normal chow-fed animals, removing extracellular calcium reduced cell surface GLUT4myc labeling under basal conditions and reduced the fold-increase in cell-surface labeled GLUT4myc following insulin stimulation by 50%. We next analyzed the role of intracellular calcium on GLUT4myc insertion by treating cells with 1μM ionomycin (IO) or 100μM thapsigargin (TH), which increase intracellular Ca2+ by increasing uptake or releasing intracellular stores respectively. Both agents increased sarcolemmal GLUT4myc by 50% respectively. Following TH treatment, insulin further increased GLUT4 translocation but not to the same extent as insulin alone. In IO treated cells, insulin did not increase GLUT4myc further in the presence or absence of extracellular Ca2+. Thus short-term high-fat feeding disrupts glucose uptake, not by limiting insulin signaling but by reducing plasma membrane GLUT4 insertion. Moreover, we identify novel roles for insulin-mediated influx of extracellular Ca2+and release of intracellular Ca2+ in insulin-mediated GLUT4 translocation.
- © 2010 by American Heart Association, Inc.