Ca²⁺- and GTP[γS]-induced translocation of the glucose transporter, GLUT-4, to the plasma membrane of permeabilized cardiomyocytes determined using a novel immunoprecipitation method

Abstract

In cardiomyocytes glucose transport is activated not only by insulin but also by contractile activity that causes translocation of the glucose transporter, GLUT-4, from intracellular vesicles to the plasma membrane. The latter effect may possibly be mediated by intracellular Ca²⁺, as suggested by previous studies. To investigate the role of Ca²⁺, we permeabilized neonatal rat myocytes with α-toxin and incubated them for 1 h either at a pCa (i.e.−log₁₀ [Ca²⁺]) of 8 (control) or at a pCa of 5 in the presence of adenosine 5′-triphosphate (ATP). Translocation of GLUT-4 was then monitored by a novel immunoprecipitation method using a peptide antibody directed against an exofacial (extracellular) loop of GLUT-4 (residues 58–80). Incorporation of GLUT-4 into the plasmalemma was stimulated 1.8-fold by 10 μM Ca²⁺ and 1.7-fold by insulin (as in the case of intact cells). The insulin effect was Ca²⁺ independent, i.e. it was identical in the absence and presence of Ca²⁺ (10 μM). Guanosine 5′-O-(3-thiotriphosphate) (GTP[γS]), which was inactive in intact cells, also caused translocation of GLUT-4 in permeabilized cardiomyocytes. Thus, incorporation of GLUT-4 into the plasma membrane was enhanced 2.5-fold by 200 μM GTP[γS] in the virtual absence of Ca²⁺ (pCa 8) and even 3.5-fold at 10 μM free Ca²⁺. We conclude that an increase in intracellular Ca²⁺ concentration increases GLUT-4 translocation of (permeabilized) cardiomyocytes to a similar extent as do insulin and GTP[γS] in the absence of Ca²⁺, but that the effects of Ca²⁺ and GTP[γS] may be additive.