Insulin-induced Recruitment of Glucose Transporter 4 (GLUT4) and GLUT1 in Isolated Rat Cardiac Myocytes

Abstract

Using isolated rat cardiomyocytes we have examined: 1) the effect of insulin on the cellular distribution of glucose transporter 4 (GLUT4) and GLUT1, 2) the total amount of these transporters, and 3) the co-localization of GLUT4, GLUT1, and secretory carrier membrane proteins (SCAMPs) in intracellular membranes. Insulin induced 5.7-and 2.7-fold increases in GLUT4 and GLUT1 at the cell surface, respectively, as determined by the nonpermeant photoaffinity label [ 3 H]2-N-[4(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-yloxy)propyl-2-amine. The total amount of GLUT1, as determined by quantitative Western blot analysis of cell homogenates, was found to represent a substantial fraction (ϳ30%) of the total glucose transporter content. Intracellular GLUT4-containing vesicles were immunoisolated from low density microsomes by using monoclonal anti-GLUT4 (1F8) or anti-SCAMP antibodies (3F8) coupled to either agarose or acrylamide. With these different immunoisolation conditions two GLUT4 membrane pools were found in nonstimulated cells: one pool with a high proportion of GLUT4 and a low content in GLUT1 and SCAMP 39 (pool 1) and a second GLUT4 pool with a high content of GLUT1 and SCAMP 39 (pool 2). The existence of pool 1 was confirmed by immunotitration of intracellular GLUT4 membranes with 1F8-acrylamide. Acute insulin treatment caused the depletion of GLUT4 in both pools and of GLUT1 and SCAMP 39 in pool 2. In conclusion: 1) GLUT4 is the major glucose transporter to be recruited to the surface of cardiomyocytes in response to insulin; 2) these cells express a high level of GLUT1; and 3) intracellular GLUT4-containing vesicles consist of at least two populations, which is compatible with recently proposed models of GLUT4 trafficking in adipocytes.

Immunogold labeling study of the distribution of GLUT-1 and GLUT-4 in cardiac tissue following stimulation by insulin or ischemia

“…To date, only the ratios of GLUT-1 and GLUT-4 have been calculated and reveal that ϳ30% of the total transporters in the myocyte are GLUT-1 (12). Whereas our studies do not enable us to confirm this ratio of the two isoforms, our data indicate the presence of a further GLUT-1 pool not previously considered in cardiac tissue.…”

“…Since we were unable to distinguish intracellular and membrane GLUT-1 in the capillary visually, we were unable to determine whether transendothelial GLUT-1 redistribution occurred in response to insulin or ischemia. We have previously assessed by Western blotting that acute ischemia does not cause GLUT-1 redistribution in isolated rat hearts (31), although GLUT-1 translocation has been reported by other groups following insulin stimulation (12), ischemia (9,40), and hypoxia (7).…”

“…Insulin-stimulated GLUT-4 translocation has been widely investigated in cardiac tissue by using immunofluorescence and Western blotting (8,12,19,27). Although these studies quantify GLUT-4 translocation following insulin stimulation, they do not differentiate between the different plasma membrane types within the myocyte.…”

Anti-hypoxic effect of ginsenoside Rbl on neonatal rat cardiomyocytes is mediated through the specific activation of glucose transporter-4 ex vivo

“…In the physiological state of the cells, GLUT-4 is found on cell membranes and in the intracellular compartment; once the cell encounters stress, such as hypoxia [19] , ischemia [20] , etc. [21][22][23] , GLUT-4 can be rapidly translocated to the cell surface, allowing the cell to transiently increase its access to extracellular glucose [24][25] . Cardiac glucose uptake and glycolysis are protective in the ischemic heart [26] , and glucose transporter deficiency decreases ischemic tolerance [27] .…”

AMP-activated protein kinase control of energy metabolism in the ischemic heart

“…Although GLUT1 primarily maintains basal glucose uptake, GLUT4 can be mobilized from intracellular compartments to the sarcolemmal membrane in response to insulin and/or contractile stimulation, thus increasing glucose uptake (although GLUT1 has also been shown to translocate to the plasma membrane upon insulin stimulation). 18 Once glucose is within the cell, it has a number of fates, including being stored as glycogen or undergoing glycolytic metabolism. An important enzyme at the interface between carbohydrate oxidation and fatty acid metabolism is pyruvate dehydrogenase (PDH), which decarboxylates pyruvate to acetyl CoA.…”