Eicosanoids participate in the regulation of cardiac glucose transport by contribution to a rearrangement of actin cytoskeletal elements.

Intact actin microfilaments are required for insulin-regulated glucose transporter isoform 4 (GLUT4) translocation to the plasma membrane. Lipoxygenase (LO) metabolites have recently been shown to contribute to the regulation of actin cytoskeleton rearrangement. In the present investigation, ventricular cardiomyocytes were used to study the effects of two structurally different LO inhibitors (esculetin and nordihydroguaiaretic acid) on insulin signalling events, glucose uptake, GLUT4 translocation and the actin network organization. Insulin stimulation increased glucose uptake 3-fold in control cells, whereas LO inhibition completely blocked this effect. This was paralleled by a slight reduction in the insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2. However, inhibition of 12-LO did not affect the association of phosphatidylinositol 3-kinase with IRS-1 and the phosphorylation of Akt/protein kinase B in response to insulin. Addition of 12(S)-hydroxyeicosatetraenoic acid almost completely restored the insulin action in cells exposed to nordihydroguaiaretic acid. Insulin stimulation increased cell surface GLUT4 2-fold in control cells, whereas LO inhibition abrogated the insulin-stimulated GLUT4 translocation. LO inhibition induced a prominent disassembly of actin fibres compared with control cells. In conclusion, we show here that 12(S)-hydroxyeicosatetraenoic acid plays a role in the organization of the actin network in cardiomyocytes. LO inhibition blocks GLUT4 translocation without affecting downstream insulin signalling. These data suggest that LO metabolites participate in the regulation of glucose transport by contributing to a rearrangement of actin cytoskeletal elements.

Regulation of subcellular distribution of GLUT4 in cardiomyocytes: Rab4A reduces basal glucose transport and augments insulin responsiveness.

Members of the Rab subfamily of small-GTP binding proteins have been suggested to be involved in insulin-regulated translocation of the glucose transporter GLUT4. To directly study this process in muscle tissue, we have established an insulin-sensitive cardiac cell line (H9K6) stably overexpressing GLUT4, which was derived from H9c2 cardiac myoblasts. H9K6-cells were transiently transfected with rab4A and rab3C with an efficiency of 65% and glucose uptake and the cellular distribution and expression of the transporter isoforms GLUT1 and GLUT4 was subsequently determined. Rab3C-overexpression caused no significant change in both basal and insulin-stimulated 2-deoxyglucose uptake compared to control cells transfected with the blank vector. Rab4A was barely detectable in membranes of H9K6 cells. However, after transient transfection this protein was expressed at a level comparable to adult cardiomyocytes. This resulted in a reduction of basal glucose uptake by 31% compared to control cells. Under these conditions insulin was able to stimulate 2-deoxyglucose uptake by 120%. Total expression of GLUT1 and GLUT4 was not affected by Rab4-overexpression. Cell surface biotinylation was used to quantify the abundance of GLUT1 and GLUT4 in the plasma membrane. A decrease of cell surface GLUT4 by about 40% compared to control cells was found in Rab4-overexpressing cells Insulin treatment increased cell surface-GLUT4 by 100% compared to only 26% in control cells. Distribution of GLUT1 was not affected under these conditions. Our data show that Rab4A but not Rab3C is able to reduce basal glucose uptake and cell surface content of GLUT4 in cardiac muscle cells. This results in an increased stimulation of glucose uptake by insulin which can be fully explained by enhanced translocation of GLUT4. We suggest that Rab4A participates in the redistribution of GLUT4 to intracellular pools and represents an essential determinant of the insulin responsiveness of GLUT4 translocation in cardiac muscle cells.