Diabetes mellitus induces long lasting changes in the glucose transporter of rat heart endothelial cells

The accumulation of glucose exerts various cytotoxic effects on endothelial and other vascular cells, and thereby contributes to the development of mi crovascular complications in diabetes. Since tissues, in which vascular com plications typically occur, do not take up glucose in an insulin regulated manner, it is an important question to know whether other mechanisms exist in these cells to restrict the uptake and the accumulation of glucose. To s tudy this question, we used microvascular endothelial cells isolated from r at heart endothelial cells (RHEC). In RHEC, the non-insulin regulated gluco se transporter (Glut-1) was detected as a broad protein band of 50-65 kD. I n contrast, the Glut-1 from rat brain, which was taken as reference, had a molecular weight of 45 kD. After treatment with endoglycosidase F, both pro teins formed a band of approximately 40 kD on SDS-PACE, demonstrating a mor e extensive glycosylation of Glut-1 in RHEC as compared to brain. Incubatio n of the cells in high glucose (22 mM, up to 10 days) did not down-regulate either Glut-1 protein or mRNA. In contrast to high glucose, deprivation of the cells from glucose led to an increase in Glut-1 mRNA and protein which is partly non-glycosylated. In cells from hearts of streptozotocin-diabeti c rats (DRHEC), Glut-1 protein, but not Glut-1 mRNA, was reduced by about 4 0%. Additionally, a significant amount of glycosyl residues was resistant t o the enzymatic treatment with N-endoglycosidase F. Both changes in Glut-1 were also observed when the cells were cultivated in low glucose (5.5 mM) f or several passages indicating a long lasting, hardly reversible modificati on of Glut-1 by diabetes. These data indicate that Glut-1 is not down-regul ated in RHEC by high glucose, and that this important mechanism to protect the endothelium against an intracellular accumulation of glucose is missing in RHEC. As a consequence, increases in blood glucose may lead to a glucos e overload with the described deleterious effects on the structure and func tion of endothelium.