Following diabetes, the heart increases its lipoprotein lipase (LPL) at the coronary lumen by transferring LPL from the cardiomyocyte to the endothelial lumen. We examined how hyperglycemia controls secretion of heparanase, the enzyme that cleaves myocyte HSPG to initiate this movement. Diazoxide (DZ) was used to decrease serum insulin and generate hyperglycemia. A modified Langendorff technique was used to separate coronary from interstitial effluent, which were assayed for heparanase and LPL. Within 30 min of DZ, interstitial heparanase increased, an effect that closely mirrored an augmentation in interstitial LPL. Endothelial cells were incubated with palmitic acid (PA) or glucose, and heparanase secretion determined. PA increased intracellular heparanase, with no effect on secretion of this enzyme. Unlike PA, glucose dose-dependently lowered endothelial intracellular heparanase, which was strongly associated with increased heparanase activity in the incubation medium. Pre-incubation with cytochalasin D or nocodazole prevented the high glucose induced depletion of intracellular heparanase. Our data suggest that following hyperglycemia, translocation of LPL from the cardiomyocyte cell surface to the apical side of endothelial cells is influenced by the ability of FA to increase endothelial intracellular heparanase followed by rapid secretion of this enzyme by glucose, which requires an intact microtubule and actin cytoskeleton.