Abstract-The diabetic heart switches to exclusively using fatty acid (FA) for energy supply and does so by multiple mechanisms including hydrolysis of lipoproteins by lipoprotein lipase (LPL) positioned at the vascular lumen. We determined the mechanism that leads to an increase in LPL after diabetes. Diazoxide (DZ), an agent that decreases insulin secretion and causes hyperglycemia, induced a substantial increase in LPL activity at the vascular lumen. This increase in LPL paralleled a robust phosphorylation of Hsp25, decreasing its association with PKC␦, allowing this protein kinase to phosphorylate and activate protein kinase D (PKD), an important kinase that regulates fission of vesicles from the golgi membrane. Rottlerin, a PKC␦ inhibitor, prevented PKD phosphorylation and the subsequent increase in LPL. Incubating control myocytes with high glucose and palmitic acid (GluϩPA) also increased the phosphorylation of Hsp25, PKC␦, and PKD in a pattern similar to that seen with diabetes, in addition to augmenting LPL activity. In myocytes in which PKD was silenced or a mutant form of PKC␦ was expressed, high GluϩPA were incapable of increasing LPL. Moreover, silencing of cardiomyocyte Hsp25 allowed phorbol 12-myristate 13-acetate to elicit a significant phosphorylation of PKC␦, an appreciable association between PKC␦ and PKD, and a vigorous activation of PKD. As these cells also demonstrated an additional increase in LPL, our data imply that after diabetes, PKD control of LPL requires dissociation of Hsp25 from PKC␦, association between PKC␦ and PKD, and vesicle fission. Results from this study could help in restricting cardiac LPL translocation, leading to strategies that overcome contractile dysfunction after diabetes. Key Words: heat shock protein Ⅲ protein kinase C Ⅲ hyperglycemia Ⅲ hyperlipidemia Ⅲ vesicles C ardiac muscle has a high demand for energy and uses multiple substrates, including fatty acid (FA), carbohydrate, amino acids, and ketones. 1 Among these substrates, carbohydrate and FA are the major sources from which the heart derives most of its energy. In a normal heart, whereas glucose and lactate account for approximately 30% of energy provided to the cardiac muscle, 70% of ATP generation is through FA oxidation. 2 FA delivery and utilization by the heart involves: (1) release from adipose tissue and transport to the heart after complexing with albumin, 3 (2) provision through breakdown of endogenous cardiac triglyceride (TG) stores, 4 (3) internalization of whole lipoproteins, 5 and (4) hydrolysis of circulating TG-rich lipoproteins to FA by lipoprotein lipase (LPL) positioned at the endothelial surface of the coronary lumen. 6 The molar concentration of FA bound to albumin is Ϸ10-fold less than that of FA in lipoprotein-TG, 7 and LPL-mediated hydrolysis of circulating TG-rich lipoproteins to FA is suggested to be the principal source of FA for cardiac utilization. 8 Coronary endothelial cells do not synthesize LPL. 9 In the heart, this enzyme is produced in cardiomyocytes and subsequently se...