Localization of Lipoprotein Lipase in the Diabetic Heart

Abstract

Abstract-Vascular endothelium-bound lipoprotein lipase (LPL) is rate limiting for free fatty acid (FFA) transport into tissues. In streptozotocin (STZ)-diabetic rats, we have previously demonstrated an increased heparin-releasable LPL activity from perfused hearts. Because heparin can traverse the endothelial barrier, conventional Langendorff retrograde perfusion of the heart with heparin could release LPL from both the capillary luminal and abluminal surfaces. To determine the precise location of the augmented LPL, a modified Langendorff retrograde perfusion was used to isolate the enzyme at the coronary lumen from that in the interstitial effluent. In response to heparin, a 4-fold increase in LPL activity and protein mass was observed in the coronary perfusate after 2 weeks of STZ diabetes. Release of LPL activity into the interstitial fluid of control hearts was slow but progressive, whereas in diabetic hearts, peak enzyme activity was observed within 1 to 2 minutes after heparin, followed by a gradual decline. Immunohistochemical studies of myocardial sections confirmed that the augmented LPL in diabetic hearts was mainly localized at the capillary endothelium. To study the acute effects of insulin on endothelial LPL activity, we examined rat hearts at various times after the onset of hyperglycemia. An increased heparin-releasable LPL activity in diabetic rats was demonstrated shortly (6 to 24 hours) after STZ injection or after withdrawal from exogenous insulin. Heparin-releasable coronary LPL activity was also increased after an overnight fast. These studies indicate that the intravascular heparin-releasable fraction of cardiac LPL activity is acutely regulated by short-term changes in insulin rather than glucose. Thus, during short periods (hours) of hypoinsulinemia, increased LPL activity at the capillary endothelium can increase the delivery of FFAs to the heart. The resultant metabolic changes could induce the subsequent cardiomyopathy that is observed in the chronic diabetic rat.

Langendorff's isolated perfused rat heart technique: a review

“…While it should be taken in account that the isolated heart is denervated this can be an advantage in this method as it allows the separation of cardiac, from sympathetic, and vagal stimulation. 15,16 Many recent studies such as Skrzypiec-Spring et al, 2007, described the general principle of Langendorff's heart preparation to involve the cannulation of the aorta, over a fixed cannula on the perfusion apparatus. This cannula is attached to the outflow of a reservoir containing an oxygenated perfusion solution maintained at 37°C and continuously gassed with a gas mixture containing 5% CO 2 and 95% oxygen for normal aerobic perfusion studies, perfusing with a physiological salt solution (KHB) containing bicarbonate which mimics the ionic content of plasma, and delivered at 37°C at a physiological pH of 7.4.…”

“…An alternative to anesthesia is cervical dislocation or concussion, but in both instances, there are major effects on catecholamine's and other circulating factors, so these are not recommended. [8][9][10]14,15 Procedure After surgical anesthesia, careful excision of aorta and cannulation are the first steps done. A set of sutures and instruments positioned close at hand and rat is placed in a dissecting tray near the isolated heart apparatus.…”

Endothelial heparanase secretion after acute hypoinsulinemia is regulated by glucose and fatty acid

“…Using retrograde perfusion of the heart with heparin to displace coronary LPL, we also found significantly elevated luminal LPL activity following diabetes (27). We determined that the increased enzyme in the diabetic heart is 1) not the result of increased gene expression (25), 2) likely unrelated to an increase in the number of capillary endothelial HSPG binding sites (25), 3) functionally relevant and capable of hydrolyzing lipoprotein-TG (30), and 4) acutely (hours) regulated by short-term changes in insulin levels (31). More recently, we have been studying the contribution of the cardiomyocyte in enabling this increased enzyme at the vascular lumen using an acute model of hypoinsulinemia induced with diazoxide (DZ) (16,17).…”

“…Adult male Wistar rats (260 -300 g) were obtained from the University of British Columbia Animal Care Unit. Previously, we have reported that an acute reduction in insulin (hours) is associated with an increase in LPL at the coronary lumen (31). To examine LPL under conditions where insulin levels are rapidly manipulated, we used DZ, a selective K ϩ ATP channel opener.…”

“…The coronary effluent was collected in timed fractions over 10 min and assayed for LPL activity by measuring the hydrolysis of [ 3 H]triolein. For modified Langendorff perfusion, to separate the coronary from the interstitial effluent, a modified Langendorff retrograde perfusion technique was used (8,9,15,31). Rats were anesthetized, the thoracic cavity was opened, and the left anterior vena cava was ligated below the azygous vein followed by ligation of the right anterior vena cava.…”