Rosiglitazone treatment improves cardiac efficiency in hearts from diabetic mice.

Isolated perfused hearts from type 2 diabetic (db/db) mice show impaired ventricular function, as well as altered cardiac metabolism. Assessment of the relationship between myocardial oxygen consumption (MVO(2)) and ventricular pressure-volume area (PVA) has also demonstrated reduced cardiac efficiency in db/db hearts. We hypothesized that lowering the plasma fatty acid supply and subsequent normalization of altered cardiac metabolism by chronic treatment with a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist will improve cardiac efficiency in db/db hearts. Rosiglitazone (23 mg/kg body weight/day) was administered as a food admixture to db/db mice for five weeks. Ventricular function and PVA were assessed using a miniaturized (1.4 Fr) pressure-volume catheter; MVO(2) was measured using a fibre-optic oxygen sensor. Chronic rosiglitazone treatment of db/db mice normalized plasma glucose and lipid concentrations, restored rates of cardiac glucose and fatty acid oxidation, and improved cardiac efficiency. The improved cardiac efficiency was due to a significant decrease in unloaded MVO(2), while contractile efficiency was unchanged. Rosiglitazone treatment also improved functional recovery after low-flow ischemia. In conclusion, the present study demonstrates that in vivo PPARgamma-treatment restores cardiac efficiency and improves ventricular function in perfused hearts from type 2 diabetic mice.

Mechanisms responsible for enhanced fatty acid utilization by perfused hearts from type 2 diabetic db/db mice.

The aim of this study was to determine the biochemical mechanism(s) responsible for enhanced FA utilization (oxidation and esterification) by perfused hearts from type 2 diabetic db/db mice. The plasma membrane content of fatty acid transporters FAT/CD36 and FABPpm was elevated in db/db hearts. Mitochondrial mechanisms that could contribute to elevated rates of FA oxidation were also examined. Carnitine palmitoyl transferase-1 activity was unchanged in mitochondria from db/db hearts, and sensitivity to inhibition by malonyl-CoA was unchanged. Malonyl-CoA content was elevated and AMP kinase activity was decreased in db/db hearts, opposite to what would be expected in hearts exhibiting elevated rates of FA oxidation. Uncoupling protein-3 expression was unchanged in mitochondria from db/db hearts. Therefore, enhanced FA utilization in db/db hearts is most likely due to increased FA uptake caused by increased plasma membrane content of FA transporters; the mitochondrial mechanisms examined do not contribute to elevated FA oxidation observed in db/db hearts.

Modulation of potassium currents by angiotensin and oxidative stress in cardiac cells from the diabetic rat.

Diabetes induces oxidative stress and leads to attenuation of cardiac K+ currents. We investigated the role of superoxide ions and angiotensin II (ANG II) in generating and linking oxidative stress to the modulation of K+ currents under diabetic conditions. K+ currents were measured using patch-clamp methods in ventricular myocytes from streptozotocin (STZ)-induced diabetic rats. Superoxide ion levels, indicating oxidative stress, were measured by fluorescent labelling with dihydroethidium (DHE). ANG II content was measured using enzyme-linked immunosorbent asssay (ELISA). The results showed DHE fluorescence to be significantly higher in cells from diabetic males, compared to controls. Relief of stress by the NADPH oxidase inhibitor apocynin or by superoxide dismutase (SOD) but not by catalase reversed the attenuation of K+ currents and reduced DHE fluorescence. In cells from diabetic females, neither apocynin nor SOD augmented K+ currents, ANG II was not elevated and DHE fluorescence was significantly weaker than in cells from males. Reduced glutathione (GSH) also augmented K+ currents in cells from diabetic males but not females. In ovariectomized diabetic females K+ currents were augmented by GSH and apocynin. Current augmentation and the attenuation of DHE fluorescence by apocynin were significantly blunted by excess ANG II (300 nm). Diabetic male rats pretreated with the angiotensin-converting enzyme (ACE) inhibitor quinapril were hyperglycaemic, but their cellular ANG II levels and DHE fluorescence were significantly decreased. In cells from these rats, K+ currents were insensitive to apocynin. In conclusion, diabetes-related oxidative stress attenuates K+ currents through ANG II-generated increased superoxide ion levels. When ANG II levels are lower, as in diabetic females or following ACE inhibition in males, oxidative stress is reduced, with blunted alterations in K+ currents.

Time-dependent systolic and diastolic function in mice overexpressing calcineurin.

Echocardiograms have been assessed only at 56 days in mice overexpressing calcineurin (CN mice). Age-dependent echocardiographic changes were evaluated because the development of sudden death is time dependent. Because cyclosporin A (CsA) reverses hypertrophy in CN mice, its effects on the time course of the development of sudden death and cardiac dysfunction were assessed. In wild-type (WT) mice, the left ventricular (LV) internal end-diastolic dimension (LVIDd) increased and the LV mass index (LVMI) decreased with age. In CN mice, two distinct phases of pathophysiology were found. After 14 days, in CN mice, the LVIDd and LVMI were significantly increased, but sudden death had not occurred. After 28 days, in CN mice, relative dilation of the left ventricle occurred, whereas the LVMI decreased. Sudden death developed during progressive dilation associated with systolic and diastolic dysfunction. CsA treatment reversed hypertrophy in CN mice but did not reverse systolic and diastolic dysfunction and exaggerated sudden death. Sudden cardiac death was associated with systolic and diastolic dysfunction but was not related to isolated cardiac hypertrophy in CN mice.

Chylomicron metabolism by the isolated perfused mouse heart.

The catabolism of rat chylomicrons, labeled in their triacylglycerol (TG) component, was investigated using perfused working mouse hearts. Perfusion of mouse hearts with heparin increased lipoprotein lipase (LPL) activity in the perfusate. This heparin-releasable LPL pool remained constant over a variety of experimental conditions, including workload and fatty acid concentrations, making the mouse heart a suitable model to study chylomicron catabolism. Endothelium-bound LPL hydrolyzed radiolabeled (3)H-labeled chylomicrons (0.4 mM TG); the fate of LPL-derived (3)H-labeled fatty acids was split evenly between oxidation (production of (3)H(2)O) and esterification (incorporation into tissue lipids, mainly TG). In comparison, the oxidation of 0.4 mM [(3)H]palmitate complexed to albumin was fourfold greater than esterification into tissue lipids. Surprisingly, the addition of unlabeled palmitate (0.4 or 1.2 mM) to perfusions with (3)H-chylomicrons did not affect the fate (either oxidation or esterification) of LPL-derived (3)H-fatty acids. These results suggest that fatty acids produced from lipoprotein hydrolysis by the action of LPL and fatty acids from a fatty acid-albumin complex do not enter a common metabolic pool in the heart.

Peroxisome proliferator-activated receptor-alpha ligands inhibit cardiac lipoprotein lipase activity.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that regulate gene expression of lipoprotein lipase (LPL) in liver and adipose tissue. We examined the direct effect of PPAR-alpha ligands on LPL catalytic activity in cultured cardiomyocytes from adult rat heart. After overnight culture (16 h), 1 microM Wy-14643 and 10 microM BM-17.0744 decreased total cellular LPL activity to approximately 50% of control with no change in enzyme synthesis or mass; as a consequence, PPAR-alpha activation produced a significant decrease in LPL specific activity (mU/ng LPL protein). Wy-14643 and BM-17.0744 also reduced heparin-releasable LPL activity and mass in the culture medium. Inhibition of LPL activity by Wy-14643 did not reduce the ability of insulin plus dexamethasone to stimulate cellular and heparin-releasable LPL activities. A similar inhibitory effect on cellular and heparin-releasable LPL activity was observed when cardiomyocytes were cultured with 60 microM linoleic acid. In conclusion, two different PPAR-alpha ligands (Wy-14643 and BM-17.0744) inhibited cellular LPL activity in cultured cardiomyocytes by a posttranscriptional and posttranslational mechanism.

Glucose metabolism in perfused mouse hearts overexpressing human GLUT-4 glucose transporter.

Glucose and fatty acid metabolism was assessed in isolated working hearts from control C57BL/KsJ-m+/+db mice and transgenic mice overexpressing the human GLUT-4 glucose transporter (db/+-hGLUT-4). Heart rate, coronary flow, cardiac output, and cardiac power did not differ between control hearts and hearts overexpressing GLUT-4. Hearts overexpressing GLUT-4 had significantly higher rates of glucose uptake and glycolysis and higher levels of glycogen after perfusion than control hearts, but rates of glucose and palmitate oxidation were not different. Insulin (1 mU/ml) significantly increased glycogen levels in both groups. Insulin increased glycolysis in control hearts but not in GLUT-4 hearts, whereas glucose oxidation was increased by insulin in both groups. Therefore, GLUT-4 overexpression increases glycolysis, but not glucose oxidation, in the heart. Although control hearts responded to insulin with increased rates of glycolysis, the enhanced entry of glucose in the GLUT-4 hearts was already sufficient to maximally activate glycolysis under basal conditions such that insulin could not further stimulate the glycolytic rate.

Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice.

Contractile function and substrate metabolism were characterized in perfused hearts from genetically diabetic C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice and their non-diabetic lean littermates. Contractility was assessed in working hearts by measuring left ventricular pressures and cardiac power. Rates of glycolysis, glucose oxidation, and fatty acid oxidation were measured using radiolabeled substrates ([5-(3)H]glucose, [U-(14)C]glucose, and [9,10-(3)H]palmitate) in the perfusate. Contractile dysfunction in db/db hearts was evident, with increased left ventricular end diastolic pressure and decreased left ventricular developed pressure, cardiac output, and cardiac power. The rate of glycolysis from exogenous glucose in diabetic hearts was 48% of control, whereas glucose oxidation was depressed to only 16% of control. In contrast, palmitate oxidation was increased twofold in db/db hearts. The hypothesis that altered metabolism plays a causative role in diabetes-induced contractile dysfunction was tested using perfused hearts from transgenic db/db mice that overexpress GLUT-4 glucose transporters. Both glucose metabolism and palmitate metabolism were normalized in hearts from db/db-human insulin-regulatable glucose transporter (hGLUT-4) hearts, as was contractile function. These findings strongly support a causative role of impaired metabolism in the cardiomyopathy observed in db/db diabetic hearts.

Glucose and fatty acid metabolism in the isolated working mouse heart.

Although isolated perfused mouse heart models have been developed to study mechanical function, energy substrate metabolism has not been examined despite the expectation that the metabolic rate for a heart from a small mammal should be increased. Consequently, glucose utilization (glycolysis, oxidation) and fatty acid oxidation were measured in isolated working mouse hearts perfused with radiolabeled substrates, 11 mM glucose, and either 0.4 or 1.2 mM palmitate. Heart rate, coronary flow, cardiac output, and cardiac power did not differ significantly between hearts perfused at 0.4 or 1.2 mM palmitate. Although the absolute values obtained for glycolysis and glucose oxidation and fatty acid oxidation are significantly higher than those reported for rat hearts, the pattern of substrate metabolism in mouse hearts is similar to that observed in hearts from larger mammals. The metabolism of mouse hearts can be altered by fatty acid concentration in a manner similar to that observed in larger animals; increasing palmitate concentration altered the balance of substrate metabolism to increase overall energy derived from fatty acids from 64 to 92%.

Lipoprotein lipase activity is stimulated by insulin and dexamethasone in cardiomyocytes from diabetic rats.

Type 1 diabetes mellitus reduces lipoprotein lipase (LPL) activity in the heart. The diabetic phenotype of decreased LPL activity in freshly isolated cardiomyocytes persisted after overnight culture (16 h). Total cellular LPL activity was 311+/-56 nmol oleate released x h(-1) x mg(-1) cell protein in diabetic cultured cardiomyocytes compared with 661+/-81 nmol oleate released x h(-1) x mg(-1) cell protein for control cultured cells. Diabetes also resulted in lower heparin-releasable (HR) LPL activity compared with control cells (111+/-25 vs. 432+/-63 nmol x h(-1) x mg(-1) cell protein). In kinetic experiments, the reduction in total cellular LPL and HR-LPL activities in cultured cells from diabetic hearts was due to a decrease in maximal velocity, with no change in apparent Km for substrate (triolein). LPL activity in primary cultures of cardiomyocytes from control rats is stimulated by the combination of insulin (Ins) and dexamethasone (Dex). Overnight treatment of cultured cardiomyocytes from diabetic rats with Ins+Dex elicited an 84% increase in cellular LPL activity (to 572+/-65 nmol x h(-1) x mg(-1) cell protein) and a 194% increase in HR-LPL activity (to 326+/-46 nmol x h(-1) x mg(-1) cell protein). This stimulation occurred at subnanomolar concentrations of the hormones, but neither hormone was effective alone. The amount of immunoreactive LPL protein mass in cultured cardiomyocytes from diabetic hearts was unchanged by Ins+Dex treatment. Addition of oleic acid (60 microM) to the overnight culture medium inhibited the already reduced HR-LPL activity in diabetic cultured cells by 73% (to 30+/-4 nmol x h(-1) x mg(-1) cell protein). The presence of oleic acid also reduced hormone-stimulated HR-LPL activity. Increasing the glucose concentration in the culture medium to 26 mM had no effect on total cellular LPL or HR-LPL activities.

The isolated working mouse heart: methodological considerations.

Our aim was to develop a working isolated murine heart model, as the extensive use of genetically engineered mice in cardiovascular research requires development of new miniaturized technology. Left ventricular (LV) function was assessed in the isolated working mouse heart perfused with recirculated oxygenated Krebs-Henseleit bicarbonate buffer (37 degrees C pH 7.4) containing 11.1 mM glucose and 0.4 mM palmitate bound to 3% albumin. The hearts worked against an afterload reservoir at a height equivalent to 50 mmHg, and heart rate was controlled by electrical pacing of the right atrium. LV pressure was measured with a micromanometer connected to a small steel cannula inserted through the apex of the heart. The experimental protocol consisted of two interventions. First, following instrumentation and stabilization, the preload reservoir was raised from a pressure equivalent of 7 to 22.5 mmHg, while pacing at 390 beats.min-1. Thereafter the height of the preload reservoir was set to 10 mmHg, and the pacing rate was varied from 260 to 600 beats.min-1. Aortic and coronary flows were measured by timed collections of effluent from the afterload line and that dripping from the heart, respectively [aortic+coronary flow=cardiac output (CO)]. Elevation of LV end-diastolic pressure (LVEDP) from approximately 5 to 10 mmHg resulted in a twofold increase in average cardiac power [product of LV developed pressure (LVDevP) and CO], whereas myocardial contractility (first derivative of LV pressure, dP/dt) and LVDevP (LV systolic pressure-LVEDP) increased only minimally (5-10%). Measured LVEDP was lower than the equivalent height of the preload reservoir by an amount that was related to the heart rate. Cardiac power, LVDevP and dP/dt were stable at heart rates up to 400 beats.min-1, but declined markedly with higher rates, consistent with the decrease in LVEDP. Thus, cardiac power was reduced to 50% of its maximum value when stimulated at approximately 500 beats.min-1, and at even higher rates there was little ejection. By systematic manipulation of the height of the preload reservoir and heart rate, we conclude that LV afterload and preload can be assessed only by high-fidelity measurement of intraventricular pressures. The heights of the afterload column and the preload reservoir are unreliable and potentially misleading indicators of LV afterload and preload.

Insulin and dexamethasone stimulation of cardiac lipoprotein lipase activity involves the actin-based cytoskeleton.

Lipoprotein lipase (LPL) activity in cultured ventricular cardiomyocytes from adult rat hearts was stimulated by the combination of insulin (100 nM) and dexamethasone (100 nM) during an overnight (16 h) incubation. Wortmannin (100 nM), rapamycin (30 ng/ml) or PD98059 (50 microM) did not prevent this stimulation, suggesting that phosphatidylinositol 3-kinase, p70 S6 kinase and the mitogen-activated protein kinase cascade are not involved in transducing the hormonal signal. In contrast, cytochalasin D (2 microM) completely abolished the stimulatory effect of insulin and dexamethasone on both heparin-releasable LPL and total cellular LPL activities. The potential role of the actin cytoskeleton in the stimulation of LPL activity by insulin and dexamethasone appears to be distal to the initial signalling events since cytochalasin D is still effective in preventing the stimulation when added 2 h after the hormones.

Depression of excitability by sphingosine 1-phosphate in rat ventricular myocytes.

Sphingosine 1-phosphate (S-1-P) is a bioactive sphingolipid that is released from activated platelets. Extracellular S-1-P augments an inwardly rectifying potassium conductance in cultured atrial preparations, but the electrophysiological effects of this compound in the ventricle are unknown. The electrophysiological effects of S-1-P were examined in single myocytes from rat ventricular muscle. Action potential waveforms and underlying ionic currents in the presence and absence of 3 microM S-1-P (1-6 min) were recorded. S-1-P increased the minimum stimulus current needed to elicit an action potential by approximately 100 pA. Pertussis toxin or preexposure to S-1-P did not alter this effect. The action potential waveform was unchanged by S-1-P. The inward sodium current (INa) was examined in a range of membrane potentials just negative to the potential for firing an action potential. S-1-P reversibly inhibited peak INa by approximately 50 pA, whereas the inward rectifier potassium current was not significantly changed. The results of this study suggest that S-1-P inhibits rat ventricular excitability by reducing INa.

Heparin-releasable lipoprotein lipase activity is increased in cardiomyocytes after culture.

The activity of lipoprotein lipase (LPL) in adult rat heart cardiomyocytes after overnight culture on laminin-coated plates for 18-22 h was compared with enzyme activity in freshly isolated cardiomyocytes. LPL activity in cellular homogenates from cultured cardiomyocytes and freshly isolated cells was 240 and 233 nmol oleate released h-1.mg-1 protein, respectively. LPL specific activity (mU/ng LPL protein) was 0.07 in cultured cells compared with 0.42 in freshly isolated cells, indicating an increased content of inactive LPL mass after overnight culture. The heparin-induced release of LPL activity into the medium of cultured cardiomyocytes (198 nmol.h-1.mg-1) was much greater than heparin-releasable LPL (HR-LPL) activity (59 nmol.h-1.mg-1) from freshly isolated cells. HR-LPL activity from cultured cardiomyocytes was dependent on serum (16.3-fold activation) and was inhibited by high ionic strength (1 M NaCl) and by a polyclonal antibody to LPL. Cultured cardiomyocytes also had more immunodetectable LPL on the cell surface compared with freshly isolated cardiomyocytes, consistent with increased HR-LPL activity. Therefore, overnight culture may permit cardiomyocytes time to recover from the stress of isolation by increasing the content of LPL on the cell surface.

Metabolic fate of exogenous diacylglycerols in A10 smooth muscle cells.

The metabolic fate of exogenous diacylglycerols, 1-palmitoyl-2-[1-14C]oleoyl-sn-glycerol (2-[14C]POG) and 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycerol (2-[14C]SAG), was determined after incubation of A10 smooth muscle cells with liposomal suspensions. Hydrolysis through a diacylglycerol (DG) lipase pathway was the predominant metabolic fate; more than 80% of cell-associated radioactivity from 2-[14C]POG and 2-[14C]SAG was recovered in lipolytic products, monoacylglycerol (MG) and fatty acids (FA), which were present in the incubation medium. Hydrolysis of 2-[14C]POG was reduced completely by tetrahydrolipstatin, a lipase inhibitor. Very little radioactivity from either 2-[14C]POG or 2-[14C]SAG was incorporated into triacylglycerol or phospholipids. DG lipase and kinase activities were measured by in vitro enzyme assays. 1-[1-14C]Palmitoyl-2-oleoyl-sn-glycerol (1-[14C]POG) was phosphorylated (kinase activity) to a greater extent than 2-[14C]SAG in assays with both soluble and particulate subcellular fractions from A10 cells. DG lipase activity (hydrolysis of 1-[14C]POG and 2-[14C]SAG) was markedly stimulated by the addition of 20 mM MgCl2 and 20 mM ATP to the assay. Under optimal assay conditions, DG lipase activity exhibited little substrate specificity. Our findings indicate that exogenous DG are mainly hydrolyzed by DG and MG lipases in A10 smooth muscle cells; as a result, signalling mechanisms responding to DG second messengers will be attenuated.

Lipoprotein lipase activity in rat cardiomyocytes is stimulated by insulin and dexamethasone.

Lipoprotein lipase (LPL) activity was studied in rat cardiomyocytes after overnight culture (16 h) in the presence of insulin (100 nM) and/or dexamethasone (100 nM). Insulin in combination with dexamethasone (INS/DEX) increased heparin-releasable LPL activity by 71% over the control level (566+/-85 versus 331+/-48 nmol/h.mg cell protein). This was accompanied by a 61% increase in total cellular LPL activity (914+/-89 versus 567+/-64 nmol/h.mg cell protein). The increase in LPL activity occurred at sub-nanomolar concentrations of the hormones, but neither hormone was effective alone. LPL protein mass, quantified by ELISA, was the same in both control and INS/DEX-treated cells (27.7 versus 28.6 ng/mg cell protein, respectively), thus LPL specific activity in cardiomyocytes was increased by INS/DEX treatment (0.113 versus 0.069 mU/ng LPL protein). These findings emphasize the importance of hormonal interactions in the regulation of LPL in heart tissue.

Fatty acids reduce heparin-releasable LPL activity in cultured cardiomyocytes from rat heart.

Varying glucose and fatty acid (FA) concentrations in the medium of cultured cardiomyocytes from adult rat hearts were tested for effects on lipoprotein lipase (LPL) activity. Glucose (5.5, 11, and 25 mM in the culture medium for 18-22 h) had no effect on either heparin-releasable LPL (HR-LPL) or on cellular LPL (C-LPL) activities. When cardiomyocytes were cultured overnight with 60 microM oleate, HR-LPL activity was reduced to 20% of control, with no change in C-LPL activity or total C-LPL mass. Similar results (HR-LPL and C-LPL activities) were obtained with 60 microM concentrations of palmitate and myristate; linoleate and eicosapentaenoate did reduce C-LPL activity, but the decrease in HR-LPL activity was much greater. Oxfenicine, an FA oxidation inhibitor, did not alter the inhibitory effect of 60 microM oleate on HR-LPL. Short-term incubations (1 and 3 h) of cultured cardiomyocytes with 60 microM oleate did not displace LPL into the medium. Immunodetectable LPL on the cell surface of oleate-treated cultured cardiomyocytes was increased compared with control cells, but heparin treatment released the same amount of LPL mass that had reduced catalytic activity.

Diacylglycerol metabolism in SM-3 smooth muscle cells.

The metabolism of endogenous and exogenous diacylglycerol (DG) was studied in SM-3 cells derived from rabbit aortic smooth muscle to determine the biochemical step(s) responsible for degrading DG second messengers. Incubation of growth-arrested SM-3 cells with [3H]myristate for 2 h resulted in selective labelling of cellular phosphatidylcholine (PC). Addition of bacterial PC-specific phospholipase C (PC-PLC, 20 U) to [3H]myristate-labelled SM-3 cells resulted in a 7.4-fold increase in endogenous radiolabelled DG and activation of the epsilon isoform of protein kinase C. Subsequent incubation of PC-PLC-treated SM-3 cells resulted in the incorporation of radioactivity primarily into products of a lipase pathway, monoacylglycerol and fatty acid. The hydrolysis of endogenous PC-derived DG was inhibited by 0.25 microM tetrahydrolipstatin, a DG lipase inhibitor. Similar results were obtained when SM-3 cells were incubated with 1,2-dioctanoyl-[2-3H]glycerol, a cell-permeable DG analog; radioactivity was mainly recovered in the glycerol product of the lipase pathway. Therefore, hydrolysis by a lipase pathway represents the principal metabolic fate of both endogenous and exogenous DG in SM-3 smooth muscle cells.

Endothelial binding sites for lipoprotein lipase are not diminished in perfused hearts from diabetic rats.

The possibility that diabetes reduces functional, heparin-releasable lipoprotein lipase (HR-LPL) activity on the coronary vasculature of perfused hearts by altering endothelial binding sites for the enzyme was examined by measuring the binding and subsequent heparin-induced release of exogenous lipoprotein lipase purified from bovine milk (mLPL). Rat hearts were first perfused with heparin (5 U/mL) for 5 min to displace endogenous HR-LPL into the perfusate. The subsequent perfusion of control hearts with 0.05-2 micrograms/mL mLPL resulted in a progressive increase in bound exogenous enzyme that could be released by a second heparin perfusion. Induction of an acute, insulin-deficient model of diabetes (100 mg/kg streptozotocin 4-5 days prior to heart perfusions) reduced endogenous HR-LPL activity, but the binding and heparin-induced release of mLPL (0.5 microgram/mL) were the same as measured in control hearts. Therefore, diabetes does not alter low-affinity, high-capacity proteoglycan binding sites for mLPL on the endothelium of perfused hearts.