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Hypertension. 1998;31:878-884

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(Hypertension. 1998;31:878-884.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Streptozotocin-Induced Diabetes Enhances Cardiac Heparin-Releasable Lipoprotein Lipase Activity in Spontaneously Hypertensive Rats

Greg Shepherd; Margaret C. Cam; Nandakumar Sambandam; Mohammed A. Abrahani; ; Brian Rodrigues

From the Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.


*    Abstract
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 *Abstract
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 down arrowResults
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Abstract—Vascular endothelial-bound lipoprotein lipase (LPL), also known as heparin-releasable LPL, catalyzes the breakdown of the triglyceride component of lipoproteins and is rate-limiting for free fatty acid transport to tissues. We previously demonstrated that heparin-releasable LPL activity increases in diabetic Wistar rat hearts, whereas with the development of hypertension in spontaneously hypertensive rats (SHR), there is a concomitant and progressive reduction in LPL activity. The objective of the present study was to examine the regulation of cardiac LPL activity in SHR-diabetic rats. Heparin perfusion of the isolated Langendorff heart induced the release of LPL activity. SHR hearts demonstrated a reduction in peak heparin-releasable LPL activity, relative to Wistar controls. However, induction of streptozotocin-induced diabetes in SHR, as in Wistar rats, increased peak heparin-releasable LPL activity in perfused hearts. The elevated heparin-releasable LPL peak could not be accounted for by enhanced LPL synthesis in that both cellular and surface-bound LPL activities in myocytes from SHR-diabetic rats were low relative to control. Chronic (12-day) insulin treatment of SHR-diabetic rats reduced the augmented heparin-releasable LPL activity and increased cell-associated LPL activity. Moreover, acute (90-minute) treatment of SHR-diabetic rats with rapid-acting insulin also reduced the heparin-releasable LPL activity to normal, although it had no effect on the low cellular LPL activity. These results demonstrate that the diabetes-induced augmentation of cardiac LPL counteracts the reduction in enzyme activity associated with hypertension. This may serve to increase the delivery of free fatty acid to the heart, and the resultant metabolic changes may lead to the severe cardiomyopathy observed in the hypertensive-diabetic rat heart.


Key Words: lipoprotein lipase • rats, inbred SHR • streptozotocin • diabetes • cardiomyopathy • heparin


*    Introduction
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 *Introduction
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 down arrowResults
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Increasing evidence suggests that an altered substrate supply and utilization by cardiac myocytes could be the causal injury in the pathogenesis of diabetic cardiomyopathy.1 2 3 4 Importantly, in diabetes, glucose utilization is insignificant, and energy production is shifted almost exclusively toward ß-oxidation of FFA.5 FFAs are supplied to cardiac cells from endogenous cardiac TG stores or from exogenous sources in the blood (as free acid bound to albumin or as TG in lipoproteins). Vascular endothelium-bound LPL catalyzes the breakdown of the TG component of VLDL and chylomicrons and regulates tissue FFA supply; it is also called "functional" LPL.6 In the adult heart, LPL is synthesized and processed in myocytes and subsequently translocated onto HSPG binding sites on the luminal surface of endothelial cells.7 At this location, it actively metabolizes lipoprotein TG to FFA and 2-monoacylglycerol,8 which are transported into the heart for numerous metabolic and structural tasks. Recently, it was demonstrated that LPL activity, rather than FFA transport via the endothelial cell or sarcolemma, is the rate-limiting step in the cellular uptake of TG-derived FFA.9

The approximate contribution of endogenous or exogenous sources of FFA toward ß-oxidation in the diabetic heart is unknown. In an insulin-deficient state, hydrolysis of augmented myocardial TG stores could contribute to tissue FFA.10 11 12 In addition, adipose tissue lipolysis is enhanced, resulting in elevated circulating FFA.13 However, changes in cardiac LPL activity, which controls exogenous FFA uptake, are inconsistent.14 15 16 Recently, we demonstrated an elevated heparin-releasable LPL activity in diabetic rat hearts,17 which we hypothesized could increase FFA supply and utilization by cardiac cells.

Hypertension occurs more frequently in type 118 and type 219 diabetic patients than in the nondiabetic population of the same age and gender. An important feature of the hypertensive-diabetic condition is its synergistic effects on the development of heart failure.20 Induction of STZ-induced diabetes in the SHR also produces a more extensive cardiomyopathy and an increased mortality rate.21 We previously reported that coinciding with the onset of hypertension in the SHR is a progressive reduction in heparin-releasable endothelial LPL.22 This phenomenon may be an adaptive response in the hypertrophic heart to reduce FFA supply and shift fuel use to glucose.23 At present, the combined effects of diabetes and hypertension, which are antagonistic, on cardiac LPL activity are unknown. Thus, the diabetes-induced augmentation of cardiac LPL could counteract the reduction in enzyme activity associated with hypertension, thereby providing an excess amount of injurious FFA substrate to the heart. Hence, the objective of the present study was to examine the effect of diabetes on the regulation of cardiac LPL activity in SHR.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
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Experimental Animals
All animals were cared for in accordance with the principles promulgated by the Canadian Council on Animal Care and The University of British Columbia. Adult male SHR were obtained at 6 to 7 weeks of age (Charles River Canada, Montreal, Quebec, Canada). The rats were maintained under a 12-hour light (7:00 AM to 7:00 PM)/dark cycle and supplied with a standard laboratory chow diet (PMI Feeds) and water ad libitum. Age-matched Wistar rats (WisC and WisD) were used as normotensive controls instead of WKY rats. We have found that WKY rats are more resistant (than Wistar or Sprague-Dawley rat strains) to the ß-cytotoxic effects of STZ.24

Induction of Diabetes
Diabetes was induced in Wistar and SHR at 7 to 8 weeks of age before the development of severe hypertension in the SHR.25 SHR were randomly divided into CON and DIA groups. Halothane-anesthetized rats were injected with STZ (55 mg/kg IV; Sigma Chemical) or an equivalent volume (1 mL/kg) of saline. Glycosuria was determined 24 hours after STZ injection, and hyperglycemia was tested at 48 hours via glucometer. All STZ-treated rats displayed both glycosuria (>4+) and hyperglycemia (>13 mmol/L). Diabetic rats were kept for 2 weeks after the STZ injection, at which time they were killed and the hearts were removed. An STZ dose-titration study was also performed in some 7- to 8-week-old SHR. In this study, increasing doses of STZ (25, 35, and 45 mg/kg) were injected into SHR, and the animals were killed 2 weeks after diabetes induction.

Isolated Whole-Heart Perfusion
Rats were anesthetized with 50 mg/kg sodium pentobarbital IP, the thoracic cavity was opened, and the hearts were removed. Rats were not injected with heparin before killing because it displaces LPL bound to HSPGs on the capillary endothelium. Consequently, it was necessary to cannulate the heart quickly to avoid clotting of blood in the coronary arteries. Immediately on excision, the beating heart was immersed in cold (4°C), calcium-free Joklik minimal essential medium (pH 7.4) supplemented with 2 g/L NaHCO3, 1.2 mmol/L MgSO4, and 1 mmol/L L-carnitine. After cannulation of the aorta, the hearts were perfused retrogradely by the nonrecirculating Langendorff technique until the perfusate was clear of blood.26 This period is necessary to remove proteases released by tissue damage during the dissection that would normally reduce LPL activity subsequently measured in the hydrolysis assay. The perfusion fluid was continuously gassed with 95% O2/5% CO2 in a double-walled water-heated chamber maintained at 37°C with a temperature-controlled circulating water bath. A peristaltic pump controlled the rate of coronary flow (7 to 8 mL/min). To measure the release of LPL activity into the medium, the perfusion solution was changed to Joklik containing 1% BSA (Fraction V; Boehringer-Mannheim Biochemica), 1 mmol/L CaCl2, and heparin (5 U/mL).27 This concentration of heparin was previously shown to maximally release cardiac LPL from its binding site.25 The coronary effluent was collected in timed fractions and frozen until assayed for LPL activity.

Preparation of Cardiac Myocytes
Perfusion of the heart with heparin predominantly releases extracellular, endothelium-bound LPL; however, activity is still measurable within the heart. This heparin-nonreleasable LPL activity is located predominantly within the myocytes. To measure this fraction, calcium-tolerant myocytes were prepared from hearts (ventricles) according to a previously described procedure.26 Briefly, hearts were removed from anesthetized rats and digested through perfusion of collagenase (228 U/mL) retrogradely through the heart. Myocytes were made calcium tolerant by successive exposure to increasing concentrations of calcium. Our method of isolation yields a highly enriched population of calcium-tolerant myocardial cells that are rod shaped in the presence of 1 mmol/L Ca2+ with clear cross-striations. Intolerant cells are intact but hypercontract into vesiculated spheres. Yield of myocytes (cell number) was determined microscopically using an improved Neubauer hemocytometer. Myocyte viability (generally between 75% and 85%) was assessed through trypan blue exclusion.

Cardiac myocytes from control and diabetic rats were suspended in Joklik minimum essential medium to a cell density of 0.4x106 cells/mL and incubated at 37°C under an atmosphere of 95% O2/5% CO2. To release surface-bound LPL activity, heparin (5 U/mL) was added to the myocyte suspension. Aliquots of cell suspension (1 mL) were removed at specified intervals, and the medium was separated from cells by centrifugation (3000g for 10 seconds). The supernatant was decanted and stored at -70°C until it was assayed for LPL activity. The corresponding cell pellets were also stored frozen for measurement of intracellular LPL activity.

LPL Assay
LPL catalytic activity in coronary perfusates and incubation medium of cardiac myocytes was determined by measuring the in vitro hydrolysis of a sonicated [3H]triolein substrate emulsion. The standard assay conditions were 0.6 mmol/L glycerol tri[9,10-3H]oleate (1 mCi/mmol; 1 Ci=37 GBq), 25 mmol/L piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 7.5), 0.05% (wt/vol) albumin, 50 mmol/L MgCl2, 2% (vol/vol) heat-inactivated chicken serum (containing the LPL activator apolipoprotein CII), and 100 µL of either medium or heart perfusate in a total volume of 400 µL. The release of [3H]oleate was measured after an incubation of 30 minutes at 30°C. Hydrolyzed [3H]oleate in the medium was determined by the addition of 3 mL of a fatty acid extraction solution (methanol 38.5%, chloroform 34.2%, heptane 27.3%) and 100 µL of 0.1 mol/L NaOH.26 After vortex mixing and centrifugation, the radioactive sodium [3H]oleate in a sample (0.5 mL) of the upper phase was determined by liquid scintillation counting. All LPL assays were performed in duplicate under conditions in which the reaction rate was linear with respect to time and the volume of medium that was assayed.28 For the LPL assay, interassay and intra-assay coefficients of variation (%CV) did not exceed 10%. Results are routinely expressed as nanomoles of oleate released per hour per milliliter (coronary perfusate) or 106 cells (myocyte medium or cells).

Heparin-nonreleasable cellular LPL activity was measured through sonication of (twice at 30 seconds) the cell pellets after resuspending them in 0.2 mL of 50 mmol/L NH4Cl buffer (pH 8.0) containing 0.125% (vol/vol) Triton X-100. After sonication, the volume was adjusted to 1 mL with use of a sucrose buffer (0.25 mol/L sucrose, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 10 mmol/L HEPES, pH 7.4).29 The assay for cell sonicate LPL activity was done essentially as described above except that 20 µL of the cell sonicate was used and heparin (2 U/mL) was included in the assay.

Plasma Measurements
Blood samples from the tail vein were collected in heparinized glass capillary tubes. Blood samples were immediately centrifuged, and plasma was collected and stored at -20°C until it was assayed. Plasma glucose, TG, and cholesterol levels were measured with kits (Boehringer-Mannheim). Plasma insulin was measured using rat insulin standards (Novo) with a coefficient of variation <15% and a sensitivity to 10 µU/mL.30

Insulin Treatment
Two days after diabetes induction, some DIA rats were treated subcutaneously with a long-acting insulin (Ultralente Beef and Pork; Connaught Novo Nordisk) once daily. The insulin injection was given at 10 AM with the dose ({approx}17 to 19 U/kg) adjusted daily to achieve normoglycemia. Treatment was continued for 12 days, after which the animals were killed. The last insulin dose was given <24 hours before death. In some experiments, 2-week DIA rats were also treated with a rapid-acting insulin (Iletin Regular Beef and Pork, 15 U IV; Eli Lilly Canada) 90 minutes before the rats were killed, and cardiac heparin-releasable and cellular LPL activities were determined.

Materials
Joklik minimum essential medium was obtained from Gibco Canada. [3H]Triolein was purchased from Amersham Canada. Heparin sodium injection (Hepalean; 1000 USP U/mL) was obtained from Organon Teknika. Collagenase (CLS 2, 325 U/mg) was purchased from Worthington Biochemical Corp. All other chemicals were obtained from Sigma Chemical.

Statistical Analysis
All data are reported as mean±SEM unless otherwise stated. One-way analysis of variance followed by the Newman-Keul's test or the unpaired Student's t test was used to determine differences between group mean values. Changes in heparin-releasable and cellular LPL activity in response to heparin over time were analyzed with multivariate analysis of variance followed by the Newman-Keul's test using the Number Cruncher Statistical System. The level of statistical significance was set at P<.05.


*    Results
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 *Results
down arrowDiscussion
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General Characteristics
Induction of diabetes in SHR resulted in glycosuria (>2%) throughout the study period. We previously demonstrated that blood pressure in both CON and DIA at 9 to 10 weeks of age increased progressively over 2 weeks relative to WisC and WisD groups.25 Moreover, diabetes per se had no effect on blood pressure in either rat strain.25 Other characteristics normally associated with diabetes, such as polydipsia (TableDown) and hyperphagia (Fig 1Down), were observed in DIA rats. Despite a greater food intake, DIA rats demonstrated a significantly reduced body weight gain over 2 weeks (Fig 1Down). Plasma insulin levels were reduced at 2 weeks after diabetes induction, accompanied by marked hyperglycemia (TableDown). TG levels were elevated in DIA rats, but there was no change in plasma cholesterol levels (TableDown).


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  		Table 1. Effect of Chronic (2-Week) Insulin Treatment on Characteristics of SHR Made Diabetic With 55 mg/kg STZ



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  		Figure 1. Effect of diabetes on food intake (left) and body weight gain (right) in SHR. Food intake was measured daily, but the figure demonstrates the value obtained before death. Insulin treatment consisted of a long-acting Ultralente insulin. Results are mean±SEM of 6 to 8 rats in each group. DIA+I indicates DIA plus insulin. +Significantly different from all other groups, *significantly different from diabetics (P<.05).

Coronary Endothelial LPL
Retrograde perfusion of whole hearts from CON and 2-week DIA rats with heparin released LPL activity into the coronary perfusate (Fig 2Down). The heparin-induced LPL discharge in CON rats was rapid, and peak activity, suggested to represent LPL that is located at or near the endothelial cell surface, was observed within 0.5 to 1 minute. Relative to WisC, CON rat hearts demonstrated a decline in peak heparin-releasable LPL activity (Fig 2Down, right). As previously reported, induction of STZ diabetes caused an increase in plasma glucose levels (WisC, 6.4±0.3; WisD, 20.2±0.65). Moreover, peak LPL activity in WisD rats was almost twice as much as that in WisC (Fig 2Down, left). Similar to WisD rats, 2-week DIA rat hearts had a higher peak heparin-releasable LPL activity relative to CON (Fig 2Down, right).



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  		Figure 2. Heparin-releasable LPL activity in perfused hearts from Wistar (left) and SHR (right) control and diabetic rats 2 weeks after diabetes induction. Rat were perfused with Joklik minimum essential medium as described in the text. At the time indicated by the arrow, heparin (5 U/mL) was added to the buffer, and LPL activity was measured in the coronary perfusate, which was collected for 10 seconds at the indicated times. Results are mean±SEM of 6 to 8 rats in each group. #Significantly different from control (P<.05).

Myocyte LPL
To determine whether the increase in cardiac heparin-releasable LPL in DIA is a consequence of augmented production, myocytes were isolated at 2 weeks after diabetes induction, and LPL activity in cell sonicates was measured. There was no difference in myocyte viability (percent of live cells) or yield (total number of cellsx106) between CON and DIA rats at 2 weeks after the induction of diabetes (TableUp). DIA rats had a reduced cellular LPL activity compared with CON (Fig 3BDown). To examine whether the diminished LPL activity in D55 rat myocytes was accompanied by a parallel reduction in the secretion of LPL, myocytes were incubated in the presence of heparin to measure both surface-bound and secreted LPL. There was a significant reduction in heparin-induced release of LPL from cardiac myocytes in DIA rats.



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  		Figure 3. LPL activity in cardiac myocytes from CON and DIA. Myocytes were prepared as described in the text. LPL activity in cell homogenates was determined at time zero by removing a sample of cell suspension, followed by centrifugation, sonication of the cell pellet, and determination of intracellular LPL activity (bottom). Heparin (5 U/mL) was then added to the incubation medium at the time indicated by the arrow, and the release of surface-bound LPL activity into the medium was determined at the indicated times of incubation. Results are mean±SEM of 6 to 8 rats in each group. #Significantly different from control (P<.05).

Effects of Insulin Treatment
Chronic insulin treatment of DIA rats for 2 weeks resulted in an increase in body weight and a normalization of food and fluid intake and plasma TG and glucose levels (Figs 1Up and 4Down, inset, and TableUp). In addition, 2-week insulin treatment attenuated the rise in heparin-releasable (Fig 4ADown) and prevented the reduction in myocyte secreted LPL activities (Fig 4BDown). Intravenous injection of DIA rats with rapid-acting insulin reduced hyperglycemia within 90 minutes (Fig 5Down, inset). Acute insulin treatment also reduced peak heparin-releasable LPL activity to control levels (Fig 5ADown). However, cell-associated LPL activity remained low in acute insulin-treated DIA rats (Fig 5BDown).



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  		Figure 4. Effect of chronic insulin treatment on heparin-releasable LPL activity in perfused hearts (A) and cell surface of cardiac myocytes (B) from SHR. Insulin treatment ({approx}17 to 19 U/kg per day) with a long-acting Ultralente Insulin was initiated 2 days after diabetes induction and continued for 12 days. Results are mean±SEM of 6 to 8 rats in each group. +Significantly different from all other groups, #significantly different from control, *significantly different from diabetics (P<.05).



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  		Figure 5. Effect of acute insulin (15 U) treatment on heparin-releasable LPL activity in perfused hearts (A) and cardiac myocytes (B) from SHR. A rapid-acting Regular Insulin was injected intravenously into diabetic rats 2 weeks after diabetes induction, and the animals were killed after 90 minutes. Right, Plasma glucose levels measured in tail vein blood samples obtained at various times after insulin injection. Results are mean±SEM of 6 to 8 rats in each group. +Significantly different from all other groups (P<.05).

Dose-Response Effects of STZ
Fig 6Down shows the peak heparin-releasable LPL activity in SHR at 2 weeks after the administration of varying doses (25 to 55 mg/kg) of STZ. We previously reported that SHR injected with 25 and 35 mg/kg STZ do not show significant glucose intolerance or hyperglycemia in the fed state.25 In these rats, peak heparin-releasable LPL activity was unchanged compared with control animals. In contrast, SHR administered 45 and 55 mg/kg STZ demonstrated hyperglycemia in the fed and fasted states.25 In these animals, peak heparin-releasable LPL activity was equally augmented compared with control animals.



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  		Figure 6. Dose-response effect of STZ on heparin-releasable LPL activity in isolated hearts from SHR. Hearts from CON and 2-week DIA were removed and perfused with Joklik buffer containing heparin (% U/mL) for 10 minutes. Results are mean±SEM of 6 to 8 rats in each group.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In the heart, endothelial LPL at the capillary lumen hydrolyzes lipoprotein TG, providing FFA substrate to cardiac muscle. The important role of LPL in the hydrolysis of circulating TG is confirmed by the loss of this function when endothelial-bound enzyme is removed by heparin perfusion of the heart.31 This rapid, heparin-releasable LPL fraction is more sensitive to altered physiological (eg, feeding, fasting) and pathological (eg, diabetes, hyperthyroidsim) states than is total cellular activity. During hypertension, LPL activity is reduced in skeletal muscle and adipose tissue in human patients32 33 and in Dahl salt-sensitive rats.34 We recently reported that the heparin-releasable LPL fraction in SHR hearts is reduced relative to that of WKY rats.22 Because nifedipine and CGS-21680, vasodilators with divergent mechanisms of action, normalized enzyme release, we concluded that flow through coronary blood vessels might influence LPL activity.22 In this regard, exercise training significantly reduces LPL activity in muscle and adipose tissue as a result of increased blood flow.35 36 In the present study, a similar reduction in heparin-releasable LPL was observed in SHR hearts relative to Wistar controls.

Varying levels of cardiac LPL protein or activity have been reported in STZ-diabetic rats.14 15 16 Recently, we demonstrated an enhanced heparin-releasable LPL activity in diabetic rat hearts, even when cellular LPL activity was simultaneously reduced.17 This enhanced activity was matched by an increase in LPL protein as measured with ELISA (N. Sambandam, E. St. Pierre, M.A. Abrahani, M.C. Cam, and B. Rodrigues, unpublished observations). In this study, an elevated peak heparin-releasable LPL activity was also observed in SHR-diabetic hearts. Although the endothelial enzyme is largely derived from cardiac myocytes that synthesize and secrete LPL,37 38 the elevated heparin-releasable LPL activity in SHR diabetic rat hearts was associated with a reduced cellular pool. Previously, a decline in cellular LPL catalytic activity was suggested to result from post-transcriptional/translational mechanisms leading to an accumulation of inactive LPL protein in diabetic cardiomyocytes.29

In perfused guinea pig hearts, LPL can move from parenchymal cells to the endothelial surface within 30 minutes.39 In mouse hearts, this movement involves "jumping" of LPL between cell surface–associated heparan sulfate and other polyanions.38 Thus, the enhanced heparin-releasable LPL pool in SHR-diabetic hearts could involve an accelerated vectorial transfer of LPL from myocytes to the capillary lumen. It should be noted that endothelial LPL can also be derived from circulating LPL,40 which was demonstrated by the uptake of injected radiolabeled LPL into the rat heart.41 Whether an increased LPL uptake from the plasma occurs in the SHR diabetic rat heart has yet to be determined. However, when the heparin-releasable LPL pool was allowed to recover for 1 hour after washout of the enzyme in vitro, Wistar diabetic rats continued to demonstrate a higher peak LPL activity after a second heparin perfusion.17 Finally, endothelial cells can internalize LPL; it is either degraded or released back into the medium. Interestingly, at an acidic pH, LPL remains bound to proteoglycans, facilitating the recycling of internalized LPL molecules without degradation.42 43 Because diabetes leads to an impairment of the Na+/H+ exchanger,44 45 it is possible that changes in pH in endothelial cells may preserve LPL, which may in turn fulfill the demand for an enhanced FFA supply to the DIA heart.

Insulin administered to severely diabetic rats reverses the depleted myocyte LPL activity by enhancing protein synthesis.13 In the present study, insulin treatment for 2 weeks simultaneously prevented the lowering of cellular LPL activity and the increase in heparin-releasable LPL activity. Interestingly, although 90-minute insulin treatment of 1-week diabetic rats failed to influence the cellular LPL store, peak heparin-releasable LPL activity was reduced to control levels, suggesting insulin may displace LPL from its binding sites in vivo. In this regard, because HSPGs associate with vascular endothelial cells via their core proteins or a glycosylphosphatidylinositol linkage,46 cleavage of the glycosylphosphatidylinositol anchor by insulin-sensitive phospholipases could release HSPG and, hence, LPL.47 48

The augmented heparin-releasable LPL activity in the DIA heart could have a number of deleterious effects. An increased enzyme activity could lead to an accelerated hydrolysis of lipoprotein TG, providing excess FFA to the heart. In transgenic mouse lines overexpressing human LPL in skeletal and cardiac muscle, elevated FFA uptake induced a severe myopathy, characterized by muscle fiber degeneration, and extensive proliferation of mitochondria and peroxisomes.9 Enhanced FFA metabolism in the heart can inhibit glucose oxidation and increase oxygen requirement.49 50 51 In this regard, it was previously demonstrated that diabetes induces a greater enhancement in FFA utilization in the SHR compared with Wistar rats.52 Lipolytic products have also been shown to enhance endothelial permeability.53 54 Interestingly, DIA demonstrate greater vascular permeability than nondiabetic SHR, or diabetic normotensive rats.55 Finally, LPL has been implicated as a receptor ligand for lipoprotein removal,56 thus directly enhancing lipoprotein movement through the artery wall,57 58 which in turn can alter Ca2+ homeostasis in cardiomyoctes.59

In conclusion, these results demonstrate that the diabetes-induced augmentation of heparin-releasable LPL activity counteracts the reduction in enzyme activity associated with hypertension. We believe that an abnormally high cardiac LPL activity could increase FFA supply, which could lead to metabolic and morphological changes associated with the severe cardiomyopathy observed in this model.


*    Selected Abbreviations and Acronyms
 
LPL = lipoprotein lipase
FFA = free fatty acid
SHR = spontaneously hypertensive rat(s)
TG = triglycerides
HSPG = heparan sulfate proteoglycan
STZ = streptozotocin
CON = SHR control
DIA = SHR diabetic
WisC = Wistar control
WisD = Wistar diabetic
WKY = Wistar-Kyoto


*    Acknowledgments
 
This work was supported by an operating grant from the Heart and Stroke Foundation of BC and Yukon. The financial support of the Canadian Diabetes Association (Scholarship to Dr Rodrigues), Heart and Stroke Foundation of BC and Yukon (Research Traineeship to N. Sambandam), and the Medical Research Council of Canada (Summer Studentship to G. Shepherd) is gratefully acknowledged.


*    Footnotes
 
Reprint requests to Dr B. Rodrigues, Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, BC, Canada V6T 1Z3.

Received August 20, 1997; first decision September 11, 1997; accepted October 21, 1997.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Rodrigues B, Xiang H, McNeill JH. Effect of L-carnitine treatment on lipid metabolism and cardiac performance in chronically diabetic rats. Diabetes. 1988;37:1358–1364.[Abstract]
  2. Rodrigues B, Cam MC, McNeill JH. Myocardial substrate metabolism: implications for diabetic cardiomyopathy. J Mol Cell Cardiol. 1995;27:169–179.[Medline] [Order article via Infotrieve]
  3. Garvey WT, Hardin D, Juhaszova M, Dominguez JH. Effects of diabetes on myocardial glucose transport system in rats: implications for diabetic cardiomyopathy. Am J Physiol. 1993;264:837–844.
  4. Chatham JC, Forder JR. Relationship between cardiac function and substrate oxidation in heart of diabetic rats. Am J Physiol. 1997;42:52–58.
  5. Tahiliani AG. Myocardial fatty acid metabolism. In: Fozzard HA, ed. The Heart and Cardiovascular System, 2nd ed. New York, NY: Raven Press; 1992:1599–1620.
  6. Eckel RH. Lipoprotein lipase: a multifunctional enzyme relevant to common metabolic diseases. N Engl J Med. 1989;320:1060–1068.[Abstract]
  7. O'Brien KD, Ferguson M, Gordon D, Deeb SS, Chait A. Lipoprotein lipase is produced by cardiac myocytes rather than interstitial cells in human myocardium. Arterioscler Thromb. 1994;14:1445–1451.[Abstract/Free Full Text]
  8. Hamosh M, Hamosh P. Lipoprotein lipase: its physiological and clinical significance. Mol Aspects Med. 1983;6:199–289.[Medline] [Order article via Infotrieve]
  9. Levak-Frank S, Radner H, Walsh A, Stollberger R, Knipping G, Hoefler G, Sattler W, Weinstock PH, Breslow JL, Zechner R. Muscle-specific overexpression of lipoprotein lipase causes a severe myopathy characterized by proliferation of mitochondria and peroxisomes in transgenic mice. J Clin Invest. 1995;96:976–986.
  10. Murthy VK, Bauman MD, Shipp JC. Regulation of triacylglycerol lipolysis in the perfused hearts of normal and diabetic rats. Diabetes. 1983;32:718–722.[Medline] [Order article via Infotrieve]
  11. Kenno KA, Severson DL. Lipolysis in isolated myocardial cells from diabetic rat hearts. Am J Physiol. 1985;249:1024–1030.
  12. Chattopadhayay J, Thompson EW, Schmid HHO. Elevated levels of nonesterified fatty acids in the myocardium of alloxan diabetic rats. Lipids. 1990;25:307–310.[Medline] [Order article via Infotrieve]
  13. Rodrigues B, Braun JEA, Spooner M, Severson DL. Regulation of lipoprotein lipase activity in cardiac myocytes from control and diabetic rats by plasma lipids. Can J Physiol Pharmacol. 1992;70:1271–1279.[Medline] [Order article via Infotrieve]
  14. Veeraraghavan K, Murthy K, Bauman MD, Shipp J. Regulation of triacylglycerol lipolysis in the perfused hearts of normal and diabetic rats. Diabetes. 1983;32:718–722.
  15. Nomura T, Hagino H, Gotoh M, Iguchi A, Sakamoto N. The effects of streptozotocin diabetes on tissue specific lipase activities in the rat. Lipids. 1984;19:594–599.[Medline] [Order article via Infotrieve]
  16. O'Looney P, Maten MV, Vahouny GV. Insulin-mediated modifications of myocardial lipoprotein lipase and lipoprotein metabolism. J Biol Chem. 1983;258:12994–13001.[Abstract/Free Full Text]
  17. Rodrigues B, Cam MC, Jian K, Lim F, Sambandam N, Shepherd G. Differential effects of streptozotocin-diabetes on cardiac lipoprotein lipase activity. Diabetes. 1997;46:1346–1353.[Abstract]
  18. Chukwuma CJ. Comments on the clinical impact of hypertension in type 1 diabetes. J Diab Comp. 1992;6:197–202.[Medline] [Order article via Infotrieve]
  19. Johnston CI, Cooper ME, Nicholls GM. Meeting report of the International Society of Hypertension Conference on Hypertension and Diabetes. J Hypertens. 1992;10:393–397.[Medline] [Order article via Infotrieve]
  20. Factor SM, Minase T, Sonnenblick EH. Clinical and morphological features of human hypertensive-diabetic cardiomyopathy. Am Heart J. 1980;99:446–458.[Medline] [Order article via Infotrieve]
  21. Rodrigues B, McNeill JH. Cardiac function in spontaneously hypertensive diabetic rats. Am J Physiol. 1986;251:571–580.
  22. Sambandam N, Chen X, Cam MC, Rodrigues B. Cardiac lipoprotein lipase in the spontaneously hypertensive rat. Cardiovasc Res. 1997;33:460–468.[Abstract/Free Full Text]
  23. Christe ME, Rodgers RL. Altered glucose and fatty acid oxidation in hearts of the spontaneously hypertensive rat. J Mol Cell Cardiol. 1994;26:1371–1375.[Medline] [Order article via Infotrieve]
  24. Rodrigues B, Cam MC, Kong J, Goyal RK, McNeill JH. Strain differences in susceptibility to streptozotocin-induced diabetes: effects on hypertriglyceridemia and cardiomyopathy. Cardiovasc Res. 1997;33:460–468.
  25. Jian K, Fok E, Cam MC, Sambandam N, Yao J, Rodrigues B. Susceptibility of spontaneously hypertensive rats to the diabetogenic effects of streptozotocin. Can J Physiol Pharmacol. 1996;74:1215–1221.[Medline] [Order article via Infotrieve]
  26. Rodrigues B, Spooner M, Severson DL. Free fatty acids do not release lipoprotein lipase from isolated cardiac myocytes or perfused hearts. Am J Physiol. 1992;262:216–223.
  27. Olivecrona T, Bengtsson G, Marklund SE, Lindahl U, Hook M. Heparin-lipoprotein lipase interactions. Fed Proc. 1977;1977:36:6–65.
  28. Ramirez I, Kryski A, Jr, Ben-Zeev O, Schotz MC, Severson DL. Characterization of triacylglycerol hydrolase activities in isolated myocardial cells from rat heart. Biochem J. 1985;23:229–236.
  29. Carroll R, Liu L, Severson DL. Post-transcriptional mechanisms are responsible for the reduction in lipoprotein lipase activity in cardiomyocytes from diabetic rat hearts. Biochem J. 1995;310:67–72.
  30. Cam MC, McNeill JH. A sensitive radioimmunoassay optimized for reproducible measurement of rat plasma insulin. J Pharmacol Toxicol Methods. 1996;35:111–119.[Medline] [Order article via Infotrieve]
  31. Borensztajn J, Robinson DS. Release of clearing factor lipase by the perfused heart. J Lipid Res. 1970;6:222–227.
  32. Pollare T, Vessby B, Lithell H. Lipoprotein lipase activity in skeletal muscle is related to insulin resistance. Atheroscler Thromb. 1991;11:1192–1203.[Abstract/Free Full Text]
  33. Marotta T, Ferrara LA, DiMarino L, Mancini M, Annuzzi G, Russo O, D'Orta G, Lucarelli C, Rossi F. Factors affecting lipoprotein lipase in hypertensive patients. Metabolism. 1995;44:712–718.[Medline] [Order article via Infotrieve]
  34. Mondon CE, Plato PA, Dall'Aglio E, Sztalryd C, Reaven GM. Mechanism of hypertriglyceridemia in Dahl rat. Hypertension. 1993;21:373–379.[Abstract/Free Full Text]
  35. Osci LB, Tsika RW, Essig DA. Exercise training has a heparin-like effect on lipoprotein lipase activity in muscle. Can J Physiol Pharmacol. 1992;70:905–909.[Medline] [Order article via Infotrieve]
  36. Pond CM, Mattacks CA, Sadler D. The effects of exercise and feeding on the activity of lipoprotein lipase in nine different adipose depots of guinea pigs. Int J Biochem. 1992;24:1825–1831.[Medline] [Order article via Infotrieve]
  37. Camps L, Reina M, Lobera M, Vilard S, Olivecrona T. Lipoprotein lipase: cellular origin and functional distribution. Am J Physiol. 1990;258:673–681.
  38. Blanchette-Mackie EJ, Masuno H, Dwer NK, Olivecrona T, Scow RO. Lipoprotein lipase in myocytes and capillary endothelium of heart: immunocytochemical study. Am J Physiol. 1989;256:818–828.
  39. Liu G, Olivecrona T. Pulse-chase study on lipoprotein lipase in perfused guinea pig heart. Am J Physiol. 1991;261:2044–2050.
  40. Olivecrona T, Bengtsson-Olivecrona G. Lipoprotein lipase and hepatic lipase. Curr Opin Lipidol. 1993;4:187–196.
  41. Hultin M, Bengtsson-Olivecrona G, Olivecrona T. Release of lipoprotein lipase to plasma by triacylglycerol emulsions: comparison to the effect of heparin. Biochim Biophys Acta. 1992;1125:97–103.[Medline] [Order article via Infotrieve]
  42. Saxena U, Klein MG, Goldberg IJ. Metabolism of endothelial cell-bound lipoprotein lipase: evidence for heparin sulfate proteoglycan-mediated internalization and recycling. J Biol Chem. 1990;265:12880–12886.[Abstract/Free Full Text]
  43. Saxena U, Klein MG, Goldberg IJ. Transport of lipoprotein lipase across endothelial cells. Proc Natl Acad Sci U S A. 1991;88:2254–2258.[Abstract/Free Full Text]
  44. Pierce GN, Ramjiawan B, Dhalla NS, Ferrari R. Na+-H+ exchange in cardiac sarcolemmal vesicles isolated from diabetic rats. Am J Physiol. 1990;258:255–261.
  45. Khandoudi N, Bernard M, Cozzone P, Feuvray D. Intracellular pH and the role of Na+/H+ exchange during ischemia and reperfusion of normal and diabetic rat hearts. Cardiovasc Res. 1990;24:873–878.[Medline] [Order article via Infotrieve]
  46. Yanagishita M, Hascall VC. Cell surface heparan sulfate proteoglycans. J Biol Chem. 1992;267:9451–9454.[Free Full Text]
  47. Spooner PM, Chernick SS, Garrison MM, Scow RO. Insulin regulation of lipoprotein lipase activity and release in 3T3–L1 adipocytes. J Biol Chem. 1979;254:10021–10029.[Free Full Text]
  48. Chan BL, Lisanti MP, Rodriguez-Boulan E, Saltiel AR. Insulin stimulated release of lipoprotein lipase by metabolism of its phosphatidylinositol anchor. Science. 1988;241:1670–1672.[Abstract/Free Full Text]
  49. Randle PJ, Hales CN, Garland PB, Newsholme EA. The glucose fatty acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet. 1963;1:785–789.[Medline] [Order article via Infotrieve]
  50. Rodrigues B, McNeill JH. The diabetic heart: metabolic causes for the development of a cardiomyopathy. Cardiovasc Res. 1992;26:913–922.[Medline] [Order article via Infotrieve]
  51. Saddik M, Lopaschuk GD. Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts. J Biol Chem. 1991;266:8162–8170.[Abstract/Free Full Text]
  52. Christe ME, Rodgers RL. Cardiac glucose and fatty acid oxidation in the streptozotocin-induced diabetic spontaneously hypertensive rat. Hypertension. 1995;25:235–241.[Abstract/Free Full Text]
  53. Hennig B, Shasby DM, Fulton AB, Spector AA. Exposure to free fatty acids increases the transfer of albumin across cultured endothelial monolayers. Arteriosclerosis. 1984;4:489–497.[Abstract/Free Full Text]
  54. Rutledge JC, Woo MM, Rezai AA, Curtiss LK, Goldberg IJ. Lipoprotein lipase increases lipoprotein binding to the artery wall and increases endothelial layer permeability by formation of lipolysis products. Circ Res. 1997;80:819–828.[Abstract/Free Full Text]
  55. Kubota I, Fukuhara T, Kinoshita M. Permeability of small coronary arteries and myocardial injury in hypertensive diabetic rats. Int J Cardiol. 1990;29:349–355.[Medline] [Order article via Infotrieve]
  56. Goldberg IJ. Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis. J Lipid Res. 1996;37:693–707.[Abstract]
  57. Rutledge JC, Goldberg IJ. Lipoprotein lipase (LpL) affects low density lipoprotein (LDL) flux through vascular tissue: evidence that LpL increases LDL accumulation in vascular tissue. J Lipid Res. 1994;35:1152–1160.[Abstract]
  58. Fernandez-Borja M, Bellido D, Vilella E, Olivecrona G, Vilaro S. Lipoprotein lipase-mediated uptake of lipoprotein in human fibroblasts: evidence for an LDL receptor-independent internalization pathway. J Lipid Res. 1996;37:464–481.[Abstract]
  59. Liu K, Pierce GN. The effects of low density lipoprotein on Ca2+ transients in isolated rabbit cardiomyocytes. J Biol Chem. 1993;268:3767–3775.[Abstract/Free Full Text]



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