Treatment of type 2 diabetic db/db mice with a novel PPAR{gamma} agonist improves cardiac metabolism but not contractile function

Andrew N. Carley,1 Lisa M. Semeniuk,1 Yakhin Shimoni,2 Ellen Aasum,3 Terje S. Larsen,3 Joel P. Berger,4 and David L. Severson1

1Department of Pharmacology and Therapeutics and 2Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada; 3Department of Medical Physiology, University of Tromsø, N-9037 Tromso, Norway; and 4Department of Metabolic Disorders, Merck Research Laboratories, Rahway, New Jersey 07065

Submitted 17 July 2003 ; accepted in final form 26 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hearts from insulin-resistant type 2 diabetic db/db mice exhibit features of a diabetic cardiomyopathy with altered metabolism of exogenous substrates and reduced contractile performance. Therefore, the effect of chronic oral administration of 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid (COOH), a novel ligand for peroxisome proliferator-activated receptor-{gamma} that produces insulin sensitization, to db/db mice (30 mg/kg for 6 wk) on cardiac function was assessed. COOH treatment reduced blood glucose from 27 mM in untreated db/db mice to a normal level of 10 mM. Insulin-stimulated glucose uptake was enhanced in cardiomyocytes from COOH-treated db/db hearts. Working perfused hearts from COOH-treated db/db mice demonstrated metabolic changes with enhanced glucose oxidation and decreased palmitate oxidation. However, COOH treatment did not improve contractile performance assessed with ex vivo perfused hearts and in vivo by echocardiography. The reduced outward K+ currents in diabetic cardiomyocytes were still attenuated after COOH. Metabolic changes in COOH-treated db/db hearts are most likely indirect, secondary to changes in supply of exogenous substrates in vivo and insulin sensitization.

diabetic cardiomyopathy; cardiac metabolism


PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPARs) are ligand-activated transcription factors within the nuclear receptor superfamily (11). PPARs heterodimerize with retinoid X receptors and bind to specific DNA response elements in the promoter region of target genes.

Three PPAR isoforms ({alpha}, {beta}/{delta}, {gamma}) can be distinguished by expression patterns and activation by relatively selective ligands. PPAR{alpha} and PPAR{beta}/{delta} are widely expressed (13, 28). PPAR{alpha} is activated by hypolipidemic fibrate drugs (23). PPAR{alpha} regulates a wide variety of target genes involved in catabolism of lipids (5). PPAR{alpha} is highly expressed in the heart; Barger and Kelly (5) have suggested that cardiac PPAR{alpha} plays a role in metabolic remodeling. In general, cardiac PPAR{alpha} expression correlates with capacity for fatty acid (FA) oxidation (26). Studies with cultured cardiomyocytes incubated with selective PPAR{alpha} ligands have revealed specific target genes that regulate FA uptake and intracellular binding and cellular FA oxidative metabolism. As a consequence, PPAR{alpha} activation by selective ligands enhanced rates of FA oxidation (14, 24, 47). Because FA are endogenous ligands for cardiac PPAR{alpha}, oxidative capacity of the heart can be regulated according to FA delivery. Interestingly, activation of cardiac PPAR{beta}/{delta} also stimulated expression of FA-metabolizing genes (24).

PPAR{gamma} expression is more restrictive (11). In adipose tissue, PPAR{gamma} promotes differentiation and lipid storage (37). PPAR{gamma} agonists augment insulin sensitivity (35, 48); consequently, thiazolidinediones that activate PPAR{gamma} are used to treat insulin resistance in type 2 diabetes.

In contrast to PPAR{alpha} and {beta}/{delta}, PPAR{gamma} expression in cardiomyocytes is extremely low (12, 13). Consequently, a PPAR{gamma} ligand had no direct effect on FA oxidation in cultured cardiomyocytes (24). Therefore, cardiac effects of PPAR{gamma} administered in vivo to diabetic animals will presumably be indirect, secondary to changes in plasma glucose and lipid concentrations and insulin sensitization.

Diabetic db/db mice provide an animal model of obese, insulin-resistant type 2 diabetes (20, 31). Isolated perfused working hearts from db/db mice exhibit features of a diabetic cardiomyopathy (39); cardiac metabolism is altered, with reduced glucose utilization (glycolysis and glucose oxidation), enhanced FA oxidation, and decreased contractile performance (1, 2, 6, 38). Cardiomyocytes from db/db hearts also exhibit electrophysiological alterations, with attenuated outward K+ currents (41).

Administration of PPAR{gamma} agonists (thiazolidinedione and novel nonthiazolidinedione ligands) to db/db mice produced insulin sensitization, resulting in reductions in elevated plasma glucose and lipid concentrations (9, 10). Therefore, the aim of this study was to determine whether chronic treatment of db/db mice with a novel nonthiazolidinedione PPAR{gamma} agonist, 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid (COOH) (29), could normalize the metabolism of glucose and a FA (palmitate) and improve the contractile performance of isolated perfused working hearts.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
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Experimental animals. Male C57BL/KsOlaHsd-leprdb/leprdb (db/db) and their lean, nondiabetic heterozygote littermates (db/+) were purchased from Harlan Laboratories (Indianapolis, IN). Mice arrived at 5-6 wk of age and were allowed to acclimatize for 1 wk. Animals were given ad libitum access to food and water and housed under a 12:12-h light-dark cycle. All experiments were approved by the University of Calgary Animal Welfare Committee.

Treatment protocol. After the 1-wk acclimatization period, db/+ and db/db mice were randomly divided into four groups. A group each of db/+ and db/db mice received powdered chow with and without the PPAR{gamma} ligand COOH as a food admixture. COOH is an indoleacetic acid-derived PPAR{gamma} agonist (Fig. 1) from Merck (transactivation EC50s on PPAR{gamma}/{delta}/{alpha} = 180/>3,000/>3,000 nM). The drug was formulated into powdered chow (Prolab RMH 2500/5P14; PMI International, Brentwood, MO) to attain a daily dosage of 30 mg/kg body wt. Food intake was monitored daily, and body weight was monitored weekly. Content of drug in the powdered chow was adjusted as needed to maintain the desired dosing and ranged from 0.2 to 0.3 mg/g chow. Untreated db/+ and db/db groups received regular mouse chow. Animals were treated for 6 wk (to 12 wk of age), at which time they were subjected to echocardiographic analysis of cardiac function and then killed for ex vivo heart perfusions and isolation of cardiomyocytes. The treatment period commenced at 6 wk of age, when db/db mice exhibit normal cardiac function, and was concluded at 12 wk, when altered metabolism and contractile dysfunction are evident in hearts from untreated db/db mice (2, 38).



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Fig. 1. Structure of 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid (COOH), a nonthiazolidinedione peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma})-selective ligand.

 

Glucose concentrations were measured routinely by the tail prick method (ONETOUCH glucose meter, Lifescan) with mice that had been heparinized before heart perfusions (see Isolated heart perfusions). Because heparin treatment produces artifactual changes in plasma lipids because of the release of lipoprotein lipase into the circulation, plasma nonesterified FA and triacylglycerol (TG) concentrations were measured in plasma samples from nonheparinized mice by means of assay kits from Wako Pure Chemical Industries and Sigma, respectively.

Assessment of cardiac function by echocardiography. Echocardiograms (M-mode measurements) to assess systolic function were obtained from conscious mice, as described by Semeniuk et al. (38). Septal wall thickness (SWT), posterior wall thickness (PWT), left ventricular (LV) internal dimensions in systole (LVIDs) and diastole (LVIDd) were determined from LV M-mode scans by use of a Hewlett-Packard Sonus 5500 ultrasound machine with a 15-MHz linear transducer. Values for heart rate (HR) were obtained from Doppler measurements of LV outflow tract velocities.

Fractional shortening (FS) and the velocity of circumferential fiber shortening (Vcf) were calculated as indexes of systolic function: FS (%) = [(LVIDd - LVIDs)/LVIDd] x 100; Vcf = FS/ET. LV mass was calculated from the following equation (38): LV mass (mg) = [(LVIDd + SWT + PWT)3 + LVIDd3] x 1.055, where 1.055 is the density of the myocardium.

Isolated heart perfusions. Mice received 100 U of heparin via intraperitoneal injection 20 min before intraperitoneal injection of 10 mg of pentobarbital sodium. Hearts were mounted on a perfusion apparatus and placed in working mode by the protocol of Belke and colleagues (7, 8). Briefly, this involved cannulation of the left atrium to control preload and cannulation of the aorta to set afterload in working (LV ejecting) mode. Hearts were perfused with a modified Krebs-Henseleit bicarbonate buffer consisting of (in mM): 118.5 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 0.5 EDTA, containing 0.4 mM [9,10-3H]palmitate (specific activity = 2.2 x 109 dpm/mmol) bound to 3% BSA and 11 mM [U-14C]glucose (specific activity = 9.1 x 106 dpm/mmol). The total FA concentration in the perfusate was ~0.7 mM because the BSA used was not essentially FA free (endogenous FA content was 0.25-0.3 mM). Although oleate is the predominant FA in rodent circulation, palmitate was utilized as the exogenous substrate to be consistent with previous studies that measured palmitate oxidation by perfused mouse hearts (1, 2, 7, 8). Evans and Wang (21) have reported that oxidation rates for palmitate and oleate by perfused rat hearts were identical. The perfusate was continually gassed with 95% O2-5% CO2.

Hearts were perfused for 40 min (preload pressure of 15 mmHg; afterload pressure of 50 mmHg) with functional measurements and the withdrawal of perfusate samples (2.5 ml) for metabolic analysis occurring every 10 min. Coronary and aortic flows were determined through the use of graduated cylinders placed within the working heart apparatus; heart pressures were measured via a pressure transducer placed in the aortic afterload line (7) by use of CV Works (University of Calgary). The sum of aortic and coronary flows was used to determine cardiac output. The pressure signal was used for calculation of HR. Perfused hearts were allowed to beat spontaneously.

The oxidation of glucose and palmitate was measured simultaneously in each heart during working heart perfusions. Perfusates contained [U-14C]glucose and [9,10-3H]palmitate, as described by Belke and colleagues (7, 8). Trapping of 14CO2 in the perfusate was used to determine the rate of glucose oxidation; the release of 3H2O into the perfusate was used to determine the rate of palmitate oxidation. Steady-state rates of metabolism were determined by averaging the results from perfusate samples removed at the four time points (0-10, 10-20, 20-30, 30-40 min) for each heart perfusion. At the end of the perfusion protocol, the atria were removed, and hearts were frozen and stored at -80°C for the determination of ventricular dry weight, which was used to normalize metabolic and flow data to correct for small variations in heart size.

Preparation of isolated cardiomyocytes. Mouse ventricular cardiomyocytes were prepared essentially as described by Belke et al. (6) with some modifications. Mice (12 wk of age) were injected with 100 U of heparin intraperitoneally 30 min before administration of pentobarbital sodium (250 mg/kg ip). The heart was rapidly excised and arrested in ice-cold buffer A, consisting of (in mM): 120 NaCl, 5.4 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 5.6 glucose, 20 NaHCO3, 0.6 CaCl2, 10 2,3-butanedione monoxime, and 5 taurine, pH 7.5. The aorta was then cannulated, and the heart was retrogradely perfused at 37°C with buffer A gassed with 95% O2-5% CO2 for 4 min, followed by 10-14 min with buffer A containing 25 µM CaCl2 and 59 U/ml type II collagenase (Worthington). The coronary flow rate was set at 2.5 ml/min. The free wall of the right ventricle was then removed and digested at 37°C for 5-10 min longer in presence of collagenase, 50 µM CaCl2, and 1% (wt/vol) FA-free BSA. Dispersed myocytes were filtered through an 85-µm mesh, gently pelleted by centrifugation, and resuspended in buffer A containing 100 µM CaCl2 and 0.6% FA-free BSA. Freshly isolated cells were then used for electrophysiological studies.

For metabolic studies, calcium concentrations were increased gradually to 1.0 mM in subsequent washings. The final viability of cardiomyocytes (percentage of rod-shaped cells that excluded trypan blue) was 75-90%, with an overall yield of 1-1.5 x 106 cells/heart. To measure glucose uptake, the cells were washed once with MEM (Sigma) containing 5% fetal serum albumin, 100 U/ml penicillin, and 100 µg/ml streptomyocin and then plated in 35-mm laminin-coated tissue culture dishes. Studies were conducted 60 min after the platedown to allow viable cells to stick to laminin so that nonviable cells could be removed before measurement of glucose uptake.

Glucose uptake by isolated cardiomyocytes. Glucose uptake assays were performed as described by Belke et al. (6). Plated cardiomyocytes were washed twice with glucose-free DMEM (GIBCO) containing 0.2% FA-free BSA and 1.0 mM pyruvate (incubation buffer). Cells were then incubated in the absence and in the presence of insulin (10 nM) in 2.0 ml of incubation buffer for 40 min at 37°C, with 95% O2-5% CO2 gassing. Twenty microliters of a 2-deoxyglucose solution containing 130 µl of glucose-free DMEM, 15 µl of a 200 mM 2-deoxyglucose solution, and 5 µCi of 2-deoxy-[3H]glucose (ICN Biomedicals) were added to the dishes, and the incubation was continued for 20 min. The buffer was then aspirated, and the cells were washed twice with cold PBS. Cells were lysed in 300 µl of 1 M NaOH at 37°C for 20 min and then washed with 200 µl of NaOH. Thirty microliters of 12 M HCl were added to 400 µl of the lysate to normalize the pH, and radioactivity was measured. Protein assay was performed with 10 µl of the lysate by use of a Micro BCA Protein Assay Kit (Pierce Chemical). Glucose uptake is expressed as pico-moles per minute per milligram of protein.

K+ current recordings. Cardiomyocytes were placed in a 1-ml chamber on the stage of an inverted microscope and perfused with a solution containing (in mM): 150 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 5 HEPES, and 5.5 glucose, brought to pH 7.4 with NaOH. The solution was bubbled with 100% O2. Currents were recorded from single cells at 20-22°C by the whole cell voltage clamp method as described previously for cardiomyocytes from mouse heart (41). The pipette solution contained (in mM): 120 K-aspartate, 30 KCl, 5 Na2ATP, 5 HEPES, 1 MgCl2, 1 CaCl2, and 10 EGTA, brought to pH 7.2 with KOH. Because currents in mouse ventricular cells are large (several nA), it was essential to minimize series resistance artifacts. This was done by using low-resistance electrodes (2-4 M{Omega}) and by active electronic compensation (60-80%). Only well-polarized cells were used, with resting potentials of at least -65 mV.

Mouse ventricle has a variety of outward K+ currents (34). Peak outward current and the sustained current at the end of a 500-ms pulse in response to voltage steps ranging from -110 to +50 mV (holding potential of -80 mV) were measured (41). The peak outward current and the sustained current are composed of a mixture of several currents from different underlying channel proteins (25, 34). Peak and sustained currents were used for comparison between groups, since these currents determine the repolarization process of the cardiac action potential. Current densities (pA/pF) were obtained by dividing current magnitudes by cell capacitance (41).

Statistical analysis. Data are expressed as means ± SE. Differences in glucose uptake, cardiac function, and substrate metabolism were determined by ANOVA with a Student-Newman-Keuls test for pairwise comparisons. Differences between means were considered statistically significant when the P values were <0.05.


    RESULTS
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 METHODS
 RESULTS
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General features of experimental animals. At 12 wk of age, diabetic db/db mice were markedly obese and hyperglycemic relative to control db/+ heterozygotes (Table 1), as shown previously (1, 2, 7). Plasma TG and FA concentrations were not elevated in diabetic mice, in contrast to previous results showing modest elevations in serum TG and FA for male (10-14 wk) db/db mice (7). Differences in background strain may account for this difference in db/db phenotype. The present investigation used db/db mice from Harlan on a C57BL/KsOlaHsd background, whereas previous studies used Jackson db/db mice with a C57BL/KsJ background. The diabetic phenotype of db/db mice is markedly influenced by background strain (20).


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Table 1. General features of experimental animals

 

Preliminary experiments showed that oral administration of COOH to db/db mice for only 10 days produced a dose-dependent decline in elevated plasma glucose and TG concentrations to near-normal levels at a dose of 30 mg/kg. In a pilot study, COOH (30 mg/kg) was administered to db/db mice (9 wk of age) daily by oral gavage. After 3 wk, glucose oxidation was increased from 0.57 ± 0.10 µmol·min-1·g dry wt-1 in perfused hearts (n = 8) from untreated db/db mice to 1.45 ± 0.20 µmol·min-1·g dry wt-1 in COOH-treated db/db hearts (n = 10). Therefore, heart metabolism was responsive to COOH administered in vivo to db/db mice. To avoid the stress of daily oral gavage, the treatment protocol was modified. 1) COOH was administered as a food admixture (30 mg/kg); 2) treatment commenced at 6 wk of age, when db/db mice exhibit modest metabolic changes but normal contractile function (2, 38); and 3) COOH was also administered to nondiabetic, db/+ control mice. The total treatment period with COOH was 6 wk (from 6 to 12 wk of age).

COOH treatment of db/db mice reduced the elevated blood glucose concentrations in untreated mice (27.2 ± 1.1 mM) to normal (10.1 ± 0.6 mM) after 6 wk (Table 1) and produced a slight but significant decrease in body weight. Plasma FA in COOH-treated db/db mice was reduced significantly, but plasma TG was unchanged. Administration of COOH to control db/+ mice did not change body weight or blood glucose.

Insulin stimulation of glucose uptake. Insulin responsiveness was tested by measuring insulin-stimulated glucose uptake by isolated cardiomyocytes. Insulin (10 nM) produced a 9.3-fold stimulation of glucose uptake by cardiomyocytes from control db/+ hearts (Fig. 2). In cardiomyocytes from untreated db/db mice, insulin-stimulated glucose uptake was reduced significantly to only 2.8-fold. Treatment of db/db mice with COOH did not change basal glucose uptake, but insulin-stimulated glucose uptake was increased significantly (Fig. 2) to a value that was no longer different from insulin-stimulated glucose uptake in control cardiomyocytes. COOH treatment of control db/+ mice produced a slight but significant (P = 0.037) increase in insulin-stimulated glucose uptake. Therefore, COOH treatment did enhance cardiac insulin responsiveness.



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Fig. 2. Glucose uptake by cardiomyocytes from control db/+ (n = 5), COOH-treated db/+ (n = 4), db/db (n = 6), and COOH-treated db/db hearts (n = 5). Glucose uptake was measured in the absence and in the presence of insulin (10 nM). *Significantly different (P < 0.05) from control db/+ cardiomyocytes (+insulin); #significantly different from untreated db/+ and db/db cardiomyocytes (+insulin).

 

Metabolism of ex vivo working hearts. The metabolism of exogenous substrates (glucose and palmitate oxidation) by working perfused hearts is shown in Fig. 3. Glucose oxidation, determined by trapping 14CO2 in the perfusate, was reasonably linear during the 40-min normoxic perfusion time (Fig. 3A), permitting the calculation of steady-state rates of metabolism (Fig. 3C). As documented previously (1, 2, 7), glucose oxidation was reduced in db/db hearts (Fig. 3, A and C). By comparison, palmitate oxidation was markedly enhanced in db/db hearts (Fig. 3, B and D). COOH treatment of db/db mice produced an increase in glucose oxidation (Fig. 3C) and decreased palmitate oxidation (Fig. 3D) to values that were no longer significantly different from control untreated db/+ hearts. On the other hand, administration of COOH to control db/+ mice had no effect on the metabolism of exogenous substrates by perfused working hearts (Fig. 3).



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Fig. 3. Cumulative glucose (A) and palmitate oxidation (B) and steady-state rates of glucose (C) and palmitate oxidation (D) for control untreated db/+, COOH-treated db/+, untreated diabetic db/db, and COOH-treated db/db working hearts during 40 min of aerobic perfusion. Steady-state rates were calculated as means of the 4 measurements shown in A and B: 0-10, 10-20, 20-30, and 30-40 min. Values are means ± SE; no. of hearts in each group is indicated in parentheses. *Significantly different (P < 0.05) from control db/+ hearts; #significantly different from untreated db/db hearts.

 

Contractile function of ex vivo working hearts. Working perfused mouse hearts provide an experimental model to assess contractile performance under experimental conditions where preload and afterload are carefully controlled (7, 30). Perfused working hearts from db/db mice at 12 wk of age exhibited signs of contractile dysfunction. Cardiac output was reduced significantly, due entirely to a reduction in aortic flow since coronary flow was unchanged (Fig. 4). Treatment of db/db mice with COOH, despite producing normalization of glucose and palmitate oxidation (Fig. 3), did not improve either aortic flow or cardiac output (Fig. 4). Interestingly, COOH did produce a reduction in HR. Administration of COOH to control db/+ mice (n = 7) had no effect on any parameters of contractile function (HR, 322 ± 24 beats/min; cardiac output, 336 ± 26 ml·min-1·g-1).



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Fig. 4. Function of perfused working hearts from db/+ (n = 10), db/db (n = 14), and COOH-treated db/db mice (n = 13). A: aortic flow; B: coronary flow; C: cardiac output; D: heart rate. Results are means ± SE. *Significantly different from db/+; $P = 0.052 relative to db/+ hearts; #significantly different from untreated db/db hearts.

 

Cardiac function in vivo by echocardiography. Systolic function in conscious mice at 12 wk of age was assessed in vivo by echocardiography, essentially as described by Semeniuk et al. (38). Diabetic db/db mice exhibited reduced contractile performance (Table 2), with decreased HR and systolic dysfunction (decreased %FS and Vcf), as observed previously (38). COOH treatment did not alter cardiac function in db/+ mice and did not improve the depressed systolic function in db/db mice.


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Table 2. Assessment of cardiac function in vivo by echocardiography with control db/+, COOH-treated db/+, diabetic db/db, and COOH-treated db/db mice

 

Outward K+ currents in cardiomyocytes. Current densities for both peak and sustained outward K+ currents were attenuated in db/db cardiomyocytes (Table 3), as reported previously (41). COOH treatment of db/db mice did not restore currents to the normal values measured in control db/+ cardiomyocytes. In fact, administration of COOH to db/db mice produced a further decline in the sustained current (Table 3).


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Table 3. Outward K+ currents in cardiomyocytes from control db/+, diabetic db/db, and COOH-treated db/db mice

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
COOH is a nonthiazolidinedione PPAR{gamma}-selective agonist (29). Preliminary experiments established that COOH administration was effective in lowering the hyperglycemia that characterizes db/db mice, a type 2 diabetes model (20, 31). Therefore, chronic administration of COOH to both control db/+ and db/db mice for 6 wk was utilized as the main experimental model. COOH treatment completely normalized blood glucose in db/db mice (Table 1). Unexpectedly, COOH also produced a slight decrease in body weight of treated db/db mice. Laplante et al. (29) observed adipose depot-specific effects of COOH administered to rats; subcutaneous fat deposition was increased but visceral fat was reduced. Therefore, COOH-induced adipose tissue remodeling in db/db mice could account for this slight reduction in body weight. Interestingly, administration of COOH to control db/+ mice had no effect on body weight or on blood glucose (Table 1). COOH administration to db/db mice also produced a significant fall in plasma FA, although TG concentrations were unchanged (Table 1).

Ex vivo perfused hearts provide an experimental system that permits control over the supply of exogenous substrates in the perfusate. Consequently, substrate metabolism can be measured concomitantly with indexes of contractile performance (8). Perfused hearts from diabetic db/db mice show early and dramatic changes in substrate utilization (1, 2, 7). Rates of glycolysis and glucose oxidation are reduced, with enhanced rates of FA oxidation. Thus FA oxidation becomes almost the exclusive source of ATP for db/db hearts (7). Reduced carbohydrate metabolism is also a feature of perfused hearts from Zucker diabetic fatty (ZDF) rats (17), another monogenic model of type 2 diabetes (31).

The profound insulin resistance observed in db/db mice in vivo from assessments of glucoregulation (20) also extends to the heart, as insulin-stimulated glucose uptake was reduced in cardiomyocytes from db/db hearts. COOH treatment enhanced insulin-stimulated glucose uptake into db/db cardiomyocytes (Fig. 2), indicating that improvement in overall diabetic status in treated db/db mice (normalization of hyperglycemia) correlated with increased cardiac insulin responsiveness. Sidell et al. (43) reported that rosiglitazone treatment restored to normal insulin-stimulated glucose uptake by perfused hearts from insulin-resistant ZDF rats.

Administration of COOH normalized the altered pattern of metabolism in db/db hearts by increasing glucose oxidation and reducing FA oxidation (Fig. 3). Given the low level of cardiac PPAR{gamma} expression and the absence of direct effects of PPAR{gamma} ligands on either gene expression or FA oxidation in cultured cardiomyocytes (24), the COOH-induced changes in perfused heart metabolism will most likely be an indirect mechanism, secondary to altered substrate supply to the heart in vivo (glucose- and FA-lowering actions). The observation that COOH treatment of control db/+ mice had no effect on cardiac glucose and palmitate oxidation (Fig. 3) is consistent with this conclusion. Increased cardiac insulin responsiveness in vivo may also be a contributory mechanism for metabolic changes. A key objective of future studies will be to determine the molecular mechanism(s) responsible for the alteration in cardiac metabolism induced by COOH treatment of db/db mice.

Hearts from db/db mice also exhibit contractile dysfunction, evident from studies with ex vivo perfused hearts (1, 2, 7) and in vivo assessment by echocardiography (38). A number of factors could contribute to the pathogenesis of contractile dysfunction (diabetic cardiomyopathy) in db/db hearts. First, sustained hyperglycemia in vivo could increase nonenzymatic glycation of interstitial proteins, which will produce myocardial stiffness and impaired cardiac contractility (3, 16), along with other glucotoxicity mechanisms (32, 36, 42). Second, altered metabolism of exogenous substrates in db/db hearts could contribute to contractile dysfunction, an example of metabolic maladaptation (45). Reduced glycolytic ATP generation may impair the function of key ion channels and pumps (44), and increased shunting of glycolytic intermediates through the hexosamine pathway can lead to increased O-glycosylation and altered function of intracellular proteins in db/db hearts (49). In addition, the enhanced utilization of lipids by db/db hearts (1, 7, 33) could also contribute to contractile dysfunction by a lipotoxicity mechanism (19, 22, 46, 50). However, these potential mechanisms for reducing contractile dysfunction in db/db hearts can be excluded, because contractile dysfunction persisted in COOH-treated db/db hearts (Fig. 4 and Table 2), despite normalization of hyperglycemia in vivo (Table 1) and normalization of exogenous substrate metabolism by perfused hearts (Fig. 3). In this regard, these results with COOH are very similar to observations with BM 17.0744, a PPAR{alpha} ligand that produced metabolic changes (increased glucose oxidation and decreased palmitate oxidation) in perfused db/db hearts without any improvement in contractile function (1). It is of interest that pharmacological interventions with agonists for two different PPARs with very different mechanisms of action have such similar effects on the phenotype of db/db hearts. An important objective for future investigations will be to determine the causative factors that produce contractile dysfunction in db/db hearts.

Cardiomyocytes from db/db hearts also exhibit electrophysiological changes, with reductions in repolarizing outward K+ currents so that action potential duration is increased markedly (41). Administration of COOH to db/db mice did not enhance K+ currents (Table 3); however, other ion currents and many other parameters of cardiac function have not been measured. Nevertheless, the cardiac effects of COOH may be restricted to metabolic changes.

The inability of COOH to influence contractile function of db/db hearts can be contrasted to results with type 2 diabetic ZDF rats (50). Chronic administration (13 wk) of troglitazone, a thiazolidinedione PPAR{gamma} agonist, to ZDF rats prevented loss of systolic function, which was assessed by echocardiography. There are several possible explanations for this discrepancy. First, the type 2 diabetic ZDF rat has less systolic dysfunction (50) compared with db/db hearts. Consequently, it may be easier to prevent reductions in contractile performance in ZDF rat hearts with a PPAR{gamma} ligand. Second, ZDF rats have less hyperglycemia but pronounced dyslipidemia compared with the db/db mice used in this study. Therefore, there are significant differences in diabetic features for these two monogenic models of type 2 diabetes (31). Third, troglitazone was administered to ZDF rats (50) for a longer period of time (from 7 to 20 wk of age), compared with the 6-wk treatment of db/db mice with COOH. And finally, part of the beneficial effects of thiazolidinediones could be due to PPAR{gamma}-independent effects (4, 15, 18) that may be absent in experiments with COOH, a nonthiazolidinedione ligand. Experiments with ex vivo perfused hearts from troglitazone-treated ZDF rats have not been conducted to determine whether substrate metabolism was altered. Another thiazolidinedione, rosiglitazone, did not influence contractile function of insulin-resistant Zucker rat hearts during normoxic perfusions but did improve recovery after ischemia-reperfusion (43). Cardioprotective effects of thiazolidinediones on postischemic functional recovery have also been observed with insulin-deficient type 1 diabetic hearts (27, 40).


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This study was supported by an operating grant from the Canadian Diabetes Association.


    ACKNOWLEDGMENTS
 
The technical assistance of Rogayah Carroll and Mariette Chuang is gratefully acknowledged.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. L. Severson, Dept. of Pharmacology & Therapeutics, Faculty of Medicine, 3330 Hospital Dr. NW, Calgary, AB T2N 4N1 Canada (E-mail: severson{at}ucalgary.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 REFERENCES
 

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