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Am J Physiol Heart Circ Physiol 281: H888-H894, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 2, H888-H894, August 2001

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

Rogayah Carroll and David L. Severson

Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada T2N 4N1


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 µM Wy-14643 and 10 µM BM-17.0744 decreased total cellular LPL activity to ~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 µM 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.

lipoprotein metabolism; fatty acids; Wy-14643; BM-17.0744


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PEROXISOME PROLIFERATOR-ACTIVATED receptors (PPARs) are ligand-activated transcription factors in the nuclear receptor superfamily that regulate gene expression by heterodimerizing with retinoid X receptors and then binding to peroxisome proliferator response elements in the promoter region of target genes (17, 19). Three distinct PPAR gene products have been identified, with distinct tissue expression patterns (19). PPAR-alpha is highly expressed in liver and, to a lesser extent, in heart, skeletal muscle, and kidney (16, 31). PPAR-gamma is predominantly expressed in adipose tissue (2, 24); PPAR-delta is ubiquitously expressed. Biological roles for PPAR-alpha and PPAR-gamma have been revealed, because the availability of selective ligands allows experiments to be performed that measure specific cellular responses. PPAR-alpha is the molecular target of fibrates, drugs used to reduce serum triacylglycerols, and Wy-14643 (15, 18). Antidiabetic thiazolidinediones, which improve insulin resistance, are PPAR-gamma ligands (2, 24).

Lipoprotein lipase (LPL, EC 3.1.1.34) hydrolyzes the triacylglycerol component of circulating lipoproteins; the fatty acid (FA) product is thus made available for tissue utilization, for example, oxidation in the heart or esterification in adipose tissue (9). LPL is a PPAR target, on the basis of identification of a peroxisome proliferator response element in the LPL promoter (29) and observations that PPAR ligands increase LPL expression measured in vivo and in cultured cells. Selective PPAR-alpha and PPAR-gamma ligands increase LPL expression in liver and adipose tissue, respectively (2, 16, 21, 24, 29, 31). However, Ranganathan and Kern (27) recently reported that direct PPAR-gamma activation with thiazolidinediones actually reduced LPL activity in cultured adipocytes by a posttranslational inhibitory mechanism; LPL mRNA levels were unchanged in differentiated adipocytes after incubation with inhibitory concentrations of thiazolidinediones. Thus PPAR regulation of LPL activity involves a complex interplay of different mechanisms, with tissue-specific direct actions evident from experiments using cultured cells plus indirect effects that may contribute when PPAR ligands are administered in vivo.

PPAR-alpha is highly expressed in hearts (5, 18, 29). PPAR-alpha activation in cultured neonatal cardiomyocytes increases transcription of genes involved in FA transport and metabolism (6, 32, 33), but direct effects of PPAR-alpha ligands on terminally differentiated cardiomyocytes from adult hearts have not been investigated. Previously, we examined the regulation of LPL activity in cultured cardiomyocytes from adult rat hearts (1, 12, 14). The present study was designed, therefore, to examine the direct effect of PPAR-alpha ligands on LPL activity in cultured adult cardiomyocytes. Wy-14643 is a selective PPAR-alpha ligand that has been used extensively as an experimental tool (15, 18, 35). BM-17.0744 is a novel PPAR-alpha ligand (25) that decreases plasma lipids and improves insulin sensitivity in diabetic animals (26). Because FA such as linoleate are direct PPAR-alpha activators (15, 20, 35) and because we observed previously that FA can regulate cardiac LPL activity (1), comparative experiments were conducted with linoleate added to the culture medium. Incubation of cultured cardiomyocytes with Wy-14643, BM-17.0744, or linoleate inhibited LPL activity by a posttranscriptional and posttranslational mechanism.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental animals. Adult male Sprague-Dawley rats (200-220 g) were obtained from local breeding sources at the University of Calgary, housed under a 12:12-h light-dark cycle, and allowed access to tap water and standard laboratory chow ad libitum. All procedures involving animals were approved by the Committee on Animal Bioethics and Care at the University of Calgary.

Materials. Joklik minimal essential medium and penicillin-streptomycin were purchased from GIBCO Canada (Burlington, ON, Canada), collagenase from Worthington Biochemical (Lakewoood, NJ), heparin (1,000 U/ml, Hepalean) from Organon Teknika (Toronto, ON, Canada), laminin from Becton-Dickinson Labware (Bedford, MA), and insulin, dexamethasone, and essentially FA-free BSA from Sigma Chemical (St. Louis, MO). Wy-14643 was obtained from Biomol (Plymouth Meeting, PA), and BM-17.0744 was obtained from Dr. Johannes Pill (Roche Diagnostics, Mannheim, Germany). Linoleic acid was obtained from Doosan Serdary Research Lab (Englewood Cliffs, NJ), glycerol-[9,10-3H]trioleate ([3H]triolein) from Amersham Canada (Oakville, ON, Canada), and trans-35S label ([35S]methionine and [35S]cysteine) and DMEM from ICN (Costa Mesa, CA). The monoclonal antibody to LPL (5D2) used for immunoprecipitations was a generous gift from Dr. J. D. Brunzell (University of Washington, Seattle, WA). Affigel-10 was obtained from Bio-Rad (Hercules, CA).

Preparation and incubation of cardiomyocytes. Ventricular cardiomyocytes from adult rat hearts were isolated under aseptic conditions by collagenase treatment as described previously (1, 12, 14). Freshly isolated cells were suspended in culture medium (Joklik minimal essential medium supplemented with 25 mM NaHCO3, 1 mM CaCl2, 1.2 mM MgSO4, 1 mM DL-carnitine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.2% wt/vol FA-free BSA, pH 7.4) that had been filtered through a 0.22-µm filter. Cell viability was determined by trypan blue (0.4% in 0.9% NaCl) exclusion. Viable cells had a rod-shaped morphology with clear cross striations. Cell number was determined in duplicate using a hemocytometer. A preparation from a single heart yielded 7-9 × 106 viable cells, which were diluted to a density of 1.5 × 105 cells/ml.

Cardiomyocytes were cultured in laminin-coated six-well (35-mm) plates as described previously (1, 12, 14). After 3 h, the cells were provided with fresh culture medium without additions (basal conditions) or with additions as indicated, and the incubation was usually continued overnight (16 h) at 37°C under a humidified atmosphere of 95% O2-5% CO2. In some experiments, cardiomyocytes were cultured for shorter times (1, 2, and 4 h) in the absence and presence of additions. At the end of this culture period, the cells were treated with fresh culture medium (1 ml/well) with or without 5 U/ml heparin for 15 min. The medium from heparin-treated cells was removed for subsequent determinations of heparin-releasable LPL (HR-LPL) activity. HR-LPL activity represents the fraction of total cellular LPL (C-LPL) that is bound to the cell surface of cardiomyocytes (9). Cardiomyocytes incubated in the absence of heparin were scraped in lysis buffer (50 mM ammonia buffer, pH 8.0, containing 0.05% Triton X-100), sonicated, and diluted in buffer containing 10 mM HEPES (pH 7.5), 0.25 M sucrose, 1 mM EDTA, and 1 mM dithiothreitol (DTT) before assay for total C-LPL activity.

Assay of LPL activity. LPL activities were determined by measuring hydrolysis of a sonicated [3H]triolein substrate emulsion (1, 10). The assay contained 0.1 mM glycerol-[9,10-3H]trioleate (6 mCi/mmol), 25 mM PIPES (pH 7.5), 0.05% (wt/vol) essentially FA-free BSA, 50 mM MgCl2, and 3% heat-inactivated chicken serum as the LPL activator. For assay of cell lysates, heparin (2 U/ml) was also present. Medium (100 µl) or cell lysate (25 µl) was incubated in a final volume of 400 µl for 30 min at 37°C; [3H]oleate generated by the action of LPL was measured by liquid-liquid extraction and radioactive scintillation counting. All assays were performed in duplicate; LPL activity is expressed as nanomoles of oleate released per hour per milligram of protein in the sonicated cell extracts. Protein concentration was measured using a Coomassie blue spectrophotometric assay (30) with BSA as standard. In some experiments, LPL activity was determined in assays with [3H]triolein substrate concentrations ranging from 0.025 to 0.6 mM (13).

ELISA for LPL mass. The ELISA for LPL was performed as described previously (12). Samples were prepared by lysing cells from 10-cm dishes in 0.2 ml of 25 mM NH4Cl, 5 mM EDTA, 0.8% (wt/vol) Triton X-100, 0.04% (wt/vol) SDS, 33 µg/ml heparin, and 10 µg/ml leupeptin (pH 8.2). Dishes were washed once with 0.4 ml of buffer containing 250 mM sucrose, 1 mM EDTA, 1 mM DTT, and 20 mM HEPES (pH 7.4), and this wash was combined with the lysate and sonicated. LPL mass is expressed as nanograms per milligram of cell protein; LPL specific activity is expressed as milliunits per nanogram of LPL protein, where 1 mU is defined as the amount of enzyme catalyzing the release of 1 nmol oleate/min. To measure HR-LPL mass, 2 ml of heparin-treated media were lyophilized and resuspended in 0.2 ml of H2O for ELISA.

LPL synthesis. The synthesis of LPL in cultured cardiomyocytes was determined by measuring the incorporation of [35S]methionine into immunoprecipitable LPL protein, essentially as described previously by Carroll et al. (10). After 3 h incubation in 10-cm dishes, cardiomyocytes were additionally incubated at 37°C for 6 h with [35S]methionine (0.2 mCi/ml), or with 0.1 mCi/ml overnight, in DMEM buffer without methionine and cysteine but containing 1 mM glutamine and 0.2% (wt/vol) FA-free BSA. Dishes were washed with 5 ml of buffer containing 20 mM HEPES (pH 7.4), 250 mM sucrose, 1 mM EDTA, and 1 mM DTT and lysed in 1.2 ml of lysis buffer as for ELISA. The lysates were then sonicated twice and centrifuged at 15,000 g for 20 min; 0.5 mg of supernatant proteins was incubated overnight with 50 µg of anti-LPL (5D2) antibody coupled to Affigel-10 beads (0.7 µg antibody/µl gel matrix) in the presence of 1 M NaCl. A second immunoprecipitation of the lysates did not reveal any additional radiolabeled LPL. The immune complexes were washed with PBS containing 1% (wt/vol) Triton X-100 and 0.1% (wt/vol) SDS and subjected to SDS-PAGE (10). LPL was identified as a protein band with an apparent molecular weight of 56,000 on the fluorograms. 35S-labeled LPL bands were dissolved in 50% hydrogen peroxide, and radioactivity was determined by scintillation spectrometry. Total 35S-labeled proteins were determined by trichloroacetic acid precipitation and liquid scintillation spectrometry.

Statistics. Values are means ± SE; n is the number of individual cultured cardiomyocyte preparations. Comparisons were made using the Student's unpaired or paired t-test (Table 1) or one-way analysis of variance followed by Dunnett's multiple comparisons test (see Figs. 4 and 5), with statistical significance corresponding to P < 0.05. 

                              
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  		Table 1.   Effect of insulin and dexamethasone on LPL activity in cultured cardiomyocytes without and with Wy-14643


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

LPL catalytic activity. The effect of Wy-14643 on LPL activity after overnight culture of cardiomyocytes is shown in Fig. 1. Wy-14643 produced a concentration-dependent inhibition of C-LPL and HR-LPL activities; greater inhibition of HR-LPL than of C-LPL activity was evident at every concentration of Wy-14643. The EC50 for transcriptional effects of Wy-14643 is typically observed at ~2 µM (17, 18). Therefore, the inhibition of LPL activity in cultured cardiomyocytes with an EC50 of <1 µM (Fig. 1) is a very sensitive response to Wy-14643.


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  		Fig. 1.   Effect of Wy-14643 on lipoprotein lipase (LPL) activities in cultured cardiomyocytes. Cardiomyocytes were cultured overnight in the presence of the indicated concentrations of Wy-14643, and cellular LPL (C-LPL) and heparin-releasable LPL (HR-LPL) activities were determined. Results are from a single experiment; similar results were obtained in a second experiment with a different cultured cardiomyocyte preparation.

The time course for LPL inhibition by Wy-14643 was investigated next. C-LPL and HR-LPL activities were not reduced significantly (<20% inhibition) after cultured cardiomyocytes were incubated with 1 µM Wy-14643 for 1, 2, and 4 h; extending the incubation to 16 h (overnight) reduced C-LPL and HR-LPL activities to 41 and 33% of control, respectively (mean of 2 experiments). Therefore, LPL inhibition by Wy-14643 is a relatively slow response.

Previous investigations have reported that the combination of 50 nM insulin and 100 nM dexamethasone (Ins-Dex) stimulated C-LPL and HR-LPL catalytic activities in cultured cardiomyocytes with no change in total cellular mass (12, 14), so that incubation with hormones increased C-LPL specific activity. The addition of Ins-Dex to the overnight culture medium increased C-LPL and HR-LPL catalytic activities by 1.75- and 1.53-fold, respectively (Table 1), consistent with previous reports (12, 14). Wy-14643 (1 µM) inhibited C-LPL and HR-LPL activities to 55 and 34% of control, but Ins-Dex still produced a significant increase in enzyme activity (1.95- and 2.24-fold stimulation, respectively). Therefore, the ability of Ins-Dex to stimulate LPL catalytic activity was still evident in the presence of an inhibitory concentration of Wy-14643 (Table 1).

Inhibition of LPL activity required a higher concentration of BM-17.0744 than of Wy-14643. Overnight culture with 1 and 10 µM BM-17.0744 reduced C-LPL activity to 99 and 59% of control, respectively; HR-LPL activity was reduced to 84 and 33% of control, respectively, by these same concentrations of BM-17.0744. Therefore, in subsequent experiments, 10 µM BM-17.0744 was used to investigate the mechanism of the inhibitory effect on LPL activity.

Anderson et al. (1) reported previously that overnight culture of rat heart cardiomyocytes with several FA reduced HR-LPL and C-LPL activities. Consistent with these previous results (1), overnight culture with linoleate (60 µM, 2:1 molar ratio to albumin in the culture medium) produced a greater and more consistent reduction in C-LPL activity (to 39 ± 8% of control, n = 3) than 60 µM oleate (82 ± 15% of control, n = 3).

The kinetic mechanism responsible for LPL inhibition was investigated in experiments that were conducted with various concentrations of the triolein substrate (Fig. 2). The Michaelis-Menten constant (Km) and maximal reaction velocity (Vmax) were 91 µM and 540 nmol · h-1 · mg-1 for control HR-LPL (Fig. 2A) and 84 µM and 610 nmol · h-1 · mg-1 for control C-LPL (Fig. 2B), respectively, consistent with previous results from this laboratory (13). Wy-14643 (1 µM) reduced Vmax of HR-LPL and C-LPL to 22 and 46% of control, respectively, without a significant change in Km. A similar reduction in Vmax was observed with 10 µM BM-17.0744 and 60 µM linoleate.


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  		Fig. 2.   Kinetic mechanism for the inhibition of LPL by Wy-14643, BM-17.0744, and linoleic acid. Cardiomyocytes were cultured overnight in the presence of no additions (control, open circle ), 1 µM Wy-14643 (), 10 µM BM-17.0744 (black-triangle), or 60 µM linoleic acid (), and HR-LPL and C-LPL activities were determined in assays at triolein substrate concentrations of 0.025-0.6 mM. Results are from a single experiment; similar results were obtained in a second experiment.

LPL synthesis. The mechanism responsible for the inhibition of C-LPL activity in cultured cardiomyocytes by Wy-14643, BM-17.0744, or linoleate was investigated further by measuring LPL synthesis from the incorporation of [35S]methionine into immunoprecipitable LPL protein (10). Rates of LPL synthesis are lower in cardiomyocytes than in adipocytes (10). Also, because the inhibitory action of Wy-14643, BM-17.0744, and linoleate on C-LPL activity was a relatively slow response, effects of 1 µM Wy-14643, 10 µM BM-17.0744, and 60 µM linoleate on LPL synthesis were examined after a 6-h pulse incubation with [35S]methionine. A typical autoradiogram showing the incorporation of radioactivity into LPL protein (~56 kDa) is shown in Fig. 3A. None of the additions reduced LPL synthesis (Fig. 3B) or the synthesis of total protein.


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  		Fig. 3.   Effect of Wy-14643, BM-17.0744, and linoleic acid on LPL synthesis. Cultured cardiomyocytes were incubated with [35S]methionine for 6 h without additions (control) and with 1 µM Wy-14643 (Wy), 10 µM BM-017.0744 (BM), or 60 µM linoleic acid (LA). A: typical fluorogram after immunoprecipitation, SDS-PAGE, and autoradiography. Arrow, position of [35S]LPL relative to molecular weight marker proteins. LPL synthesis (B) and catalytic activity (C), expressed as a percentage of control (no additions), were determined after pulse incubations of 6 h. Values are means ± SE (n = 3 different cultured cardiomyocyte preparations).

The incubation time of 6 h in these pulse experiments was insufficient to produce a significant reduction in LPL activity (Fig. 3C). Therefore, pulse incubations were extended to 16 h (overnight) so that synthesis could be examined when there was a concomitant reduction in LPL catalytic activity. Wy-14643 or linoleate still did not reduce LPL synthesis significantly (87 ± 4 and 92 ± 5% of control, respectively) under these experimental conditions, even though C-LPL activity was reduced to 70 ± 3 and 56 ± 6% of control, respectively (n = 4 experiments).

LPL mass and specific activity. The inhibitory mechanism of Wy-14643, BM-17.0744, and linoleate was investigated further by measuring LPL catalytic activity (nmol · h-1 · mg cell protein-1) and mass (ng LPL/mg cell protein), so that LPL specific activity (mU/ng LPL) could be calculated. Overnight culture with 1 µM Wy-14643 reduced C-LPL activity to 47% of control, but LPL mass was unchanged (Fig. 4). As a consequence, Wy-14643 reduced LPL specific activity from 0.201 ± 0.024 to 0.109 ± 0.013 mU/ng LPL protein (P < 0.05). For HR-LPL, 1 µM Wy-14643 decreased catalytic activity to 28% of control (Fig. 5). The mass of LPL displaced into the medium by heparin was also reduced significantly, but only to 46% of control. Because the reduction in mass was less than the inhibition of catalytic activity (Fig. 5), HR-LPL specific activity was significantly reduced from 0.453 ± 0.049 to 0.279 ± 0.037 mU/ng LPL (P < 0.05).


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  		Fig. 4.   Effect of Wy-14643, BM-17.0744, and LA on C-LPL catalytic activity, mass, and calculated specific activity. Cardiomyocytes were cultured overnight without additions (control) and with 1 µM Wy-14643, 10 µM BM-17.0744, and 60 µM linoleic acid, and C-LPL catalytic activity (A) and mass (B) were determined and LPL specific activity was calculated (C). Values are means ± SE (n = 4 different cultured cardiomyocyte preparations). *Significantly different (P < 0.05) from control.



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  		Fig. 5.   Effect of Wy-14643, BM-17.0744, and LA on HR-LPL catalytic activity, mass, and calculated specific activity. Cardiomyocytes were cultured overnight without additions (control) and with 1 µM Wy-14643, 10 µM BM-17.0744, and 60 µM linoleic acid, and HR-LPL catalytic activity (A) and mass (B) were determined and LPL specific activity was calculated (C). Values are means ± SE (n = 4 different cultured cardiomyocyte preparations). *Significantly different (P < 0.05) from control.

The specific activity of HR-LPL in control cultured cells is higher than the specific activity of total C-LPL because of the presence of a significant pool of inactive intracellular mass (1, 12); heparin displaces only fully active LPL from the cell surface of control cardiomyocytes.

Similar to Wy-14643, 10 µM BM-17.0744 produced a significant inhibition of C-LPL catalytic activity to 53% of control, with no change in LPL mass (Fig. 4), resulting in a significant reduction in C-LPL specific activity to 0.121 ± 0.011 mU/ng LPL protein. BM-17.0744 also reduced HR-LPL catalytic activity (to 48% of control) and mass (48% of control), with no change in HR-LPL specific activity (Fig. 5).

Linoleate (60 µM) inhibited C-LPL activity (49% of control; Fig. 4) less than HR-LPL activity (36% of control; Fig. 5), as reported previously (1). Linoleate did not change total C-LPL mass (Fig. 4), but the mass of HR-LPL in the medium was reduced significantly to 56% of control (Fig. 5). As a consequence, C-LPL specific activity was reduced significantly to 0.120 ± 0.021 mU/ng LPL protein; the specific activity of HR-LPL was unchanged.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PPARs are widely expressed, and PPAR activation produces pleiotropic effects at multiple tissue sites, but very little is known about how PPAR ligands affect cardiac function (4). PPAR-alpha is highly expressed in the heart (4, 5, 18, 29), but identification of direct PPAR-alpha targets in the heart has been restricted to experiments with cultured neonatal cardiomyocytes. For example, PPAR-alpha activators (FA and Wy-14643) increased transcription (mRNA levels) of proteins involved in FA transport (FA translocase and FA-binding protein) and metabolism (acyl-CoA synthase, long-chain acyl-CoA dehydrogenase, and carnitine palmitoyltransferase I) and uncoupling protein-2 in neonatal cardiomyocytes (6, 32, 33). These changes in gene transcription may be part of the adaptive developmental increase in cardiac FA oxidation that occurs after birth (23, 34). However, effects of PPAR-alpha activation on cardiomyocytes from adult hearts may be quite different.

A direct effect of PPAR-alpha activation on LPL activity in terminally differentiated cardiomyocytes from adult rat hearts has, therefore, been investigated. Two structurally different PPAR-alpha ligands, Wy-14643 and BM-17.0744, were potent inhibitors of C-LPL activity in cultured cardiomyocytes. Inhibition of LPL was observed at low concentrations, with an EC50 of 0.1 µM Wy-14643 for HR-LPL (Fig. 1). By comparison, most transcriptional responses in neonatal cardiomyocytes were observed at 100 µM Wy-14643 (32, 33). The high sensitivity of LPL in adult cardiomyocytes to Wy-14643 and the use of two different ligands strongly suggest that this inhibitory response is mediated by PPAR-alpha activation.

Our observation that this inhibition of C-LPL catalytic activity in adult cardiomyocytes incubated with PPAR-alpha ligands was not accompanied by any change in total cellular LPL mass or LPL synthesis implicates a posttranscriptional and posttranslational inhibitory mechanism that results in synthesis of LPL with reduced specific activity. An inhibitory mechanism involving decreased LPL transcription and (or) reduced protein synthesis would have been evident as a reduction in [35S]methionine incorporation into immunoprecipitable LPL protein; furthermore, catalytic activity and mass would have decreased, with no change in LPL specific activity.

LPL is synthesized as an inactive monomer; acquisition of catalytic activity requires dimerization after processing of N-linked oligosaccharides (9). Therefore, the reduction in C-LPL specific activity (mU/ng LPL protein) by Wy-14643 and BM-17.0744, producing a decrease in Vmax, could be caused by inhibition of LPL processing and reduced conversion of the inactive monomer to the active dimer. It should be emphasized that PPAR-alpha activators may still act by a transcriptional mechanism, but one that inhibits LPL processing. Our results indicate only that cardiac LPL is not a direct target for transcriptional upregulation by PPAR-alpha ligands, since LPL mass and rates of synthesis were not increased, despite the presence of a peroxisome proliferator response element in the LPL promoter (29). Bergö et al. (3) proposed that the fasting-induced fall in adipose tissue LPL activity is due to a similar posttranslational mechanism, with an increased proportion of inactive monomeric LPL relative to the active dimeric form. Translocation of LPL to the cell surface may also be impaired by Wy-14643 and BM-17.0744, since less LPL catalytic activity and mass were displaced into the medium by heparin. Because HR-LPL specific activity was reduced significantly by Wy-14643, heparin must have displaced inactive and active forms of LPL into the medium of cultured cardiomyocytes.

Our results showing inhibitory effects of Wy-14643 and BM-17.0744 on C-LPL catalytic activity with no change in enzyme mass or synthesis in cultured cardiomyocytes are, in fact, very similar to recent observations by Ranganathan and Kern (27) with PPAR-gamma ligands and adipocytes. Thiazolidinediones reduced LPL catalytic activity in differentiated 3T3-F442A and 3T3-L1 cells and rat adipocytes, with no change in LPL synthesis ([35S]methionine incorporation into immunoprecipitable protein) or LPL mass determined by Western blotting. A major difference was in the time course of LPL inhibition. Maximal reductions in adipocyte LPL activity in response to thiazolidinediones were obtained after only 4 h of incubation (27). In contrast, inhibition of LPL in cultured cardiomyocytes by Wy-14643 required an overnight incubation. Nevertheless, inhibition of posttranslational processing resulting in decreased LPL specific activity appears to be a common mechanism for inhibition of LPL activity by PPAR-alpha and PPAR-gamma ligands in cardiomyocytes and differentiated adipocytes, respectively. Although PPAR-gamma ligands did increase LPL mRNA in undifferentiated preadipocytes (27, 29), LPL activity was extremely low and unchanged (27). Clearly, PPAR ligands can directly regulate LPL activity in adipose tissue and heart by mechanisms other than increased LPL gene expression. Interestingly, although incubation of a cultured hepatocyte (AML-12) cell line with a PPAR-alpha ligand increased LPL mRNA, as expected, since the LPL promoter has a peroxisome proliferator response element (29), the anticipated corresponding increase in LPL catalytic activity was, in fact, not documented.

The combination of Ins-Dex increases LPL specific activity in cultured cardiomyocytes (12, 14), presumably by increasing the proportion of active dimeric LPL relative to the inactive monomeric form. A reduction in C-LPL and HR-LPL activities by Wy-14643 did not prevent the stimulatory effect of Ins-Dex. This result further illustrates the complex regulation of LPL by posttranslational mechanisms.

Because incubation of cultured cardiomyocytes with linoleate produced similar reductions in C-LPL and HR-LPL activities when compared directly with Wy-14643 and BM-17.0744, it is tempting to suggest that this inhibitory effect of linoleate is also mediated by PPAR-alpha activation. This FA-induced inhibition of cardiac LPL activity could also account for the observation that LPL activity is reduced in hearts after induction of insulin-deficient diabetes, which results in a profound elevation of plasma FA concentration (7, 8, 28). These suggestions could be tested by examining the effects of insulin-deficient diabetes on cardiac LPL activity in PPAR-alpha -null mice (11). In this regard, it is interesting that LPL overexpression in transgenic mice with presumed enhanced delivery of FA to the heart caused peroxisomal proliferation with a severe myopathy (22). Consequently, PPAR-alpha -mediated inhibition of LPL by FA could be a protective mechanism to prevent a potentially toxic oversupply of FA to the myocardium, particularly since PPAR-alpha ligands generally increase FA utilization, at least in neonatal cardiomyocytes (6, 32, 33), which can have deleterious effects on contractile function because of lipotoxicity (36).

In summary, although PPAR-alpha ligands such as Wy-14643 can stimulate gene expression in neonatal cardiomyocytes with increased mRNA levels for FA-metabolizing enzymes (6, 32, 33), PPAR-alpha activation inhibited LPL activity in cultured cardiomyocytes from adult rat heart by a posttranscriptional and posttranslational mechanism. Investigations into the biochemical basis for LPL inhibition by PPAR-alpha ligands are in progress.


    ACKNOWLEDGEMENTS

This work was supported by Medical Research Council of Canada Operating Grant MT 13227.


    FOOTNOTES

Address for reprint requests and other correspondence: D. L. Severson, University of Calgary, Faculty of Medicine, Dept. of Pharmacology and Therapeutics, 3330 Hospital Dr. NW, Calgary, AB, Canada T2N 4N1 (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.

Received 12 July 2000; accepted in final form 17 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 281(2):H888-H894
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