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J. Biol. Chem., Vol. 277, Issue 6, 4098-4103, February 8, 2002

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A Role for Peroxisome Proliferator-activated Receptor  (PPAR) in the Control of Cardiac Malonyl-CoA Levels

REDUCED FATTY ACID OXIDATION RATES AND INCREASED GLUCOSE OXIDATION RATES IN THE HEARTS OF MICE LACKING PPAR ARE ASSOCIATED WITH HIGHER CONCENTRATIONS OF MALONYL-CoA AND REDUCED EXPRESSION OF MALONYL-CoA DECARBOXYLASE^*

Fiona M. Campbell^§, Ray Kozak, Alese Wagner, Judith Y. Altarejos, Jason R. B. Dyck, Darrell D. Belke^§¶, David L. Severson^¶, Daniel P. Kelly, and Gary D. Lopaschuk^**

From the  Departments of Pharmacology and Pediatrics, University of Alberta, Edmonton, Alberta T6G 2S2, Canada, the ^¶ Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta T2N 4N1, Canada and the  Center for Cardiovascular Research, Cardiovascular Division, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, June 29, 2001, and in revised form, October 31, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Peroxisome proliferator-activated receptor  (PPAR) is a nuclear receptor transcription factor that has an important role in controlling cardiac metabolic gene expression. We determined whether mice lacking PPAR (PPAR (/) mice) have alterations in cardiac energy metabolism. Rates of palmitate oxidation were significantly decreased in isolated working hearts from PPAR (/) hearts compared with hearts from age-matched wild type mice (PPAR (+/+) mice), (62 ± 12 versus 154 ± 65 nmol/g dry weight/min, respectively, p < 0.05). This was compensated for by significant increases in the rates of glucose oxidation and glycolysis. The decreased fatty acid oxidation in PPAR (/) hearts was associated with increased levels of cardiac malonyl-CoA compared with PPAR (+/+) hearts (15.15 ± 1.63 versus 7.37 ± 1.31 nmol/g, dry weight, respectively, p < 0.05). Since malonyl-CoA is an important regulator of cardiac fatty acid oxidation, we also determined if the enzymes that control malonyl-CoA levels in the heart are under transcriptional control of PPAR. Expression of both mRNA and protein as well as the activity of malonyl-CoA decarboxylase, which degrades malonyl-CoA, were significantly decreased in the PPAR (/) hearts. In contrast, the expression and activity of acetyl-CoA carboxylase, which synthesizes malonyl-CoA and 5'-AMP-activated protein kinase, which regulates acetyl-CoA carboxylase, were not altered. Glucose transporter expression (GLUT1 and GLUT4) was not different between PPAR (/) and PPAR (+/+) hearts, suggesting that the increase in glycolysis and glucose oxidation in the PPAR null mice was not due to direct effects on glucose uptake but rather was occurring secondary to the decrease in fatty acid oxidation. This study demonstrates that PPAR is an important regulator of fatty acid oxidation in the heart and that this regulation of fatty acid oxidation may in part occur due to the transcriptional control of malonyl-CoA decarboxylase.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Peroxisome proliferator-activated receptor  (PPAR)¹ is a lipid-activated nuclear receptor (1, 2) that induces the transcription of a number of genes involved in the control of lipid metabolism. PPAR is expressed in the heart and is involved in the transcriptional regulation of a number of genes involved in cardiac fatty acid oxidation. These include medium chain acyl-CoA dehydrogenase, fatty acid binding proteins, fatty acid transporters, 3-ketoacyl-CoA thiolase, and muscle carnitine palmitoyltransferase I (3-9). PPAR increases gene transcription by binding as a heterodimer with the retinoid X receptor to PPAR response elements on the promoter regions of the target gene (2, 8, 10).

Fatty acid oxidation is an important source of energy in the heart and normally provides the majority of ATP necessary to sustain contractile function (11, 12). In many pathological states, fatty acid oxidation can dramatically increase, providing almost all of the energy requirements of the heart (reviewed in Ref. 11). For instance, in uncontrolled diabetes, fatty acid oxidation can account for almost 100% of the heart's energy production (13, 14). This unfortunately occurs at the expense of glucose metabolism, which contributes to both the development of contractile dysfunction and an increased sensitivity of the heart to injury during an ischemic insult (for a review, see Ref. 15). High rates of cardiac fatty acid oxidation are also seen in the fasting state (16). Increased expression of cardiac PPAR and activities of PPAR-controlled genes in hearts from diabetic (17) or fasted (18, 19) animals have been implicated in these high rates of fatty acid oxidation. However, a direct relationship between PPAR and fatty acid oxidation rates in the intact heart has not been established.

An important regulator of cardiac fatty acid oxidation is malonyl-CoA, which inhibits fatty acid uptake into mitochondria due to a potent inhibition of carnitine palmitoyltransferase I (20). Decreasing malonyl-CoA levels are accompanied by significant increases in fatty acid oxidation; conversely, increases in malonyl-CoA can result in substantial decreases in fatty acid oxidation (21). Despite the importance of malonyl-CoA in regulating fatty acid oxidation, the role of PPAR in regulating the expression and activity of the enzymes involved in controlling malonyl-CoA levels has received little attention. Malonyl-CoA is synthesized in the heart by acetyl-CoA carboxylase (ACC) (21) and is degraded by malonyl-CoA decarboxylase (MCD) (22). ACC is in turn regulated by 5'-AMP-activated protein kinase (AMPK), a key fuel-sensing kinase that can phosphorylate and inhibit ACC (23). Recent evidence also suggests that in skeletal muscle AMPK can phosphorylate and activate MCD (24). A recent study has shown that treatment of rats with a PPAR agonist, Wy-14643, increases MCD mRNA expression in the heart, although the effects of this treatment on malonyl-CoA levels and fatty acid oxidation rates have not been determined (25).

Glucose metabolism in the heart consists of two main pathways, glycolysis and glucose oxidation. Although activators of PPAR improve insulin sensitivity and hyperglycemia in rodent models of insulin resistance (26, 27), it is unclear if this is a direct effect on glucose metabolism or whether this occurs indirectly through the effects of PPAR activation on fatty acid oxidation. Long-chain fatty acids, which are activators of PPAR, have been shown to decrease mRNA levels of GLUT4 level in vitro (28). Whether this effect is mediated by PPAR remains to be determined.

The availability of mice that lack PPAR provides a useful model to examine the role of PPAR in the regulation of fatty acid and glucose metabolism in the heart. Several studies have shown that hearts from PPAR (/) mice show decreased expression of a number of enzymes involved in fatty acid oxidation including MCAD (6, 19, 29, 30). This is associated with decreased rates of fatty acid oxidation in isolated cardiac myocytes from these animals (30). However, direct determination of metabolism in the intact hearts of these animals is lacking. We recently developed an isolated working mouse heart model that allows us to directly measure energy metabolism in the intact heart under conditions of physiologically relevant levels of metabolic demand (31, 32). The aim of this study was to use this experimental model to determine what effects PPAR has on fatty acid and glucose metabolism in the heart. We also sought to determine whether concentrations of malonyl-CoA were altered in these hearts and whether PPAR mediated control of fatty acid oxidation involves transcriptional regulation of the key genes involved in the control of malonyl-CoA levels.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals-- PPAR (/) mice from a Sv/129 genetic background were produced as described (19, 29, 33). Wild type Sv/129 mice (PPAR (+/+) mice) were used as controls.

Heart Isolation and Perfusion Conditions-- Mouse working heart perfusions were carried out using the methods and apparatus previously described (31, 32). Adult PPAR (/) and PPAR (+/+) controls between 25 and 40 g in body weight were heparinized (100 units intraperitoneal) 10 min prior to anesthesia with 10 mg of intraperitoneal Na⁺ pentobarbitol. Hearts were then excised and placed in ice-cold Krebs-Henseleit bicarbonate solution (118 mM NaCl, 25 mM NaHCO₃, 4.7 mM KCl, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, and 5 mM glucose, gassed with 95% O₂, 5% CO₂ (pH 7.4)). Hearts were then cannulated as working hearts and perfused at a 7-mm Hg left atrial preload and 50-mm Hg aortic afterload with Krebs-Henseleit solution containing 5 mM glucose, 0.4 mM palmitate prebound to 3% bovine serum albumin, and 100 microunits/ml insulin. Glucose and palmitate were appropriately radiolabeled for energy metabolic measurements, as described below.

Spontaneously beating hearts were perfused for a 30-min aerobic period, and heart rate pressure measurements were recorded using a pressure transducer (Harvard Apparatus). Data were collected using an MP100 system from AcqKnowledge (BIOPAC Systems, Inc.). Cardiac output and aortic flows were obtained by monitoring the flow into the left atria and from the afterload line using Transonic flow probes, respectively. Coronary flow was calculated from the difference of the cardiac output and aortic flows. Cardiac work was calculated as the product of peak systolic pressure and cardiac output.

At the end of the 30-min perfusion, the ventricle from the heart was frozen immediately in liquid N₂, and the mass was recorded. Hearts were then ground using a mortar and pestle cooled to the temperature of liquid N₂. A portion of this powdered tissue (~20 mg) was weighed (wet weight) and then dried at 60 °C overnight to remove all water (dry weight). The ratio of this sample (dry/wet weight) was used to calculate the total dry mass of the heart. Metabolic rates were calculated using total dry mass of the heart to correct for variations in heart size.

Measurement of Fatty Acid Oxidation, Glucose Oxidation, and Glycolysis-- Metabolism of glucose and palmitate in PPAR (/) and PPAR (+/+) mouse hearts was measured according to the methods outlined by Saddik and Lopaschuk (12). All determinations of substrate metabolism were made in duplicate. Rates of glucose oxidation, glycolysis, and palmitate oxidation were measured in separate series of hearts. Steady state metabolic rates were calculated as the mean values when perfusate samples were removed from the working heart apparatus. Values obtained for the metabolic pathways were normalized for heart mass (dry weight). Glucose oxidation rates were determined by trapping and measuring ¹⁴CO₂ released by the metabolism of [U-¹⁴C]glucose. Collection of ¹⁴CO₂ released during glucose oxidation was performed as described previously (31). Glycolytic flux was determined as previously described (31) by measuring the amount of ³H₂O released from the metabolism of [5-³H]glucose by the triosephosphate isomerase and enolase steps of the glycolytic pathway (12). Palmitate oxidation rates were measured by quantitatively collecting ¹⁴CO₂ produced from hearts perfused with [1-¹⁴C] palmitate as described previously (12, 31).

Biochemical Measurements-- Detection and quantification of CoA esters were performed by extracting CoA esters from powdered tissue into 6% perchloric acid and measuring with a modified high pressure liquid chromatography procedure, as described previously (34).

MCD activity was measured in extracts isolated from mouse heart tissue as described previously (22). To measure AMPK and ACC activity, powdered heart tissue was homogenized in 0.05 M Tris, 0.25 M mannitol, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM sodium pyrophosphate, 10% glycerol, 1 mM dithiothreitol, pH 7.5, with the addition of Sigma protease inhibitor mixture for mammalian cells. The homogenate was centrifuged for 10 min at 800 × g, and the supernatant was used to perform the ACC and AMPK assays as described previously (34, 35).

Protein expression of MCD, ACC, the ₁ and ₂ subunits of AMPK, GLUT1, and GLUT4 were measured by Western blot analysis. Samples were subjected to SDS-PAGE and transferred to nitrocellulose as described (34). Membranes were immunoblotted with polyclonal antibodies raised to rat liver MCD (36). Polyclonal antibodies to the ₁ and ₂ subunits of AMPK were a gift from Dr. David Carling (Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom). GLUT1 and GLUT4 polyclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Chemicon International, respectively. Horseradish peroxidase-linked streptavadin was used for detection of ACC as described (34). The blots were then developed using the Amersham Biosciences, Inc. enhanced chemiluminesence Western blotting detection system.

The expression of MCD mRNA was determined by Northern blotting. RNA was extracted from mouse heart tissue with Trizol reagent (Invitrogen) according to the manufacturer's protocol. Total RNA was then subjected to Northern blotting. A probe corresponding to nucleotides 676-1073 of the rat glyceraldehyde-3-phosphate dehydrogenase coding sequence was prepared by RT-PCR of mouse heart RNA using a Qiagen OneStep RT-PCR kit and the specific primers, 5'-AGAACATCATCATCCCTGATCC-3' (forward) and 5'-TTACTCCTTGGAGGCCATGT (reverse). A 308-base pair probe to mouse MCD (accession number NM019966) was prepared by RT-PCR of mouse heart RNA using primers 5'-GCAGAATGGGGCTGTGATAG-3' (forward) and 5'-TTTCTAAGAAGGGGCCCAAG-3' (reverse). Labeling and detection was carried out using the North2South^® Direct HRP Labeling and Detection kit from Pierce, as described in the kit protocol.

Statistical Analysis-- Data are expressed as mean ± S.E. Comparisons between two group means were performed using the Student's t test. Where appropriate, analysis of variance followed by the Neuman-Keuls test was used to determine statistical significance when more than two group means were involved. Significance was set at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cardiac Function-- Various parameters of heart function in the PPAR (/) and PPAR (+/+) mouse hearts perfused at a 7-mm Hg preload and 50-mm Hg afterload are shown in Table I. Heart function throughout the 30-min perfusion period was stable in all hearts (data not shown). Under the perfusion conditions used, there was no difference in function between the PPAR (/) mouse hearts and the PPAR (+/+) mouse hearts. As a result, any differences in metabolism between these two groups could not be explained by differences in contractile function.

View this table: [in this window] [in a new window]   Table I Parameters of cardiac function Functional parameters were measured in isolated working hearts from PPAR (/) and wild type (+/+) controls perfused in 7-mm Hg preload and 50-mm Hg afterload for 30 min.

Glucose and Palmitate Metabolism-- Steady state rates of palmitate oxidation, glycolysis, and glucose oxidation in PPAR (/) and PPAR (+/+) hearts are shown in Fig. 1. Rates of palmitate oxidation, glycolysis, and glucose oxidation were linear in all hearts throughout the perfusion period (data not shown). Palmitate oxidation in PPAR (/) hearts was significantly lower than in PPAR (+/+) hearts. This decrease in palmitate oxidation was accompanied by a significant increase in both glucose oxidation and glycolysis.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Glycolysis, glucose oxidation, and palmitate oxidation in isolated working hearts from PPAR (/) and PPAR (+/+) mouse hearts. Steady state rates of glycolysis, glucose oxidation, and palmitate oxidation were measured in isolated working hearts as described under "Experimental Procedures." Values represent the mean ± S.E. of nine PPAR (/) and six PPAR (+/+) hearts for glycolysis; eight PPAR (/) and nine PPAR (+/+) hearts for glucose oxidation; and eight PPAR (/) and five PPAR (+/+) hearts for palmitate oxidation. *, significantly different from PPAR (+/+) hearts, p < 0.05.

The contribution of both palmitate oxidation and glucose oxidation to tricarboxylic acid cycle acetyl-CoA production is shown in Fig. 2. In PPAR (+/+) hearts, 58.5% of the tricarboxylic acid cycle acetyl-CoA was derived from glucose oxidation, whereas 41.5% originated from palmitate oxidation. These values are similar to our previously published values in mouse hearts perfused under these conditions (31). Overall tricarboxylic acid cycle activity did not differ between the PPAR (/) and PPAR (+/+) hearts. However, the contribution of palmitate oxidation to tricarboxylic acid cycle activity in PPAR (/) hearts was markedly reduced compared with (+/+) hearts. This decrease was compensated by an increased production of acetyl-CoA from glucose oxidation. Glycolytic rates were also significantly increased in PPAR (/) hearts relative to PPAR (+/+) hearts.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Relative contributions of palmitate and glucose oxidation to the production of acetyl-CoA for the tricarboxylic acid cycle in PPAR (/) and PPAR (+/+) hearts. Tricarboxylic acid cycle activity was calculated from the glucose and palmitate oxidation rates shown in Fig. 2, using a value of 8 mol of acetyl-CoA for every 1 mol of palmitate oxidized and 2 mol of acetyl CoA for every 1 mol of glucose oxidized.

Levels of CoA Esters in PPAR (/) and PPAR (+/+) Hearts-- Malonyl-CoA levels in tissue from PPAR (/) and PPAR (+/+) hearts frozen following perfusion are shown in Fig. 3. Malonyl-CoA levels were significantly higher in the PPAR (/) hearts compared with the PPAR (+/+) hearts. In contrast, there was no difference between the two groups in the levels of other CoA esters measured. Acetyl-CoA levels were 12.18 ± 3.51 and 9.40 ± 2.73 nmol/g dry weight, in PPAR (/) and PPAR (+/+), respectively (p not significant). Free CoA, acetyl-CoA, succinyl-CoA, and glutathione CoA levels were also not different between these two groups (data not shown).

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Malonyl-CoA levels in PPAR (/) and PPAR (+/+) mouse hearts. Values represent the mean ± S.E. of four PPAR (/) and three PPAR (+/+) hearts. *, significantly different from PPAR (+/+) hearts, p < 0.05.

Expression and Activity of Enzymes Controlling Cardiac Malonyl-CoA-- To determine the mechanism responsible for the increase in malonyl-CoA levels in the PPAR (/) hearts, we measured the expression and activity of enzymes known to regulate malonyl-CoA levels in the heart (ACC, AMPK, and MCD). In PPAR (/) hearts, a significant decrease in both MCD protein levels (Fig. 4A) and activity (Fig. 5A) was observed compared with PPAR (+/+) hearts. MCD mRNA levels in PPAR (/) were also significantly reduced compared with PPAR (+/+) hearts (Fig. 6). In contrast, there was no difference in the protein expression (Fig. 4B) or activity (Fig. 5B) of ACC between hearts from the two groups. The relative expression of the two isoforms of ACC in wild type and PPAR (/) null hearts was similar (the ratio of protein expression of ACC II compared with ACC I was 1.69 ± 0.61 and 1.66 ± 0.76, respectively, n = 4; values ± S.E.). Protein levels of both the ₁ and ₂ catalytic subunits of AMPK were also not different in the PPAR (/) and PPAR (+/+) hearts (Fig. 4C). Similarly, no difference in AMPK activity was observed between these two groups of hearts (Fig. 5C).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Expression of malonyl-CoA decarboxylase (A), acetyl-CoA carboxylase (B), and the 1 and 2 catalytic subunits of AMP-activated protein kinase (C) proteins in PPAR (/) and PPAR (+/+) mouse hearts. Values represent the mean ± S.E. of three PPAR (/) and three PPAR (+/+) hearts for MCD; four PPAR (/) and four PPAR (+/+) hearts for ACC; and four PPAR (/) and three PPAR (+/+) hearts for AMPK. *, significantly different from PPAR (+/+) hearts, p < 0.05.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Activities of malonyl-CoA decarboxylase (A), acetyl-CoA carboxylase (B), and the 1 and 2 catalytic subunits of AMP-activated protein kinase (C) in PPAR (/) and PPAR (+/+) mouse hearts. Values represent the mean ± S.E. of five PPAR (/) and four PPAR (+/+) hearts for MCD; four PPAR (/) and three PPAR (+/+) hearts for ACC; and four PPAR (/) and three PPAR (+/+) hearts for AMPK. *, significantly different from PPAR (+/+) hearts, p < 0.05.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Expression of malonyl-CoA decarboxylase mRNA in PPAR (/) and PPAR (+/+) mouse hearts. Values represent the mean ± S.E. of three PPAR (/) and three PPAR (+/+) hearts. Blots were stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). MCD expression is presented as a percentage of the glyceraldehyde-3-phosphate dehydrogenase expression to compensate for RNA loading differences. *, significantly different from PPAR (+/+) hearts, p < 0.05.

Expression of GLUT1 and GLUT4-- To determine whether the increased glycolysis and glucose oxidation rates observed in the PPAR (/) hearts could be due to differences in glucose transporter expression, GLUT1 and GLUT4 levels were measured in hearts from PPAR (/) and PPAR (+/+) mice (Fig. 7). No differences in GLUT1 and GLUT4 expression were observed between these two groups.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Expression of glucose transporters GLUT1 and GLUT4 in PPAR (/) and PPAR (+/+) mouse hearts. Values represent the mean ± S.E. of four PPAR (/) and four PPAR (+/+) hearts. *, significantly different from PPAR (+/+) hearts, p < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It has been well documented that PPAR controls the expression of genes important for fatty acid oxidation (3-9). In this study, we demonstrate that a deficiency of PPAR results in a dramatic decrease in fatty acid oxidation in the intact working heart. Our results also suggest that PPAR regulates malonyl-CoA levels in the heart. In hearts from PPAR (/) mice, malonyl-CoA levels were elevated, thereby contributing to the low palmitate oxidation rates observed in these hearts. This increase in malonyl-CoA is not due to an increase in ACC expression or activity or to alterations in AMPK control of ACC activity. However, the increase in malonyl-CoA levels can be explained by the decreased expression and activity of MCD in the hearts of mice lacking PPAR, suggesting that MCD is under the transcriptional control of PPAR. While the human (37) and rodent (36, 38) MCD cDNA sequence has been identified, it has not yet been determined whether a PPAR response element exists in the promoter region of this gene.

Malonyl-CoA levels in the heart rapidly change in response to changes in metabolic demand (21, 39). For instance, if glucose supply and oxidation rates are high, malonyl-CoA levels increase, resulting in a decrease in fatty acid oxidation rates (21). In contrast, in the absence of glucose, malonyl-CoA levels decrease, and fatty acid oxidation rates increase (21). Malonyl-CoA levels also decrease in the presence of increased metabolic demand that occurs during increases in cardiac work (39). In the PPAR (/) hearts, a number of enzymes involved in fatty acid oxidation decrease, which probably contributes to the low fatty acid oxidation rates observed in these hearts. In these hearts, there are three possible scenarios as to how malonyl-CoA levels are controlled. The first scenario is that malonyl-CoA levels are not under PPAR control and that malonyl-CoA decreases in the PPAR (/) hearts to compensate for the decrease in fatty acid oxidation. The second scenario is that malonyl-CoA levels are not under PPAR control, but malonyl-CoA levels increase secondary to the increase in glucose oxidation (which we have shown increases malonyl-CoA levels (21)). The third scenario is that like other fatty acid oxidation enzymes, PPAR also controls the enzymes involved in regulating malonyl-CoA levels and that malonyl-CoA levels would increase in the PPAR (/) hearts. The first scenario is unlikely since malonyl-CoA levels increased rather then decreased in the PPAR (/) hearts. The second scenario is also unlikely, since glucose oxidation increases malonyl-CoA by increasing the supply of acetyl-CoA available for malonyl-CoA synthesis by ACC (21). In PPAR (/) hearts, the increase in glucose oxidation was not accompanied by an increase in acetyl-CoA levels, suggesting that the observed increase in glucose oxidation is not responsible for the increase in malonyl-CoA levels. Our data support the third scenario and suggest that malonyl-CoA levels in the heart are under PPAR control. We also demonstrate that PPAR may control MCD expression/activity. This is supported by the recent observation that feeding rats the PPAR ligand Wy-14643 results in an increase in heart mRNA levels for MCD (25).

Unlike MCD, neither ACC expression nor activity was altered in the PPAR (/) hearts. Similarly, neither AMPK expression nor activity was different between PPAR (/) and PPAR (+/+) hearts. These data do not support a role for PPAR regulating either ACC or AMPK expression/activity in the heart. However, it cannot be ruled out that stimulating PPAR or overexpressing PPAR may alter the expression or activity of either ACC or AMPK. This possibility is presently being explored.

The decreased rate of fatty acid oxidation in PPAR (/) mice was compensated by an increase in glucose oxidation rates such that cardiac function is unchanged in these hearts. The observation that overall tricarboxylic acid cycle activity did not change in the PPAR (/) hearts despite a dramatic decrease in fatty acid oxidation suggests that glucose oxidation increased in response to the decrease in fatty acid oxidation. The increase in glycolysis also probably occurred because of the decrease in fatty acid oxidation rates. However, direct effects of PPAR on glucose metabolism cannot be ruled out. To determine whether PPAR has any direct effects on glucose transport, GLUT1 and GLUT4 levels were measured in the PPAR (/) and PPAR (+/+) hearts. The lack of effect on glucose transporter expression suggests that PPAR effects on glucose metabolism were not direct but rather were through alterations in fatty acid oxidation. Even if PPAR were to alter glucose transporter content, this could not explain the increase in glucose oxidation observed in this PPAR (/) hearts, since studies with perfused mouse hearts overexpressing GLUT4 showed increased rates of glycolysis but not of glucose oxidation (40). As a result, the increase in glucose oxidation (and probably glycolysis) in the PPAR (/) hearts was most likely occurring secondary to the decrease in fatty acid oxidation.

In conclusion, we demonstrate that knocking out PPAR alters fatty acid oxidation in the heart, indicating the role that this nuclear receptor plays in the regulation of fatty acid metabolism. In addition to directly controlling a number of enzymes involved in fatty acid oxidation, we show that PPAR may also control fatty acid oxidation by altering malonyl-CoA levels, secondary to controlling MCD expression and activity.

	FOOTNOTES

^* This work was funded by grants from the Canadian Diabetes Association and the Canadian Institutes for Health Research.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.

^§ An Alberta Heritage Foundation for Medical Research postdoctoral fellow.

^** An Alberta Heritage Foundation for Medical Research Medical Scientist. To whom correspondence should be addressed: 423 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. Tel.: 780-492-2170; Fax: 780-492-9753; E-mail: gary.lopaschuk@ualberta.ca.

Published, JBC Papers in Press, December 4, 2001, DOI 10.1074/jbc.M106054200

	ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; ACC, acetyl-CoA carboxylase; AMPK, 5'-AMP-activated protein kinase; MCD, malonyl-CoA decarboxylase.

	REFERENCES

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