A potent PPARalpha agonist stimulates mitochondrial fatty acid beta -oxidation in liver and skeletal muscle

Anne Minnich, Nian Tian, Lisa Byan, and Glenda Bilder

Department of Cardiovascular Biology, Aventis Pharmaceuticals Research and Development, Collegeville, Pennsylvania 19426-0994


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The proposed mechanism for the triglyceride (TG) lowering by fibrate drugs is via activation of the peroxisome proliferator-activated receptor-alpha (PPARalpha ). Here we show that a PPARalpha agonist, ureido-fibrate-5 (UF-5), ~200-fold more potent than fenofibric acid, exerts TG-lowering effects (37%) in fat-fed hamsters after 3 days at 30 mg/kg. In addition to lowering hepatic apolipoprotein C-III (apoC-III) gene expression by ~60%, UF-5 induces hepatic mitochondrial carnitine palmitoyltransferase I (CPT I) expression. A 3-wk rising-dose treatment results in a greater TG-lowering effect (70%) at 15 mg/kg and a 2.3-fold elevation of muscle CPT I mRNA levels, as well as effects on hepatic gene expression. UF-5 also stimulated mitochondrial [3H]palmitate beta -oxidation in vitro in human hepatic and skeletal muscle cells 2.7- and 1.6-fold, respectively, in a dose-related manner. These results suggest that, in addition to previously described effects of fibrates on apoC-III expression and on peroxisomal fatty acid (FA) beta -oxidation, PPARalpha agonists stimulate mitochondrial FA beta -oxidation in vivo in both liver and muscle. These observations suggest an important mechanism for the biological effects of PPARalpha agonists.

fibrates; carnitine palmitoyltransferase I; nuclear receptors; gene expression; peroxisome proliferator-activated receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR) is so named because of its initial identification as the molecular mediator of the peroxisome proliferation response to a number of chemical stimuli, including fibrates, in mice (15, 17). The regulation of genes involved in peroxisomal fatty acid (FA) beta -oxidation by PPARalpha activators is well recognized (23). However, the peroxisomal response is likely to be rodent specific and may not occur in humans (14, 31).

Elevated plasma triglyceride (TG) concentrations constitute an independent risk factor for coronary artery disease (11, 13). Fibrate drugs and FAs are believed to be weak PPARalpha agonists, and PPARalpha is likely to mediate the hypolipidemic effects of fibrate TG-lowering therapy. One demonstrated mechanism for the TG-lowering effects of fibrates that is likely to occur in humans is via reduction of hepatic apolipoprotein C-III (apoC-III) transcription and synthesis (28). Because apoC-III is thought to inhibit very low density lipoprotein (VLDL)-TG hydrolysis by lipoprotein lipase and to inhibit uptake of VLDL remnants (1, 26, 32), a reduction in apoC-III synthesis would be predicted to result in a lowering of plasma TG concentrations.

In addition to apoC-III, molecular studies have implicated PPARalpha in the regulation of a number of genes involved in mitochondrial FA beta -oxidation; however, the PPAR responsiveness of such genes in vivo is less well studied. Carnitine palmitoyltransferase I (CPT I) catalyzes the transfer of FA from CoA to carnitine, allowing the initial transport of fatty acids into mitochondria for beta -oxidation. Its activity and expression are highly regulated and rate limiting. Eicosapentaenoic acid and fenofibrate administration increased mitochondrial CPT I and II activities in rabbits (9). Shunting of FAs toward beta -oxidation would be expected to result in decreased substrate availability for TG synthesis in liver, presumably resulting in a reduction of VLDL-TG secretion. Although fibrate drugs and FAs are believed to exert their effects on gene regulation via PPARalpha activation, TG lowering and regulation of mitochondrial FA beta -oxidation genes have not been directly demonstrated with the use of a potent, bona fide PPARalpha agonist. We used a ureido-fibrate analog (UF-5), shown to stimulate microsomal FA-omega hydroxylation and to lower VLDL cholesterol in rats (12), for this purpose.

The fat-fed hamster represents a potentially important model of nondiabetic hypertriglyceridemia. Unlike other rodents, hamsters respond to fat feeding with a greater than twofold increase in plasma TG levels (29). Lipoprotein metabolism in hamsters may more closely reflect that of humans than of rats or mice (29), but hamsters are not generally considered responsive to classical fibrates (16). Here we describe the effects of UF-5 on TG metabolism in vivo in a hamster model of hypertriglyceridemia and on FA catabolism in vitro.

PPARalpha -mediated responses have been traditionally studied in liver, but human and rat skeletal muscle expresses high levels of PPARalpha (21), and in humans, skeletal muscle may be the major site of PPARalpha expression. We therefore hypothesized that a potent PPARalpha agonist would stimulate mitochondrial FA beta -oxidation in muscle as well as in liver.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. 2-(4-(2-(N'-(4- fluorophenyl)-N-heptylureido)ethyl)phenoxy)-2-methylpropionic acid was synthesized as previously described (12). Fenofibrate was obtained by hydrolysis of fenofibrate methyl ester (Sigma, St. Louis, MO). Wy-14643 was from BioMol (Plymouth Meeting, PA). Etomoxir was kindly provided by Dr. H. P. O. Wolf (Allensbach, Germany).

In vivo protocol. Male Golden Syrian hamsters (Harlan Sprague Dawley, Madison, WI), weighing 120-135 g, were group housed with a 12:12-h light-dark cycle. Hamsters were placed on a high-fat high-cholesterol diet (0.05% cholesterol, 10% coconut oil; Dyets, Bethlehem, PA) for ~2 wk before treatment with UF-5 or vehicle and continued on this diet throughout the treatment period. UF-5 was prepared by sonication with vehicle (0.5% methylcellulose, 0.2% Tween 80) and administered twice daily by gavage in a rising-dose fashion (7.5, 15, and 30 mg/kg, n = 7 animals/group) with each dose given for a 1-wk period, resulting in a total of 3 wk of treatment with incremental doses. Vehicle was administered at 5 ml/kg. In a second study, vehicle, UF-5 (7.5, 15, and 30 mg/kg), and fenofibric acid (30, 60, and 120 mg/kg) were administered twice daily to fat-fed hamsters (n = 6/group) with a similar 3-wk rising-dose protocol. Blood samples, removed under CO2 narcosis, were obtained at specified times throughout the studies and were analyzed for triglycerides, cholesterol, and compound. All blood samples, except where indicated, were removed from animals fasted for 17 h. All samples were taken 45-60 min after dosing. Hamsters were terminated by CO2 overdose, and select tissues (liver and soleus muscle) were removed, blotted, weighed, flash-frozen in liquid N2, and frozen (-70°C) for subsequent RNA analyses. All animal protocols were approved by the Institutional Animal Care and Use Committee.

Quantitation of plasma UF-5 concentrations. Plasma samples were extracted with 0.1 µg/ml internal standard solution in acetonitrile on Porvair filtration disks. One hundred microliters of 10 mM ammonium acetate pH 3.5 were added and samples injected onto liquid chromatography-mass spectrometry/mass spectrometry in ionspray (positive) mode with magnetic resonance monitoring. Chromatography was performed on Luna C8 (2) 30 × 4.6 mm × 3 µm (Phenomenex), with 15-90% acetonitrile-ammonium acetate gradient as mobile phase with flow rate of 1 ml/min. The difluoro analog (12) of UF-5 was used as internal standard.

Quantitation of plasma TG, cholesterol, and glucose concentrations. Plasma TG concentrations were measured by the peroxidase method using the Sigma Diagnostic assay kit according to manufacturer's instructions. Plasma high-density lipoprotein (HDL) cholesterol was determined enzymatically after precipitation of apoB-containing lipoproteins by the phosphotungstic acid-Mg2+ method with Autokit Cholesterol (Wako Pure Chemical Industries, Osaka, Japan) and spectrophotometry with an automatic analyzer (Hitachi model 7050) at 600 nm. Plasma low-density lipoprotein (LDL) cholesterol was measured with the LDL Direct assay kit (Wako Pure Chemical Industries) (cholesterol esterase/cholesterol oxidase method), and the resulting H2O2 was measured colorimetrically at 585 nm. Plasma glucose was quantitated by the glucose oxidase-peroxidase method using the Sigma Diagnostic assay kit according to the manufacturer's instructions.

Cell culture. HepG2 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in DMEM-10% FCS-1% penicillin-streptomycin. Human primary skeletal muscle cells were obtained from Clonetics (San Diego, CA) and cultured in SkBM, supplemented with SkGM Singlequots.

Northern blotting. RNA was extracted from hamster soleus muscle or liver with Trizol reagent according to the manufacturer's protocol. Total RNA was subjected to Northern blotting onto Nytran membranes (Schleicher & Schuell, Keene, NH). A probe for rat apoC-III was cloned exactly as previously described (28). Human liver and muscle CPT I probes (accession nos. R28631 and W85710, respectively) were excised from pT7T3 by EcoR I/Not I and EcoR I/Pac I digestion, respectively. A probe corresponding to nucleotides 760-964 of the rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) coding sequence was cloned by PCR with primers 5'-CATCAAGAAGGTGGTGAAGC-3 (forward) and 5'-ACCCTGTTGCTGTAGCCATA-3 (reverse) into PCR2.1 and excised with EcoR I. A probe to the mouse homolog of human S10 (accession no. NM001014) was prepared by PCR of mouse liver cDNA using primers corresponding to nucleotides 17-170. Full-length human PPARalpha was subcloned from pSG5-hPPARalpha (a kind gift from Dr. Bart Staels) into pcDNA3.1. To create pcDNA3.1-hPPARgamma , Bluescript SK-, containing a full-length hPPARgamma -1 (ATCC), was digested with Asp718, filled in, digested with Not I, and then ligated into pcDNA3.1 digested with Not I/Kpn I (filled in). Probes for hPPAR isoforms were excised from pcDNA3.1. Probes were labeled with the Random Primers DNA Labeling System (Life Technologies, Rockville, MD) and [alpha 32P]dCTP (Amersham, Buckinghamshire, England). Blots were hybridized with probes (as indicated in Figs. 1-5) with ExpressHyb (Clontech, Palo Alto, CA) and washed according to the manufacturer's protocol. After exposure of membranes to X-ray film, signals were quantitated by densitometry (Personal Densitometer SI, Molecular Dynamic, Sunnyvale, CA). Blots were stripped for reprobing by boiling 2 × 10 min in 0.5% SDS.


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Fig. 1.   Effect of peroxisome proliferator-activated receptor-alpha (PPARalpha ) agonist treatment on gene expression in liver and muscle in fat-fed hamsters. Liver (A) and soleus muscle (B) total RNA (15 µg) from hamsters treated with rising doses of ureido-fibrate-5 (UF-5) and vehicle (Table 1) was subjected to Northern blotting, as described in METHODS. Blots were probed with rat apolipoprotein C-III (apoC-III, A) and human liver carnitine palmitoyltransferase I (CPT I) and human muscle CPT I (B) and then stripped and probed with rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A: lanes 1-7, vehicle; lines 8-14, UF-5. B: lanes 1-5 and 6-12 are from vehicle- and UF-5-treated hamsters, respectively.



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Fig. 2.   Effect of short-term UF-5 treatment on liver and muscle gene expression in hamsters. A: hepatic apoC-III and CPT I mRNA levels. B: soleus muscle CPT I mRNA levels. A and B: lanes 1-5, vehicle; lanes 6-10, UF-5 15 mg/kg; lanes 11-15, UF-5, 30 mg/kg.



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Fig. 3.   Comparison of UF-5 and fenofibric acid (FF) on gene expression in hamsters. A: apoC-III and B: muscle CPT I mRNA levels relative to GAPDH were quantitated by Northern blotting, as in Fig. 1 and METHODS. Lanes 1-6, vehicle; lanes 7-12, UF-5; lanes 13-18, FF.



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Fig. 4.   Expression of PPARalpha mRNA and protein in human liver and skeletal muscle tissue and cells. A: total RNA (15 µg) from human liver and skeletal muscle (SkM) (Clontech), HepG2 cells, or human skeletal muscle cells (hSKMC) at passage 3 was subjected to Northern blotting and probed with 32P-labeled cDNAs corresponding to human PPARs alpha  and gamma  (indicated left). Approximate transcript size is indicated right. B: pellets (P) and supernatants (S) of protein extracts from the indicated cell types were subjected to Western blotting, as described in METHODS. Protein molecular mass standards in kDa are indicated on right. TNTalpha and TNTgamma , in vitro translated human PPARalpha and -gamma , respectively (arrows). Duplicate blots were probed with a 1:2,000 dilution of rabbit anti-human PPARalpha (left) and PPARgamma (right).



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Fig. 5.   PPARalpha agonists stimulate palmitate beta -oxidation in HepG2 and human primary skeletal muscle cells. Left: HepG2 cells or primary human skeletal muscle cells (hSKMC) were incubated for 48 h with concentrations of UF-5 indicated on the X-axis. Palmitate beta -oxidation was measured as described in METHODS. Counts/min in the absence of live cells were subtracted from raw values, which were then corrected for cell DNA content, as described in METHODS. Y-axis represents data normalized for activity in the presence of corresponding concentrations of DMSO. Each point represents >= 3 independent experiments consisting of 3 wells each. Where there are no error bars, experiments were duplicate only. *P < 0.05 vs. DMSO. Right: inhibition of palmitate beta -oxidation by etomoxir. HepG2 cells were incubated with indicated concentrations of UF-5 with or without 40 µM etomoxir, and palmitate beta -oxidation was measured as described in METHODS.

Western blotting. Cell proteins were extracted from 100-mm dishes with PBS-1% Triton X-100-5 mg/ml NaEDTA-1 mM phenylmethylsulfonyl fluoride. Extracts were sonicated and centrifuged at 20,000 g for 15 min at 4°C. Proteins were resolved by electrophoresis on 10% SDS-PAGE gels, transferred onto nitrocellulose membranes, and probed with rabbit polyclonal antibodies against human PPARalpha (6) or PPARgamma (kind gift of Bart Staels) by use of the Western Breeze (Novex, San Diego, CA) hybridization/detection system. pcDNA3.1-hPPARalpha and -gamma positive controls were transcribed and translated in vitro with the Promega (Madison, WI) system as per manufacturer's instructions. Protein was quantitated by the Bradford method with the Bio-Rad (Hercules, CA) reagent according to manufacturer's instructions, with BSA as standard.

Cellular FA beta -oxidation assay. The rate of cellular beta -oxidation of [9,10(n)-3H]palmitic acid (52 Ci/mmol, Amersham) was measured as 3H2O release, as previously described (20). For cell incubations, [3H]palmitic acid was used at a final concentration of 22 µM in Hanks' balanced salt solution-0.5% free FA-free BSA (Sigma) by dilution with unlabeled palmitic acid (Sigma). Cell DNA content was quantitated with pico Green double-strand DNA quantitation reagent (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. Linearity of palmitate beta -oxidation with time and cell number was established by plating increasing numbers of HepG2 cells and measuring cellular FA beta -oxidation after 48 h of growth. After 1- or 2-h incubation with [3H]palmitate, cell supernatants were assayed for palmitate beta -oxidation product. On the basis of these data, cells were plated at 1.2 × 105 cells/well, and a 2-h incubation with substrate was used for experiments. UF-5 was prepared as 100 mM stock in DMSO and added once every 24 h. In some experiments, 40 µM etomoxir (2) was included either as a 24-h preincubation or during the latter 24 h to inhibit CPT I.

Statistics. Data are presented as means ± SE. Means were compared with Student's t-test. In Figs. 1-5 and Tables 1 and 2, a P value of <0.05 was considered significant.

                              
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Table 1.   Effect of UF-5 on plasma triglyceride levels in the fat-fed hamster


                              
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Table 2.   Comparison of UF-5 and fenofibric acid effects on lipid metabolism in hamsters


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of UF-5 on FA and TG metabolism in fat-fed hamsters. Recent reports (4, 12) demonstrated that UF-5 is a potent PPARalpha activator with an EC50 value of 400 and 30 nM for human and mouse PPARalpha , respectively, in cell-based transactivation assays.

Administration of UF-5 at 15 or 30 mg/kg twice daily for 1 wk to hamsters fed a high-fat diet resulted in ~50 and 70% reduction in fasting and nonfasting plasma TG levels, respectively (Table 1). UF-5 concentrations well in excess of EC100 in plasma (2,639 ± 502 ng/ml, n = 7) and liver (39.9 ± 5.3 µg/g) were achieved. Hepatic apoC-III mRNA levels in hamsters treated for 3 wk with rising doses of UF-5 were reduced by 63% when corrected for GAPDH compared with vehicle-treated hamsters (Fig. 1A). To assess whether UF-5 treatment affects expression of mitochondrial FA beta -oxidation genes, blots of total RNA from liver and soleus muscle were hybridized with tissue-specific CPT I probes. These yielded bands of ~5 and 3 kb, respectively, as reported (34). UF-5 treatment significantly increased hamster hepatic CPT I expression by 1.4-fold compared with vehicle (Fig. 1B). Muscle CPT I was upregulated 2.3-fold in UF-5- compared with vehicle-treated hamsters (Fig. 1B).

Effects of treatment duration on UF-5-induced lipid metabolism changes. To see how the treatment duration affects TG lowering and gene expression, fat-fed hamsters were treated for 3 days with UF-5 or vehicle. TG lowering with 30 mg/kg treatment of the nonfasted hamster was 37% after 3 days, or ~50% of the effect of the rising-dose treatment (Table 1). However, suppression of hepatic apoC-III levels was similar to that achieved after the rising-dose treatment (56 vs. 63%; Fig. 2A), and stimulation of hepatic CPT I expression was even greater (160 vs. 37%; Fig. 2A). Muscle-type CPT I-to-GAPDH mRNA ratio was unaffected in hamsters treated for 3 days with 30 mg/kg UF-5 (Fig. 2B). To see whether any effect might have been underestimated due to upregulation of GAPDH by UF-5, blots were stripped and reprobed with a murine S10 corresponding to a ribosomal protein. Muscle CPT I/S10 mRNA levels were not significantly different among any of the treatment groups (Fig. 2B).

Effects of UF-5 compared with fenofibric acid on lipid metabolism in hamsters. Next we compared the effects of fenofibric acid (FF), a very weak PPARalpha activator (4), to UF-5 in vivo. As expected, administration of rising doses of UF-5 to fat-fed hamsters lowered plasma TG concentrations by >50%, whereas FF at the doses tested did not (Table 2). UF-5, but not FF treatment, also lowered plasma LDL cholesterol (Table 2), consistent with putative inhibitory effects on VLDL secretion. Hepatic apoC-III mRNA levels in hamsters treated for 3 wk with rising doses of UF-5 or FF were reduced by ~50% when corrected for GAPDH, compared with vehicle-treated hamsters (Fig. 3A). Soleus muscle CPT I mRNA levels were increased approximately twofold in UF-5-treated hamsters but were not affected in FF-treated animals (Fig. 3B).

To investigate whether the TG lowering and weight loss effects of UF-5 were related to anorectic effects, food consumption was measured in this study. In fact, food consumption increased significantly in UF-5-treated hamsters after 2 and 3 wk of treatment (Table 2).

UF-5 stimulates mitochondrial FA-beta oxidation in vitro. These results suggested that, in addition to previously described effects of fibrates on apoC-III expression and on peroxisomal FA beta -oxidation, PPARalpha agonists might also exert TG-lowering effects through stimulation of mitochondrial FA beta -oxidation. To further explore the ability of PPARalpha agonists to stimulate FA beta -oxidation in vivo, we studied cell types representative of tissues carrying out high rates of FA beta -oxidation, namely HepG2 and hSKMC. To assess the suitability of the cell types for these experiments, the presence of PPARalpha mRNA and protein was first established. mRNA for hPPARalpha was in greater abundance in human skeletal muscle tissue than in liver (Fig. 4A). HepG2 and hSKMC expressed PPARalpha mRNA (Fig. 4A) and also contained immunoreactive PPARalpha protein (Fig. 4B) in hSKMC until 7 passages.

[3H]palmitate beta -oxidation was linear between 1 and 2 h and over 3.0 × 104-2.5 × 105 HepG2 cells/well under the experimental conditions tested. UF-5 stimulated FA beta -oxidation in HepG2 and in human primary skeletal muscle cells (Fig. 5, left) in a dose-dependent manner. Maximal stimulation was ~2.7- and 1.6-fold in HepG2 and hSKMC, respectively. Stimulation by 200 µM FF was 9% (n = 4, P < 0.06) over DMSO in HepG2 cells. To assess the extent to which stimulation of FA beta -oxidation by UF-5 was due to an increase in mitochondrial vs. peroxisomal beta -oxidation, etomoxir, a CPT I inhibitor, was used. Etomoxir almost completely inhibited both basal and UF-5-stimulated palmitate beta -oxidation (Fig. 5, right), indicating that PPARalpha agonist-stimulated FA beta -oxidation in HepG2 cells is mainly mitochondrial.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

It is widely believed that the lipid-lowering effects of fibrates are mediated through PPARalpha (22). Fibrates have been shown retrospective of their clinical efficacy to be very weak PPARalpha agonists, with unmeasurable binding affinities to PPARalpha and with EC50 values in cell-based reporter assays in the tens of micromolar range (4). It has been hypothesized that more potent, directed PPARalpha agonists would exert more powerful TG-lowering effects (27). The data reported herein support the hypothesis with a potent bona fide PPARalpha agonist, UF-5 (4), in a fat-fed hamster model of nondiabetic hypertriglyceridemia. Greater than 50% plasma TG lowering with UF-5 was seen at doses an order of magnitude lower than those typically necessary for clinically used fibrates. These effects are likely to be mediated through PPARalpha , given that selectivity for murine PPAR isoform activation of UF-5 is 26-fold for PPARalpha over PPARgamma (4).

A further objective of this study was to explore the potential mechanism for TG lowering of PPARalpha agonists. Consistent with the effects of fenofibrate in rats (28), we observed a sharp reduction in hepatic apoC-III expression in fat-fed hamsters. In addition to effects on hepatic apolipoprotein expression, we show here that PPARalpha agonist treatment influences CPT I expression. Two CPT I isoforms, liver (3, 7) and muscle (33, 34), have been cloned and characterized. Previous molecular evidence has implicated PPARalpha in regulation of the muscle isoform. The promoter of human muscle-type CPT I is stimulated by FAs, a response mediated by a PPRE to which PPARalpha binds (19, 35). Oleate increases CPT I expression in cardiac myocytes (2). More limited information also implicates PPAR in regulation of the liver isoform; clofibrate and FAs increase CPT I mRNA expression in fetal rat hepatocytes (5). In this study, UF-5 treatment of fat-fed hamsters upregulated both hepatic and muscle CPT I.

PPARalpha is also implicated in regulation of other genes involved in mitochondrial FA beta -oxidation. The promoters of medium-chain acyl-CoA dehydrogenase and rat mitochondrial HMG-CoA synthase are PPAR responsive (10, 24). Expression of acyl-CoA synthase (18, 25) and fatty acid transfer protein (8, 18) is induced by fibrates in rats. PPAR responsiveness of these genes in vivo in mice has also been modeled with etomoxir, which inhibits CPT I and is presumed to activate PPARalpha indirectly by causing accumulation of cellular FA (2, 10). Further evidence for the importance of PPARalpha regulation of some of these genes was provided by the absence of their regulation in PPARalpha knockout mice (2). These studies, in combination with the CPT I upregulation by UF-5 in vivo, suggest that stimulation of mitochondrial FA beta -oxidation represents an additional TG-lowering mechanism of PPARalpha agonists. Consistent with this, UF-5 markedly stimulated palmitate beta -oxidation in human hepatic and skeletal muscle cells in the present study. Although UF-5 is a potent activator of human PPARgamma (4), the effects of UF-5 are not likely due to PPARgamma activation, because PPARgamma immunoreactive protein was undetectable in these cells. The reason for the high (micromolar-range) concentrations necessary to achieve this relative to PPARalpha activation potency (~50 nM for human PPARalpha ) is not clear. However, a similar dissociation between apparent activation potency and functional effects in untransfected cells has been observed for fenofibrate effects on gene regulation (28, 30).

In this study, hamster soleus muscle CPT I mRNA levels were increased 2.3-fold in hamsters treated for 3 wk with rising doses of up to 30 mg/kg UF-5, but they were unaffected in animals treated with 30 mg/kg UF-5 for only 3 days. This result suggests that, although reduction of apoC-III and increase of CPT I expression in liver may contribute to short-term TG lowering in hamsters, stimulation of muscle FA beta -oxidation does not. However, the data suggest that stimulation of muscle FA beta -oxidation contributes to the greater TG-lowering effect of 30 mg/kg UF-5 after longer-term administration. Furthermore, fenofibric acid administration at the doses tested lowered hepatic apoC-III to a similar extent as did efficacious doses of UF-5 but did not affect muscle CPT I gene expression and had no TG-lowering efficacy. These results, by dissociating TG lowering from suppression of hepatic apoC-III expression, provide further support for the importance of muscle CPT I upregulation in TG lowering in fat-fed hamsters. Increased FA beta -oxidation did not decrease glucose utilization in muscle as suggested by the lack of effect of UF-5 on plasma glucose concentrations. In fact, the decrease in body weight seen in UF-5-treated hamsters suggests that PPARalpha activation increased energy expenditure in this animal model.

The relative contribution of FA beta -oxidation and apoC-III regulation in TG lowering by PPARalpha agonists may have implications for lipoprotein and lipid metabolism. Reduction of apoC-III synthesis, by stimulating lipoprotein lipase activity, might be expected to increase the VLDL-TG fractional catabolic rate (1), thereby increasing LDL production and raising LDL levels. In contrast, stimulation of FA beta -oxidation would be expected to reduce VLDL-TG production and therefore to lower LDL levels. In fact, UF-5 treatment significantly lowered LDL cholesterol by over 50%, consistent with the importance of the latter mechanism for TG lowering.

An additional effect of UF-5 in hamsters was a large increase in liver weight (Tables 1 and 2). This increase probably does not reflect fatty liver as judged by visual observation. Rather, hepatic hypertrophy undoubtedly results from peroxisome proliferation (as measured by increased cyanide-insensitive palmitoyl-CoA oxidation and by an increased immunoreactive bifunctional enzyme, not shown). In any case, the observed hepatic hypertrophy raises the possibility that the TG-lowering effect of UF-5 occurs at least partially through increased FA utilization in liver for cell membrane lipid synthesis. It is also possible that the increased hepatic CPT I expression reflects the need for an increase in cellular ATP production associated with liver hypertrophy. However, the following observations argue against these mechanisms: 1) UF-5 stimulated FA beta -oxidation directly in liver cells with no associated cellular proliferation or hypertrophy; 2) the TG-lowering effects of less potent, clinically administered PPARalpha agonists such as fenofibrate are dissociated from liver hypertrophy in humans; and 3) this possibility does not apply to UF-5-induced increase in muscle FA beta -oxidation, because UF-5 did not cause muscle hypertrophy (as judged by soleus muscle weights).

In summary, the present study supports the hypothesis that a potent PPARalpha agonist exerts marked TG-lowering effects. Stimulation of mitochondrial FA beta -oxidation in liver and muscle appears to contribute to maximal hypolipidemic effects of PPARalpha activation. These results should be considered in the design and monitoring of similar pharmacological agents.


    ACKNOWLEDGEMENTS

We thank Drs. Linda Merkel and Mark Perrone for helpful discussions; Colleen Charsky, Charles Hanning, and Tambra Peters for excellent technical assistance; and Elizabeth O'Connor, Kevin Darlington, and Henry Sarau for assistance with animal studies. We thank Dr. Ken Page for analysis of UF-5 levels and Dr. Zaid Jayyossi and Margaret Muc for measurements of liver peroxisomal enzymes. We are grateful to Drs. Robert Groneberg for chemical syntheses and Hong Zhu for providing the rat GAPDH probe. We acknowledge MSD Panlabs (Bothell, WA) for plasma HDL and LDL cholesterol determinations.


    FOOTNOTES

Present addresses: Anne Minnich and Nian Tian, Division of Respiratory Disease and Rheumatoid Arthritis, Aventis Pharmaceuticals, Bridgewater, NJ 08807; Lisa Byan, Guilford Pharmaceuticals, Baltimore, MD 21205; Glenda Bilder, Valleybrooke Corporate Center, Malvern, PA 19355.

Address for reprint requests and other correspondence: A. Minnich, Aventis Pharmaceuticals, Rt. 202/206, Bridgewater, NJ 08807 USA (E-mail: anne.minnich{at}aventis.com).

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 19 May 2000; accepted in final form 20 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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