37 17- upregulates the expression of peroxisome proliferator-activated  and lipid oxidative in skeletal muscle

S E Campbell1,2, K A Mehan3, R J Tunstall3, M A Febbraio1,4, and D Cameron-Smith3 1Exercise Physiology and Metabolism Laboratory, Department of Physiology, University of Melbourne, Parkville, Victoria 3010, Australia 2Department of Kinesiology, University of Waterloo, Ontario N2 L 3G1, Canada 3School of Health Sciences, Deakin University, Burwood, Victoria 3125, Australia 4School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia

(Requests for offprints should be addressed to D Cameron-Smith; Email: [email protected])

Abstract

β This study examined the actions of 17 -estradiol (E2) and on the regulation of the peroxisome proliferator-activated receptors (PPARα and PPARγ) family of nuclear factors and the mRNA abundance of key enzymes involved in fat oxidation, in skeletal muscle. Specifically, carnitine palmitoyltransferase I (CPT I), β-3-hydroxyacyl CoA dehydrogenase (β-HAD), and pyruvate dehydrogenase kinase 4 (PDK4) were examined. Sprague–Dawley rats were ovariectomized and treated with placebo (Ovx), E2, progesterone, or both in combination (E+P). Additionally, sham-operated rats were treated with placebo (Sham) to serve as controls. (or vehicle only) delivery was via time release pellets inserted at the time of surgery, 15 days prior to analysis. E2 treatment increased PPARα mRNA expression and content (P<0·05), compared with Ovx < γ treatment. E2 also resulted in upregulated mRNA of CPT I and PDK4 (P 0·05). PPAR mRNA expression < was also increased (P 0·05) by E2 treatment, although protein content remained unaltered. These data α demonstrate the novel regulation of E2 on PPAR and genes encoding key that are pivotal in regulating skeletal muscle lipid oxidative flux. Journal of Molecular Endocrinology (2003) 31, 37–45

Introduction Beckett et al. 2002). The mechanism by which E2 regulates the activity of lipid oxidative enzymes in The ovarian hormones exert significant control on type I oxidative skeletal muscle remains unclear. cellular lipid homeostasis in metabolically active However, in -deficient (ArKO) mice, tissues such as the liver and skeletal muscle (for exogenous E2 is necessary to maintain the review see Campbell & Febbraio 2001a). In skeletal expression and enzyme activity of the hepatic muscle, ovariectomy suppresses the maximal -oxidation pathway (Nemoto et al. 2000, Toda activity of both carnitine palmitoyltransferase I et al. 2001), suggesting a capacity for E2 to regulate (CPT I) and -3-hydroxyacyl CoA dehydrogenase the expression of genes involved in lipid oxidation. (-HAD), key enzymes in lipid oxidation. The The actions of E2 and progesterone on the activity of these enzymes is up-regulated following expression of genes encoding proteins of particular treatment with 17-estradiol (E2) in red gastroc- importance in influencing the flux of fatty acids nemius (type I muscle fiber), but not white and glucose through the mitochondrial pathway gastrocnemius or vastus medialis (predominantly in type I muscle fibers has yet to be analyzed. type II muscle fibers) (Campbell & Febbraio 2001b, Therefore, this study aimed to examine the mRNA

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abundances of CPT I, -HAD, and pyruvate ad libitum and rats were given 20 g standard rat dehydrogenase kinase 4 (PDK4) following ovari- chow per day, to control food intake. All ectomy with subcutaneous infusion of E2, progester- experimental interventions were formally approved one and combined E2 and progesterone (E+P) in by the Animal Research Ethics Committee, red gastrocnemius muscle. PDK4 is of interest as University of Melbourne. the protein product of this gene phosphorylates and Rats were bilaterally ovariectomized or sham- inactivates the pyruvate dehydrogenase complex, a operated under sodium brietal anesthesia (60 mg/ key regulatory site for glucose oxidative flux kg, i.p.). Groups of ovariectomized rats were (Sugden et al. 2000). treated immediately with time release hormone Skeletal muscle contains both functional pellets (Innovative Research of America, Sarasota, (Katzenellenbogen et al. 1993) and progesterone FL, USA) inserted subcutaneously, administering (Shughrue et al. 1988) receptors. These receptors, as either 17-estradiol (E2) (2·5 µg/day), progesterone has been described in non-classical sex steroidal (1·5 mg/day), or both hormones in combination target organs including vascular and adrenal tissue, (E+P). Groups of ovariectomized (Ovx) and influence the expression of a variety of genes sham-operated (Sham) rats were treated with (Gargett et al. 2002, Serova et al. 2002). However, vehicle-only placebo pellets. The Sham group rats one characteristic feature of the genes involved in were tested on varying days of their estrous cycle to fatty acid oxidation is their sensitivity and get an inclusive group of ‘control’ animals. Rats regulation by the peroxisomal proliferator-activated were treated for fourteen full days post-operation, receptor (PPAR) family of transcription factors and on the morning of day 15 they were suffocated (Gulick et al. 1994, Mascaro et al. 1998, Wu et al. with CO2 (80:20, CO2:O2). A midline incision was 1999). Both the PPAR and PPAR isoforms, made and the diaphragm was cut to ensure death. which are present in skeletal muscle (Lemberger A cardiac puncture was performed, the blood spun et al. 1996, Loviscach et al. 2000), contribute to at 7000 g for 2 min, and the plasma removed and regulating the expression of many genes necessary stored at 80 C for analysis of the sex . for lipid uptake and oxidation in skeletal muscle Efficacy of surgical ovariectomy and (Camirand et al. 1998, Minnich et al. 2001, Muoio treatments were determined with the analysis of et al. 2002b). Cross-talk between the nuclear plasma E2 and progesterone using RIA kits receptors is not uncommon, and it might be (Diagnostic Products Corporation, Los Angeles, speculated that the ovarian hormones may interact CA, USA) at the end of the treatment period. with the PPARs in their regulation of cellular lipid Furthermore, plasma insulin was also measured metabolism. Indeed, in estrogen sensitive tissue, E2 using a commercially available RIA kit (Pharmacia has been demonstrated to induce the formation of a and Upjohn Diagnostics AB, Uppsala, Sweden) prostaglandin D2 metabolite capable of acting as a and free fatty acids (FFA) were determined by for PPAR (Ma et al. 1998), suggesting that spectrophotometric assay analysis for free fatty ff the e ects of E2 on lipid metabolism could be acids (FFA-C test kit, Wako Chemicals, Neuss, realized through this signaling pathway. Therefore, Germany). The muscles of the hindlimb were a second aim of the current study was to examine rapidly exposed, the gastrocnemius removed and the actions of E2 and progesterone on skeletal the red portion dissected out. Tissues were snap   muscle gene expression and protein abundance of frozen in liquid N2 and stored at 80 C for PPAR and PPAR. further analysis.

Materials and methods Real-time PCR Tissue RNA was extracted using a modified Experimental design acid guanidinium thiocyanate–phenol–chloroform Female Sprague–Dawley rats, 12–15 weeks old and extraction method for total RNA (Chomczynski & weighing 203·14·5 g (meanS.E.) were used in Sacchi 1987). Samples (0·5 µg/µl RNA) were these experiments. All animals were housed in a reverse transcribed to cDNA using the commer- temperature-controlled room (212 C) with a cially available Promega Reverse Transcription Kit light/darkness cycle of 12 h. Water was available (Promega, USA). The primers were designed using

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Table 1 GenBank accession numbers and sequences for PCR primers

GenBank locus Primer sequence

Gene β-actin X00351 Forward: 5′-GAC AGG ATG CAG AAG GAG ATT ACT-3′ Reverse: 5′-TGA TCC ACA TCT GCT GGA AGG T-3′ β-HAD AF095449 Forward: 5′-TCG TGA CCA GGC AAT TCG T-3′ Reverse: 5′-CCG ATG ACC GTC ACA TGC T-3′ PDK4 AF034577 Forward: 5′-GGG ATC TCG CCT GGC ACT TT-3′ Reverse: 5′-CAC ACA TTC ACG AAG CAG CA-3′ PPARα NM_013196 Forward: 5′-TGG AGT CCA CGC ATG TGA AG-3′ Reverse: 5′-CGC CAG CTT TAG CCG AAT AG-3′ PPARγ NM_013124 Forward: 5′-CAT GAC CAG GGA GTT CCT CAA-3′ Reverse: 5′-GCA AAC TCA AAC TTA GGC TCC ATA A-3′ CPTI AF029875.1 Forward: 5′-TTT GAG ATG CAC GGC AAG AC-3′ Reverse: 5′-CTG GAC AAG AGG CGA ACA CA-3′

the Primer Express software package version 1·0 IGEPAL (Sigma, USA), and Complete Protease (Applied Biosystems, Foster City, CA, USA) using Inhibitor Cocktail (Sigma), then homogenized gene sequences obtained from GenBank (see Table with a Polytron PT 1200 (Kinematica, Littau- 1 for GenBank accession numbers and sequences). Lucerne, Switzerland). Homogenates were spun at BLAST search for each primer confirmed homolo- 10 000 r.p.m. (4 C, 15 min) and the supernatants gous binding to the desired mRNA. Real-time removed and analyzed for total protein (BCA PCR was used to quantify mRNA expression using protein assay kit, Pierce, Rockford, IL, USA). SYBR Green technology. Briefly, 12 ng cDNA Denatured total proteins, 120 µg from each sample, were amplified with forward and reverse primers were separated by electrophoresis on a 10% SDS- and SYBR Green Universal PCR Master Mix polyacrylamide gel and transferred to a nitrocellu- (Applied Biosystems) for 40 cycles of PCR using lose membrane for 2 h at 50 mA/membrane (Nova the GeneAmp 5700 sequence detection system Blot, Pharmacia Biotech, USA). Membranes were (Applied Biosystems). Direct detection of PCR blocked for 1 h with 5% skim milk in Tris-buffered product was monitored by measuring the increase saline (50 mM Tris–HCl, 750 mM NaCl, 0·25% in fluorescence caused by the binding of SYBR Tween), and were incubated overnight at 4 C Green to double stranded DNA. Tissue mRNA with either a polyclonal anti-PPAR or anti- levels were quantified using critical threshold PPAR antibody (1 in 200 dilutions) (Santa emission increases above a threshold level (10 times Cruz Biotechnology, Santa Cruz, CA, USA). the S.D. of the background). Non-specific changes in Membranes were washed (45 min), followed by gene expression were corrected for by running the a 60-min incubation with anti-rabbit IgG conju- constitutively expressed -actin mRNA (Whitley gated to horse-radish peroxidase (1 in 8000 et al. 2000), and results were corrected with dilution), then washed again. Immunoreactive reference to the amount of -actin mRNA. End bands were detected using enhanced chemilumi- products were checked for contamination and nescence (Super-Signal, Pierce). An internal control ‘mispriming’ by verifying the denaturing peaks and of previously extracted rat muscle was used in ensuring the presence of only one product. each gel to normalize for variation in signal observed across the membranes. The resulting autoradiographs were analyzed by laser scan- Immunoblotting ning densitometry (GS710 Calibrated Imaging Samples were thawed in ice-cold lysis buffer Densitometer, BioRad, USA) and quantified with (40 µl/mg muscle) containing 1% SDS, 1% QuantityOne (BioRad), version 4·1. www.endocrinology.org Journal of Molecular Endocrinology (2003) 31, 37–45

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Table 2 Characteristics of the animals in the various experimental groups. Results are means±S.E.M. (n=8 per group)

Sham Ovx E2 Progesterone E2+Progesterone

Final body weight (g) 218±4 219±4 216±4 216±5 218±4 Plasma FFA (mmol/l) 0·69±0·06 0·70±0·04 0·76±0·06 0·64±0·05 0·73±0·04 Plasma estradiol (pg/ml) 34·9±4·2 14·4±1·0* 45·0±2·4*† 15·5±0·7* 49·9±1·9*† Plasma progesterone (ng/ml) 25·0±6·3 7·5±0·6* 5·6±0·9* 44·3±1·1*† 44·5±1·9*† Plasma insulin (pmol/l) 44·9±4·1 51·0±3·5 46·6±3·5 52·1±2·3 44·6±2·9

*P<0·05 compared with Sham; †P<0·05 compared with Ovx.

Statistics

All data are reported as meansS.E.M. Statistical comparisons were performed using ANOVA, with a Neuman–Keuls F test for post hoc comparison. Significance was accepted at P,0·05.

Results Animal characteristics The animal characteristics are presented in Table 2 (n=8 per group). Plasma E2 and progesterone concentrations confirmed the efficacy of the surgeries and sex steroid treatments. No effort was made to control for the ovarian status of the sham operated (Sham) animals, thus considerable vari- ation is demonstrated in both the plasma E2 and progesterone values from this group. No significant differences were found among the mean values of either initial or final body weight. Plasma insulin levels were also similar between all treatment groups (Table 2).

PPAR gene expression and protein abundance The gene expression and protein abundance of PPAR in the various treatment groups are presented in Fig. 1. The absence of the ovarian hormones in the ovariectomized (Ovx) rats lowered (P,0·05) the mRNA expression of PPAR compared with sham-operated (Sham) rats. Figure 1 Effect of ovariectomy and hormone Administration of E2 to ovariectomized animals treatments on PPARα mRNA expression (A) and protein markedly increased PPAR mRNA abundance in abundance (B) in red gastrocnemius muscle. Animal comparison with both the Ovx and Sham rats groups were Sham operated placebo (S), (P,0·05). Similarly, administration of progester- ovariectomized placebo (O), or ovariectomized and β one, or a combination of both hormones, resulted treated with 17 -estradiol (E), progesterone (P) or estradiol and progesterone (E+P). *P<0·05 compared in elevated (P,0·05) PPAR mRNA levels with Sham (S); †P<0·05 compared with Ovx (O). (B) compared with ovariectomy, although not to the Representative immunoreactive bands shown for same extent. The protein abundance of PPAR PPARα protein.

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Downloaded from Bioscientifica.com at 09/28/2021 08:10:16PM via free access Ovarian hormones and muscle gene expression · S E CAMPBELL and others 41 followed a very similar pattern to its mRNA expression, although the only significant increase in protein was observed in the E2-treated rats compared with Ovx rats (P,0·05). There was, however, a trend for lower PPAR protein content in Ovx rats compared with Sham rats (P=0·073).

PPAR gene expression and protein abundance The gene expression and protein abundance of PPAR in the various treatment groups are presented in Fig. 2. E2 treatment resulted in greater (P,0·05) PPAR gene expression, compared with both Ovx and Sham rats. However, PPAR mRNA levels were similar between Ovx and Sham rats, and in progesterone and E+P rats. Interest- ingly, this increase in gene expression did not translate into an increase in protein abundance, indicating that post-transcriptional regulation may ff attenuate any e ect of E2 on PPAR. Protein abundance of PPAR was similar across all treatment groups.

CPT I, -HAD and PDK4 gene expression The gene expressions of CPT I, -HAD, and PDK4 are presented in Fig. 3. The pattern of mRNA expression of all three enzymes was similar to that of both PPAR mRNA expression and Figure 2 Effect of ovariectomy and hormone protein abundance, providing further evidence of treatments on PPARγ mRNA expression (A) and protein their PPAR responsiveness. Specifically, treatment abundance (B) in red gastrocnemius muscle. Animal of ovariectomized rats with E2 resulted in a groups were Sham operated placebo (S), sevenfold increase (P,0·05) in CPT I mRNA ovariectomized placebo (O), or ovariectomized and β expression compared with both the Ovx and Sham treated with 17 -estradiol (E), progesterone (P) or estradiol and progesterone (E+P). *P<0·05 compared groups. Although progesterone and E+P rats also with Sham (S); †P<0·05 compared with Ovx (O). (B) appear to increase mRNA expression of CPT I Representative immunoreactive bands shown for these values were not statistically significant PPARγ protein. (P=0·29 and P=0·39 respectively). There were also no significant differences in expression of -HAD mRNA. Conversely, PDK4 gene expression was addition to enhancing the expression of genes decreased (P,0·05) in Ovx rats compared with all critical in the regulation of lipid oxidation in treatment groups, while E2 rats had increased skeletal muscle. The marked actions of E2 are (P,0·05) mRNA abundance compared with all exemplified by the dramatic increases in CPT I and treatment groups, but in particular, a 23-fold PDK4 mRNA. The importance of the ovarian increase compared with Ovx rats. hormones in maintaining PPAR levels is further emphasized by the decrease in mRNA expression, Discussion and a trend for lower protein content, in Ovx rats compared with Sham rats. Conversely, the results ff The results from this study demonstrate that E2 is a of progesterone treatment alone did not di er from potent regulator of PPAR mRNA and protein, in Sham rats; however, when given in combination www.endocrinology.org Journal of Molecular Endocrinology (2003) 31, 37–45

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with E2 it did suppress the up-regulation of mRNA expression and protein content of PPAR observed in E2-treated rats. This is similar to previously observed effects of progesterone in skeletal muscle lipid metabolism (Campbell & Febbraio 2001b), and indicates that these differences may result from transcriptional regulation. Furthermore, these results highlight the interaction between E2 and progesterone in vivo, suggesting that cross-talk between the ovarian hormones likely moderates the ff e ect of E2 on metabolism. Together, these data demonstrate significant regulation of skeletal muscle lipid oxidation genes and PPAR by the ovarian hormones. Little is known about the regulation of the expression of the genes encoding for PPAR and PPAR. Expression of PPAR is strongly induced at the transcriptional level by glucocorticoids (Lemberger et al. 1994), although this is attenuated by the presence of insulin (Steineger et al. 1994). However, the pathway involved in this regulation is still unknown. Importantly, changes in this study occurred in the absence of changes in insulin levels, and thus are more likely to be due to a direct effect of the ovarian hormones. Alternatively, the expression of PPAR is thought to be trans- activated by the CCAATT enhancer binding protein alpha (C/EBP) (Hamm et al. 2001). E2 has previously been linked with C/EBP in the control of some of its target genes (Calkhoven et al. 1997). An interesting finding of the current study was the specific control by E2 on protein levels of the PPARs in skeletal muscle. Despite similar enhance- ment in the gene expression of both PPAR and PPAR,E2 resulted in a selective up-regulation of the protein abundance of PPAR alone. Recent data suggests that PPAR ligand binding, which initiates dimerization with the nuclear binding partner (RXR), minimizes protein degradation by stabilizing the PPAR protein (Hirotani et al. 2001). It is tempting to speculate that in skeletal muscle E2 may generate a PPAR specific ligand, in contrast to the previous evidence for a PPAR ligand in the mallard duck Figure 3 Effect of ovariectomy and hormone treatments on mRNA expression of CPT I (A), β-HAD uropygial gland (Ma et al. 1998). Further studies to (B), and PDK4 (C) in red gastrocnemius muscle. Animal examine the nature of E2 activated PPAR ligands groups were Sham operated placebo (S), in skeletal muscle are required, particularly given ovariectomized placebo (O), or ovariectomized and the clinically important ability of PPAR ligands to treated with 17β-estradiol (E), progesterone (P) or decrease insulin resistance (Ye et al. 2001). estradiol and progesterone (E+P). *P<0·05 compared with Sham (S); †P<0·05 compared with Ovx (O). Continuous administration of E2 up-regulated the expression of all analyzed genes, with the

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Downloaded from Bioscientifica.com at 09/28/2021 08:10:16PM via free access Ovarian hormones and muscle gene expression · S E CAMPBELL and others 43 exception of -HAD. Indeed, E2 administration the actions of progesterone appear not to be increased the gene expression of PDK4 (25-fold entirely inhibitory as progesterone administration increase) and CPT I (7-fold increase) compared alone tended to result in greater mRNA levels than with Ovx rats. These two enzymes represent in both the sham and ovariectomized groups. Thus, important regulatory sites in the coordinated these data demonstrate that the relationship control of lipid oxidative pathways in skeletal between the ovarian hormones and lipid oxidative muscle. PDK4 functions by phosphorylating and genes is complex, suggesting either a complex inactivating pyruvate dehydrogenase (PDH), which interplay between the ovarian hormones them- catalyzes the oxidation of pyruvate to acetyl-CoA selves or the involvement of additional factors (Sugden & Holness 1994). Previous studies have regulating the expression of the analyzed genes. demonstrated regulation of PDK4 gene expression Despite the significant impact of the ovarian in response to exercise (Muoio et al. 2002a), hormones in determining gene abundance, caution starvation (Sugden et al. 2000, Holness et al. 2002b), is necessary when extrapolating from alterations in and insulin treatment (Wu et al. 1999), in parallel to mRNA expression to protein content. Many modifications in skeletal muscle lipid oxidation factors, including mRNA stability, translation elicited by these treatments. Concomitantly, CPT I efficiency, post-translational modifications and catalyzes the initial and rate-limiting step in the abundance of relevant ligands, exert significant transport of fatty acid into mitochondria, thus control on the final abundance and activity of the activation of this gene increases fatty acid oxidation gene product (Orphanides & Reinberg 2002). by promoting their entry into the mitochondria. However, the changes in CPT I mRNA are Up-regulation of both of these enzymes will allow broadly consistent with the previously observed for a greater lipid oxidative capacity and provide a changes in maximal enzyme activities follow- molecular mechanism for the impact of E2 on lipid ing identical treatment protocols (Campbell & oxidation observed previously in rodent studies Febbraio 2001b). While previously our data examining lipid oxidation either at rest or during demonstrated significant suppression of CPT I in exercise with hormonal treatment (Campbell & Ovx rats compared with the sham-operated Febbraio 2001b). However, these actions of E2 controls (Sham), the gene responses despite being cannot be generalized to other tissues and may vary lowered by Ovx failed to reach statistical markedly depending upon the administered E2 significance. In the present study, no significant dosage. Normalized expression of lipid metabolic alterations in -HAD gene abundances were genes has previously been demonstrated in the liver demonstrated, despite our previous demonstration of estrogen-deficient aromatase knockout mice of suppressed maximal enzyme activity following supplemented with exogenous E2 (Nemoto et al. Ovx treatment when compared with either Sham 2000, Toda et al. 2001). In contrast, higher doses of or E2 treatment. Little is known of the transcrip- E2 delivered via subcutaneous pellet at markedly tional control of -HAD, with the absence of higher doses than those used in the present study significant alteration in gene expression suggesting (5 mg/day vs 2·5 µg/day) have been shown to regulation of this gene is exerted predominantly via suppress liver CPT I mRNA (Gower et al. 2002). It post-translational mechanisms. is unclear whether these results demonstrate There are several possible mechanisms by which tissue-specific control or are due to a greater E2 treatment may mediate the observed alterations infused dose. in the expression of PPAR, PDK4 and CPT I. In While these data demonstrate a significant addition to acting directly via the receptors located response to E2 alone, the data suggest complex within skeletal muscles (Katzenellenbogen et al. interplay in the gene expression response of E2 in 1993), there is the possibility of indirect regulation the presence of progesterone. In the sham-treated through alterations in nutrients and/or hormones. animals, despite comparable E2 levels (see Table 2), PDK4 has been shown to be regulated by the presence of approximately 4·5-fold higher alterations in fatty acid availability, induced by progesterone levels appears to markedly alter the either high fat feeding, starvation or fat infusion mRNA response. Sham-treated animals had lower (Holness et al. 2002b), in addition to body mass levels of all genes, although only PDK4 and reduction, insulin availability and insulin resistance PPAR were significantly suppressed. However, (Majer et al. 1998, Rosa et al. 2003). However, in www.endocrinology.org Journal of Molecular Endocrinology (2003) 31, 37–45

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the present study no alterations in either plasma Acknowledgement fatty acids or insulin were demonstrated. Given the substantial role that PPAR plays in the regulation The authors acknowledge the technical assistance of the analyzed genes and lipid metabolism of Ms Janelle Mollica. (Mascaro et al. 1998, Wu et al. 1999, Minnich et al. 2001, Ye et al. 2001, Muoio et al. 2002b), it is tempting to attribute the effects seen in this study References as due, in part, to the elevated levels of PPAR Beckett T, TchernofA&TothMJ2002Effect of ovariectomy and following E2 treatment. However, recent data estradiol replacement on skeletal muscle enzyme activity in female demonstrate that this relationship is complex. First, rats. Metabolism 51 1397–1401. Calkhoven CF, SnippeL&AbG1997Differential stimulation by the actions of E2 on lipid metabolism may act CCAAT/enhancer-binding protein alpha isoforms of the independently of PPAR (Djouadi et al. 1998). 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