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Endocrinology Vol. 144, No. 1 101-109
Copyright © 2003 by The Endocrine Society

ARTICLE

Sex Difference in Hepatic Peroxisome Proliferator-Activated Receptor {alpha} Expression: Influence of Pituitary and Gonadal Hormones

Masoumeh Jalouli1, Linda Carlsson1, Caroline Améen, Daniel Lindén, Anna Ljungberg, Liliane Michalik, Staffan Edén, Walter Wahli and Jan Oscarsson

Department of Physiology and Pharmacology (M.J., L.C., C.A., D.L., A.L., S.E., J.O.), Göteborg University, Göteborg S-405 30, Sweden; and Institute de Biologie Animale (L.M., W.W.), Faculté des Sciences, Lausanne CH-1015, Switzerland

Address all correspondence and requests for reprints to: Jan Oscarsson, Department of Physiology, Göteborg University, Box 434, S-405 30 Göteborg, Sweden. E-mail: Jan.Oscarsson{at}fysiologi.gu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peroxisome proliferator-activated receptor (PPAR) {alpha} is a nuclear receptor that is mainly expressed in tissues with a high degree of fatty acid oxidation such as liver, heart, and skeletal muscle. Unsaturated fatty acids, their derivatives, and fibrates activate PPAR{alpha}. Male rats are more responsive to fibrates than female rats. We therefore wanted to investigate if there is a sex difference in PPAR{alpha} expression. Male rats had higher levels of hepatic PPAR{alpha} mRNA and protein than female rats. Fasting increased hepatic PPAR{alpha} mRNA levels to a similar degree in both sexes. Gonadectomy of male rats decreased PPAR{alpha} mRNA expression to similar levels as in intact and gonadectomized female rats. Hypophysectomy increased hepatic PPAR{alpha} mRNA and protein levels. The increase in PPAR{alpha} mRNA after hypophysectomy was more pronounced in females than in males. GH treatment decreased PPAR{alpha} mRNA and protein levels, but the sex-differentiated secretory pattern of GH does not determine the sex-differentiated expression of PPAR{alpha}. The expression of PPAR{alpha} mRNA in heart or soleus muscle was not influenced by gender, gonadectomy, hypophysectomy, or GH treatment. In summary, pituitary-dependent hormones specifically regulate hepatic PPAR{alpha} expression. Sex hormones regulate the sex difference in hepatic PPAR{alpha} levels, but not via the sexually dimorphic GH secretory pattern.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PEROXISOME PROLIFERATOR-activated receptors (PPARs) belong to the nuclear steroid receptor superfamily (for review, see Refs.1, 2, 3). Three different subtypes of the receptor ({alpha}, ß/{delta}, and {gamma}) with different tissue distribution have been identified. PPAR{alpha} is mainly expressed in liver, heart, skeletal muscle, kidney, and intestine. PPAR{gamma} is mainly expressed in adipose tissue and the immune system, whereas PPAR{delta} is widely distributed (4). The PPARs heterodimerize with the 9-cis-retinoic acid receptor (retinoid X receptor) and bind to specific DNA-responsive elements (peroxisome proliferator response elements) in the promoter of their target genes (1, 5). Ligands for PPAR{alpha} include fibrates, long-chain unsaturated fatty acids, and eicosanoids along with leukotriene B4 (6, 7, 8, 9, 10). PPAR{alpha} plays an important role in regulating the lipid homeostasis, especially fatty acid oxidation and various aspects of lipoprotein metabolism (for review, see Refs.2, 3 and11). Despite great knowledge about the roles of PPAR{alpha}, little is known about the regulation of the PPAR{alpha} expression in the liver and other tissues.

The PPAR{alpha} expression in the liver is developmentally regulated. Transcripts of PPAR{alpha} mRNA are first detected on d 13 (12). PPAR{alpha} expression increases during the suckling period followed by a decrease post suckling (13). Fibrates have been shown to increase PPAR{alpha} mRNA (14), indicating that PPAR{alpha} regulates its own expression. Intake of high amounts of dietary triglycerides containing polyunsaturated fatty acids for several weeks has also been shown to increase PPAR{alpha} mRNA levels (15), probably via a similar positive regulation by the receptor itself. Glucocorticoids increase PPAR{alpha} mRNA levels in vivo (16) and in vitro (17, 18), and PPAR{alpha} mRNA and protein levels follow the diurnal variation of corticosterone in the rat (16). Hypophysectomy has been shown to increase PPAR{alpha} mRNA in both female (19) and male rats (20). These studies indicate that the sum of the effects of pituitary-dependent hormones on PPAR{alpha} mRNA expression is inhibitory. GH is one of the pituitary-dependent hormones that inhibit PPAR{alpha} mRNA expression (19, 20). GH has also been shown to decrease PPAR{alpha} mRNA in cultured hepatocytes (19, 21), indicating a direct effect via the GH receptor of hepatocytes.

Several studies have reported that, in rats, females are less responsive than males to various effects of fibrates, including increased liver weight, peroxisome proliferation, and peroxisomal ß-oxidation, as well as changes in various enzyme activities (22, 23, 24, 25, 26, 27, 28, 29). Studies in mice also indicate that males are more responsive than females to peroxisome proliferators (30). Rats and humans show a similar sex difference with respect to the effect of ethanol consumption on hepatic {omega}oxidation. These studies show that ethanol consumption results in larger {omega}-oxidation in males than in females; a difference that may be due to a sex difference in PPAR{alpha} expression (31, 32).

GH may play a role in the sexually dimorphic regulation of PPAR{alpha}-sensitive functions. Indeed, GH given as a continuous infusion and estrogen treatment were shown to prevent the induction of peroxisomal ß-oxidation by clofibrate, whereas hypophysectomy and testosterone treatment of female rats had the opposite effect (25, 29). These findings indicate that both sex hormones and GH play a role in the sex difference in response to peroxisome proliferators in the liver.

Sex differences in the liver may be due to a direct effect of sex steroids or to an indirect effect of these hormones via their regulation of the secretory pattern of GH (33, 34). In female rats, GH is secreted irregularly but continuously, whereas in males, GH is secreted episodically with low or undetectable levels between peaks (35). In the rat, several sexually dimorphic hepatic functions are regulated by the secretory pattern of GH (for review, see Refs.33, 34 and36). In terms of fatty acid metabolism, a few sexually dimorphic liver functions are regulated by the secretory pattern of GH. These functions include expression of cytochrome P450 enzymes (33, 37, 38, 39), fatty acid composition of phosphatidylcholine (40), triglyceride synthesis, and very low density lipoprotein secretion (41).

The primary aim of the present study was to investigate whether the differences in peroxisome proliferator responses on several PPAR{alpha}-regulated hepatic functions is correlated to a sex difference in PPAR{alpha} expression. The second aim was to evaluate the importance of the secretory pattern of GH and gonadal hormones for the sex difference in PPAR{alpha} expression. The third aim was to investigate whether gender, hypophysectomy, gonadectomy, or GH could influence PPAR{alpha} expression in heart or soleus muscle, other tissues with a high degree of PPAR{alpha} expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female and male Sprague Dawley rats from Møllegaard Breeding Center (Ejby, Denmark) were used. Hypophysectomy was performed at 42–43 d of age by Møllegaard Breeding Center. Gonadectomy was performed at 35 d of age. Age-matched female and male rats served as controls. The rats were maintained under standardized conditions of temperature (24–26 C) and humidity (50–60%) and with lights on between 0500 and 1900 h. The rats had free access to standard laboratory chow (rat and mouse standard diet, B&K Universal Ltd., Sollentuna, Sweden) and water, if not stated otherwise. Hormonal treatment started 7–10 d after hypophysectomy. The hypophysectomized rats were given cortisol phosphate (400 µg/kg·d; Solu-Cortef, Upjohn, Puurs, Belgium) and L-thyroxin (10 µg/kg·d; Nycomed, Oslo, Norway) diluted in saline and given as a daily sc injection at 0800 h (41, 42). Recombinant bovine GH was a generous gift from Dr. Parlow, NIH (Torrance, CA). The hormone was diluted in 0.05 M phosphate buffer, pH 8.6, with 1.6% glycerol and 0.02% sodium azide. Bovine GH (0.5–2 mg/kg·d) was given either continuously by means of Alzet osmotic mini-pumps (Model 2001, Alza Corp., Palo Alto, CA), or as two daily sc injections at 12-h intervals (0800 and 2000 h) (39, 42, 43). Testosterone (1 mg/kg·d) and 17ß-estradiol (1 mg/kg·d) were diluted in propylene glycol and given by means of Alzet osmotic mini-pumps (Model 2001, Alza Corp.) (26, 44, 45, 46). The rats were anesthetized with a combination of ketamine hydrochloride (77 mg/kg: Ketalar, Parke-Davis, Detroit, MI) and xylazine (9 mg/kg; Rompun, Bayer Corp., Lever-Kusen, Germany) when osmotic mini-pumps were implanted sc on the back of the rats. The treatments continued for 7 d, if not stated otherwise. The rats were decapitated, trunk blood collected, and the liver, heart and soleus muscle were taken out at the end of the experiments. The tissues were cut in pieces and immediately frozen in liquid nitrogen and stored at -70 C until assays.

Measurement of PPAR{alpha} mRNA and CYP2C11 mRNA
Total RNA was isolated with TRI REAGENT according to the manufacturer’s protocol (Ambion, Inc., Austin, TX) (47). The concentration of RNA was determined spectrophotometrically at 260 nm.

PPAR{alpha} mRNA.
A 249-bp fragment, nucleotide 76 from ATG to nucleotide 324, of rat PPAR{alpha} cDNA (accession no. M88592) subcloned into pBluescript II (Stratagene, La Jolla, CA) was used to generate a biotin-labeled antisense probe (Maxiscript, Ambion, Inc.) as described previously (19).

CYP2C11mRNA.
A 205-bp fragment of rat CYP2C11 cDNA in pGEM was kindly supplied by Dr. Agneta Mode (Karolinska Institute, Stockholm, Sweden). The plasmid was linearized with BamH1 and a biotin-labeled antisense probe was generated as described for PPAR{alpha}. An 80-bp fragment of 18S (Ambion, Inc.) or a 126-bp fragment of ß-actin (Ambion, Inc.) was used as internal controls. Neither 18S nor ß-actin mRNA was regulated by the various hormonal treatments used in this study.

The RNA probes were hybridized to the sample RNA in a ribonuclease protection assay using an RPA III kit (Ambion, Inc.). The protected fragments were separated on denaturing 6% polyacrylamide Tris base, boric acid, EDTA-urea gels (Novex, San Diego, CA) and transferred to Bright Star-Plus membranes (Ambion, Inc.) by a transfer system (Transblot cell; Bio-Rad Laboratories, Inc., Hercules, CA). After the transfer, the protected fragments were cross-linked to the membrane by UV irradiation (UVC Crosslinker, Hoefer Pharmacia Biotech, San Francisco, CA). The detection was carried out using the Bright Star BioDetect Kit as described by the manufacturer (Ambion, Inc.). The chemiluminescence was detected using a Fluor-S-Multimager (Bio-Rad Laboratories, Inc.) and the band intensity was quantified with ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). The amounts of the transcripts are expressed as the ratio between the PPAR{alpha} or CYP2C11 and internal control band.

Western blot
Nuclei preparations from frozen liver tissue were performed as previously described (16). Protein concentrations were determined with RC DC protein assay kit II (Bio-Rad Laboratories, Inc.). Western blotting was performed using an enhanced chemiluminescence protocol (Amersham Biosciences, Buckinghamshire, UK). Thirty micrograms of protein were separated on 8 or 10% polyacrylamide Tris-glycine gels (Novex, San Diego, CA). After electrophoresis, the proteins were transferred to Hybond-P polyvinylidene difluoride transfer membrane (Amersham Biosciences) in transfer buffer [25 mM Bis-Tris (pH 7.6) with 192 mM glycine and 25% methanol] for 2.5–3 h at 400 mA (Transblot cell, Bio-Rad Laboratories, Inc.). Equal loading was confirmed by staining the membranes with 0.2% Ponceau S (Serva, Heidelberg, Germany). The molecular mass standard Full Range Rainbow marker (Amersham Biosciences) was used. The membrane was blocked overnight at 4 C in 50 mM Tris-buffered saline, pH 7.6, containing 0.1% Tween-20 (TBS-T) and 5% nonfat milk and then incubated for 1 h with affinity purified polyclonal PPAR{alpha} antibodies (16) diluted 1/500 or monoclonal histone H1 antibodies (sc-8030, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1/100 in TBS-T and 5% nonfat milk. The membrane was incubated for 1 h with peroxidase labeled antirabbit IgG (Amersham Biosciences) diluted 1/2500 (PPAR{alpha}) or peroxidase-labeled antimouse IgG (Amersham Biosciences) diluted 1/10000 (histone H1). Detection and development was performed using enhanced chemiluminescence detection system. The chemiluminescence was measured using a Fluor-S-Multimager (Bio-Rad Laboratories, Inc.) and the band intensity was quantified with ImageQuant software (Molecular Dynamics, Inc.). The amount of the PPAR{alpha} is expressed as the ratio between the PPAR{alpha} and histone H1 control band.

Statistics
Values are expressed as the mean ± SEM. Comparison between groups was made using either Student’s t test or one-way ANOVA followed by Bonferroni’s test between individual groups. The values were transformed to logarithms when appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sex difference in hepatic PPAR{alpha} expression
To investigate whether the sex difference in responsiveness to fibrates is correlated to a sex difference in the expression of PPAR{alpha} mRNA, the expression of PPAR{alpha} in normal intact male and female rats was measured. Two groups of male and female rats were used. One group was killed in the morning (0930–1030 h) and the other in the early afternoon (1330–1430 h) (Fig. 1AGo). The levels of PPAR{alpha} mRNA were significantly higher in males compared with females at both time points (Fig. 1AGo). No significant variation in PPAR{alpha} mRNA levels was observed between the groups killed in the morning and the early afternoon. To investigate whether hepatic PPAR{alpha} protein levels also differ between sexes, nuclear preparations from male and female rat livers were analyzed with Western blot (Fig. 1BGo). The amount of histone H1 in the nuclear preparations was not different between male and females. Therefore, the amount of PPAR{alpha} was expressed as the ratio between the intensity of the PPAR{alpha} and histone H1 bands. Male rats had markedly higher expression of the PPAR{alpha} protein than female rats (Fig. 1BGo).



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  		Figure 1. Hepatic PPAR{alpha} mRNA (A) and PPAR{alpha} protein expression (B) in the liver of female and male rats. Normal intact animals were kept under standardized conditions during 1 wk before the experiment. The rats were about 50 d of age at the end of the experiment. One group of rats was killed between 0930 and 1030 h (morning) and the other group between 1330 and 1430 h (afternoon). PPAR{alpha} mRNA was determined by a ribonuclease protection assay using 20 µg total RNA (A). PPAR{alpha} protein expression was determined in 30 µg nuclear protein using Western blot (B). PPAR{alpha} protein levels are given as the ratio between the intensity of the PPAR{alpha} and histone H1 bands. Two representative samples from each group are shown below the columns in panels A and B. Values are expressed as means ± SEM of results obtained in six animals in each group in panel A and in five to six animals in each group in panel B. Values with different superscripts were significantly different from each other (P < 0.05; panel A, one-way ANOVA followed by Bonferroni’s test; panel B, Student’s t test).

 
Sex difference in PPAR{alpha} mRNA after fasting
The induction of PPAR{alpha} mRNA expression seen in fasted animals has been shown to be of importance for metabolic adjustments during fasting (15, 48). In the next experiment, we therefore wanted to evaluate whether there is a sex difference in PPAR{alpha} mRNA expression in the rat liver after 20 h of fasting and if male and female rats respond differently to fasting with respect to PPAR{alpha} mRNA expression (Fig. 2Go). Normal intact animals were kept under standardized conditions during 1 wk, and food was withdrawn from half of the rats 20 h before the animals were killed (0900–1000 h). The results showed that fasting increased the expression of PPAR{alpha} mRNA to a similar degree in both sexes (Fig. 2Go). In males, PPAR{alpha} mRNA levels increased by 72% and in females the expression increased by 52%.



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  		Figure 2. Effect of fasting on PPAR{alpha} mRNA expression in the liver of male and female rats. Normal intact animals were kept under standardized conditions during 1 wk before the experiment. Food was withdrawn for 20 h before they were killed (fasted) at about 50 d of age. PPAR{alpha} mRNA was analyzed by ribonuclease protection assay using 20 µg total RNA. Values are expressed as means ± SEM of results obtained in five to six animals in each group. Two representative samples from each group are shown below the columns. Values with different superscripts were significantly different from each other (P < 0.05, one-way ANOVA followed by Bonferroni’s test).

 
Effects of hypophysectomy on PPAR{alpha} mRNA levels in male and female rats
To investigate whether the sex difference in PPAR{alpha} mRNA expression is dependent on pituitary dependent hormones, we compared the effect of hypophysectomy in male and female rats (Fig. 3Go). The hypophysectomized rats, treated with L-thyroxin and cortisol during 7 d, were compared with normal age matched rats. The results showed that the hypophysectomized rats of both sexes had higher PPAR{alpha} mRNA levels than their gender controls and that hypophysectomy eliminated the sex difference (Fig. 3Go). After hypophysectomy, PPAR{alpha} mRNA levels increased 3-fold in females, but only by 43% in males. This finding indicates that pituitary dependent hormones have an inhibitory effect on PPAR{alpha} expression in both males and females, but the sum of the inhibitory influences is more pronounced in female rats.



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  		Figure 3. Effect of hypophysectomy on liver expression of PPAR{alpha} mRNA in male and female rats. Female and male rats were hypophysectomized (Hx) at 42–43 d of age and hormone therapy started 7–10 d later. Normal (N) age-matched female and male rats were compared with Hx females and males treated with L-thyroxin (10 µg/kg·d) and cortisol (400 µg/kg·d) for 7 d. PPAR{alpha} mRNA was analyzed by ribonuclease protection assay using 20 µg of total RNA. There were six rats in each group. Values are expressed as means ± SEM. Two representative samples from each group are shown below the columns. Values with different superscripts were significantly different from each other (P < 0.05, one-way ANOVA followed by Bonferroni’s test).

 
Effect of different modes of GH administration on PPAR{alpha} mRNA levels
The next experiments were designed to investigate the contribution of the sexually dimorphic secretory pattern of GH on the different PPAR{alpha} mRNA expression in male and female rats (Figs. 4Go and 5Go). Hypophysectomized females were given GH for 7 d, either as a continuous infusion or as two daily injections to mimic the female and male specific secretory pattern of GH, respectively (33, 38, 39) (Fig. 4Go). The rats that were given GH as two daily injections were killed either 2–2.5 h (1000–1030 h) or 6–6.5 h (1400–1430 h) after the last injection. Continuous infusion of GH, mimicking the female secretory pattern, significantly decreased the PPAR{alpha} mRNA expression (Fig. 4Go). Two daily injections of GH, mimicking the male secretory pattern, had no significant effect. However, the effect of a continuous infusion of GH did not significantly differ from the effect of two daily injections of GH (Fig. 4Go). Similar results were obtained when hypophysectomized males were treated in the same way (data not shown).



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  		Figure 4. Effects of different modes of GH administration on PPAR{alpha} mRNA expression in the liver. Female rats were hypophysectomized (Hx) at 42–43 d of age and hormone therapy was started 7–10 d later. All Hx rats were treated with L-thyroxin (10 µg/kg·d) and cortisol (400 µg/kg·d). Bovine GH (0.7 mg/kg·d) was given for 7 d either as a continuous infusion (GH c) using osmotic mini-pumps implanted sc or as two daily sc injections (GH x 2) at 0800 h and 2000 h. The rats that were given GH as two daily injections were killed either 2–2.5 or 6–6.5 h after last injection (inj.). Values represent means ± SEM of six animals per group. Two representative samples from each group are shown below the columns. Values with different superscripts were significantly different from each other (P < 0.05, one-way ANOVA followed by Bonferroni’s test).

 


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  		Figure 5. Effects of a continuous infusion of GH to male and female rats on PPAR{alpha} mRNA (A) and CYP2C11 mRNA (B). Male and female rats were treated with a continuous infusion of bovine GH (GH c; 0.5 mg/kg·d) by means of osmotic minipumps for 1 wk. PPAR{alpha} mRNA and CYP2C11 mRNA were analyzed by ribonuclease protection assay using 20 µg of total RNA. Data in panels A and B represent means ± SEM of seven to eight animals per group. Two representative samples from each group are shown below the columns in panel B. Values with different superscripts were significantly different from each other (P < 0.05, one-way ANOVA followed by Bonferroni’s test).

 
To further elucidate the role of the secretory pattern of GH in the regulation of PPAR{alpha}, normal male and female rats were treated with a continuous infusion of GH (0.5 mg/kg·d) for 7 d (Fig. 5Go). If the female secretory pattern of GH is of importance for the lower levels of PPAR{alpha} mRNA in females, a continuous infusion of a low dose of GH would decrease the expression of PPAR{alpha} mRNA only in males. However, GH infusion had no effect in either males or females (Fig. 5AGo). To investigate whether the continuous infusion of GH to males resulted in the expected feminization of the males, CYP2C11 mRNA expression was analyzed (Fig. 5BGo). As expected (36), a continuous infusion of GH markedly decreased CYP2C11mRNA levels in the males, but not in the females. Taken together, these results suggest that the sex difference in PPAR{alpha} expression in rat liver is not regulated by the sexually dimorphic secretory pattern of GH.

The lack of a consistent effect of two daily injections of GH on the expression of PPAR{alpha} may be due to a rapid and transient effect of each injection of GH. We therefore investigated the effect of a single sc injection of GH to hypophysectomized rats and measured the PPAR{alpha} mRNA expression at several time points after the injection (Fig. 6Go). Hypophysectomized females were treated with L-thyroxin and cortisol for 3 d and thereafter given a single sc injection of GH (2 mg/kg). PPAR{alpha} mRNA levels markedly decreased 3 h after the GH injection, but the levels were not significantly affected at any other time point (Fig. 6Go). These results indicate that a single sc injection of GH rapidly and transiently decreases PPAR{alpha} mRNA levels.



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  		Figure 6. Effect of a single sc injection of GH (2 mg/kg) to hypophysectomized female rats on PPAR{alpha} mRNA expression. Female rats were hypophysectomized at 42–43 d of age, and hormone therapy was started 7–10 d later. All hypophysectomized rats received L-thyroxin (10 µg/kg·d) and cortisol (400 µg/kg·d) therapy for 3 d before the experiment. The rats were killed at the indicated times after the GH injection. PPAR{alpha} mRNA was analyzed by ribonuclease protection assay using 20 µg of total RNA. The values represent the means ± SEM of four animals per group. Two representative samples from each group are shown below the columns. Values with different superscripts were significantly different from each other (P < 0.05, one-way ANOVA followed by Bonferroni’s test).

 
Expression of PPAR{alpha} in liver, heart, and soleus muscle after hypophysectomy and GH treatment
To evaluate whether the effects of hypophysectomy and GH were specific to the liver, we also measured the levels of PPAR{alpha} mRNA in heart and soleus muscle (Table 1Go). Normal female rats were compared with hypophysectomized rats given combined L-thyroxin and cortisol treatment and with hypophysectomized rat given GH as a continuous infusion in addition to L-thyroxin and cortisol. As previously shown (19), the hypophysectomized females had markedly higher hepatic PPAR{alpha} mRNA levels and GH treatment decreased the levels back to normal (Table 1Go). However, in the heart and the soleus muscle the expression of PPAR{alpha} mRNA was not changed (Table 1Go). Therefore, the effect of hypophysectomy and GH on PPAR{alpha} mRNA levels is specific to the liver. We extended these findings by investigating the effects of hypophysectomy and GH treatment on PPAR{alpha} protein expression in nuclear preparations (Fig. 7Go). These results show that hypophysectomy and GH regulate PPAR{alpha} protein levels in parallel with the mRNA levels in the liver. The expression of histone H1 was not influenced by the various hormonal treatments and therefore used as internal control.


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  		Table 1. Effects of Hx and GH on expression of PPAR{alpha} mRNA in liver, heart, and soleus muscle in female rats

 


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  		Figure 7. Effect of hypophysectomy and GH treatment on hepatic PPAR{alpha} protein expression. Female rats were hypophysectomized (Hx) at 42–43 d of age, and hormone therapy was started 7–10 d later. All Hx rats were given L-thyroxin (10 µg/kg·d) and cortisol (400 µg/kg·d) therapy. Bovine GH (0.7 mg/kg·d) was given as GH c using osmotic mini-pumps. The rats were killed after 7 d of treatment. Liver PPAR{alpha} protein expression was determined in 30 µg nuclear protein using Western blot. PPAR{alpha} protein levels are given as the ratio between the intensity of the PPAR{alpha} and histone H1 bands. Two representative samples from each group are shown below the columns. The values are expressed as means ± SEM of four animals per group. Values with different superscripts were significantly different from each other (P < 0.05, one-way ANOVA followed by Bonferroni’s test).

 
Effects of sex steroids and gonadectomy on PPAR{alpha} mRNA levels
The next experiments were performed to evaluate the importance of gonadal steroids for the sex-specific expression of PPAR{alpha} mRNA (Fig. 8Go). Normal female rats were treated with testosterone (1 mg/kg·d) and normal male rats were treated with 17ß-estradiol (1 mg/kg·d) for 1 wk using osmotic mini-pumps. The doses of sex steroids were chosen to ensure a masculinization and feminization of lipid metabolism in the rat (44, 45, 46), including peroxisomal ß-oxidation (26). Normal age matched animals of both sexes served as controls. The statistical analysis (one-way ANOVA) showed a significant effect of the treatments on PPAR{alpha} mRNA levels (Fig. 8Go), but the post hoc test (Bonferroni) showed no significant pair-wise effects. However, the results clearly indicate opposite effects of testosterone and 17ß-estradiol on the expression of PPAR{alpha} mRNA. The male rats treated with 17ß-estradiol had a significantly lower body weight gain than untreated males and testosterone treated female rats (data not shown).



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  		Figure 8. Effects of sex steroids on the expression of PPAR{alpha} mRNA. Normal female rats were treated with testosterone (T; 1 mg/kg·d) and normal male rats were treated with 17ß-estradiol (E2; 1 mg/kg·d) for 1 wk using osmotic mini-pumps. Age-matched rats of both sexes served as controls. The rats were about 60 d of age at the end of the experiment. PPAR{alpha} mRNA was analyzed by ribonuclease protection assay using 20 µg of total RNA. Experimental data represent the means ± SEM of four to five animals per group. Comparison between means by one-way ANOVA showed that P < 0.05, but Bonferroni’s test showed no significant differences between individual groups.

 
To further evaluate the importance of the gonadal steroids for the sex difference in PPAR{alpha} mRNA levels, both male and female rats were gonadectomized (Fig. 9Go). After the operation, the animals were kept under standardized conditions during the 3 wk before they were killed. Sham-operated age-matched rats of both sexes served as controls. Gonadectomy of male rats resulted in significantly lower expression of PPAR{alpha} mRNA in the liver compared with control male rats. In female rats, gonadectomy resulted in a trend toward an increase in PPAR{alpha} mRNA expression (Fig. 9AGo). In the same experiment, we also measured the levels of PPAR{alpha} mRNA in heart and soleus muscle to study if the sex difference and effect of gonadectomy on PPAR{alpha} mRNA levels are liver-specific effects (Fig. 9Go, B and C). The results show that there was no sex difference or effect of gonadectomy on PPAR{alpha} mRNA expression in the heart or soleus muscle (Fig. 9Go, B and C). Gonadectomy of males and females did not affect their weight gain compared with sham-operated gender controls during the 3-wk period (data not shown).



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  		Figure 9. Effects of gonadectomy (Gx) on liver (A), heart (B), and soleus muscle (C) expression of PPAR{alpha} mRNA. Female and male rats were gonadectomized at 35 d of age. Sham-operated, age-matched rats of both sexes served as controls. Animals were kept under standardized conditions during 3 wk before the rats were killed. PPAR{alpha} mRNA was analyzed by ribonuclease protection assay using 20 µg of total RNA. Experimental data represent means ± SEM of six animals per group. Values with different superscripts were significantly different from each other (P < 0.05, one-way ANOVA followed by Bonferroni’s test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that the expression of PPAR{alpha} mRNA and PPAR{alpha} protein levels are higher in male than in female rat liver. It is shown that the gonads regulate the sex difference in PPAR{alpha} expression, but not via the sexually dimorphic secretory pattern of GH. Our data indicate that GH decrease PPAR{alpha} expression in a similar manner whether or not it is given in a pulsatile or a continuous manner. Therefore, the lack of GH explains the increase in expression of PPAR{alpha} in both sexes after hypophysectomy. We show that gonadectomy decreases PPAR{alpha} levels in males but rather the opposite occur in females. Moreover, testosterone treatment and estradiol treatment of intact rats reversed the sex difference. Therefore, the less marked effect of hypophysectomy in males than in females is probably due to the lack of the stimulatory influence of testosterone that nearly balances out the effect of GH deficiency.

The fact that male rats are more responsive to peroxisome proliferators than female rats (22, 24, 25, 26, 27, 28, 29) may therefore, at least in part, be explained by our finding that PPAR{alpha} is more abundant in male rats than in female rats. It has been suggested that male rats are 32- to 64-fold more sensitive than females to peroxisome proliferators (26) and an increased sensitivity in males is in line with a higher expression of the receptor. Fasting induced an increase in hepatic PPAR{alpha} mRNA levels of a similar magnitude in male and female rats. Thus, the higher expression of PPAR{alpha} in males did not result in a larger increase in PPAR{alpha} gene expression in males than in females after fasting.

We observed that PPAR{alpha} protein levels varied in parallel with the mRNA levels showing for the first time that gender, hypophysectomy, and GH have marked effects on the hepatic expression of the protein. Because the intensity of the PPAR{alpha} protein bands from female rat livers was very low the difference in expression observed between males and females may have been overestimated. However, this study and others indicate that mRNA and protein levels of PPAR{alpha} closely follow each other (13, 16, 49), indicating that the receptor expression is regulated primarily at the mRNA level.

We were not able to detect a significant difference in PPAR{alpha} mRNA levels between the rats that were killed in the morning and early afternoon in either male or female rats. This finding is in contrast with an earlier observation of an increased PPAR{alpha} mRNA expression in the afternoon compared with the morning in male rats (16). The reason for the discrepant results is unclear, but the use of different rat strains may be of importance. Another more likely possibility is the different light cycles used in the two studies. The lights were on from 0500 h in this study and from 0730 h in the study of Lemberger et al. (16). Thus, the different dark-light cycles probably resulted in comparably higher PPAR{alpha} levels in this study at 0930 h than in the other study (16).

The physiological function of a sex difference in PPAR{alpha} expression is unclear. However, studies performed in PPAR{alpha}-knockout mice clearly show that male and female mice respond differently to PPAR{alpha} deficiency. Inhibition of mitochondrial ß-oxidation with etomoxir resulted in massive hepatic lipid accumulation and hypoglycemia in PPAR{alpha} null mice of both sexes. However, 100% of the male mice died from this treatment, whereas only 25% of the females died (50). We have shown that female PPAR{alpha} null mice have higher hepatic secretion of triglycerides, as well as higher serum triglyceride and apolipoprotein B levels than male PPAR{alpha} null mice; sex differences that were not detected in wild-type mice (51). Together, our finding of a sexual dimorphism in PPAR{alpha} expression and the sexual dimorphism observed in PPAR{alpha}-deficient mice show that PPAR{alpha} and gonadal hormones interact in the regulation of metabolism.

It has been shown that the hepatic expression of PPAR{alpha} mRNA is decreased by a continuous infusion of GH in hypophysectomized female (19) and male rats (20). However, no studies have previously investigated the role of the sexually dimorphic secretory pattern of GH in the regulation of PPAR{alpha} expression. Because several sexually dimorphic functions in the liver are under the regulatory control of the male and female secretory pattern of GH (33, 34, 37, 38, 39, 40, 41), we investigated the possibility that the sex difference in PPAR{alpha} expression was regulated by the secretory pattern of GH. It has been shown previously that the male pattern of GH secretion can be mimicked in hypophysectomized rats by giving one or two daily sc injections of GH (33, 38, 39). The prerequisite for a masculinization of this mode of administration is that the levels of GH are very low between the pulses, as indicated by the findings that more frequent injections of GH result in feminization (36, 38, 39). However, the levels of PPAR{alpha} mRNA were not significantly different from hypophysectomized controls or hypophysectomized rats given a continuous infusion of GH when the rats were treated with two daily injections of GH. We therefore performed two additional experiments to rule out the possibility that the secretory pattern of GH is of importance in the regulation of PPAR{alpha}. The experiment when one injection of GH was given to hypophysectomized female rats showed that GH pulses decrease PPAR{alpha} mRNA and that the time-point of measurement is of major importance for the observed effect. Furthermore, if the continuous secretion of GH in female rats had been of importance for the lower expression of PPAR{alpha} mRNA in females, a continuous infusion of GH given to normal male rats would have decreased the expression of PPAR{alpha} mRNA in a similar manner as CYP2C11 mRNA expression. Therefore, it can be concluded that GH decreases PPAR{alpha} mRNA, but the secretory pattern of GH cannot explain the sex difference in PPAR{alpha} mRNA expression.

The single injection of GH given to hypophysectomized rats resulted in a marked decrease in PPAR{alpha} mRNA within 3 h. This rapid decrease is consistent with the marked and rapid decrease in PPAR{alpha} mRNA expression shown to occur in the late afternoon in the rat (16) and after injection of hamsters with lipopolysaccharides (52). Our results and the results of others therefore suggest that PPAR{alpha} mRNA has a short half-life. The dose given as a single injection in this experiment was about 6-fold higher than the dose given when GH was given as two daily injections or 54% higher than the diurnal total secretion of GH in the rat. We expect from the results of two daily injections of GH to hypophysectomized male and female rats (see Fig. 4Go) that there is also a transient but less marked decrease in PPAR{alpha} mRNA using a more physiological dose of GH.

It has been shown that the effect of fibrates on peroxisomal ß-oxidation is dependent on the mode of GH administration (29), indicating that the sex difference in peroxisomal ßoxidation, in contrast to PPAR{alpha} expression, is dependent on the sexually dimorphic secretory pattern of GH. It was shown that hypophysectomy enhances the fibrate induction of the hepatic peroxisomal ß-oxidation and that a continuous infusion of GH, in contrast to two daily injections of GH, suppressed the peroxisomal ß-oxidation (29). However, in these experiments it was not indicated at which time point after the last injection of GH the measurements were performed. Moreover, it was shown that testosterone enhanced the effect of fibrates (29). Our experiments also indicate that testosterone increases PPAR{alpha} mRNA expression, but the interaction between sex hormones and GH on PPAR{alpha} expression remains to be studied.

In humans, the regulation of PPAR{alpha} in the liver is much less studied. The human liver contains less PPAR{alpha} than in the rat, but the expression can vary by up to an order of magnitude between individuals (53, 54). No information is available regarding a sex difference in the PPAR{alpha} expression in human liver. However, indirect measurements suggest that men may have a higher hepatic expression of PPAR{alpha} than women. In both rats and human, males are less susceptible to ethanol with respect to accumulation fatty acids in the liver (31, 32). These studies indicate that men accumulate less hepatic fatty acids because ethanol consumption induce {omega}-oxidation and peroxisomal ß-oxidation more markedly in men than in women (32). In line with the assumption that PPAR{alpha} expression is involved in this sex difference, fibrate treatment induces CYP4A1 mRNA expression more markedly in male rats than in female rats (28).

In the present study, we conclude that the regulation of PPAR{alpha} expression by gonadectomy, hypophysectomy, and GH treatment is liver specific because there was no change in the expression of PPAR{alpha} mRNA in neither heart nor soleus muscle by these hormonal manipulations. The finding of unchanged gene expression of PPAR{alpha} in the heart after hypophysectomy and GH treatment is in line with a previous DNA microarray study (20). Interestingly, it was shown recently that fasting induces an increased expression of PPAR{alpha} mRNA only in the liver and the small intestine, in contrast to 20 other investigated rat tissues including muscle (55). Thus, the expression of PPAR{alpha} mRNA in the liver, but not in the skeletal muscle or heart, seems to be regulated by various factors including fasting and pituitary-dependent hormones. However, the effect of hormones on PPAR{alpha} expression in other tissues with a high expression of this receptor such as the small intestine remains to be investigated.


   Footnotes
 
This work was supported by Grants 8269 and 14291 from the Swedish Medical Research Council, King Gustav V’s and Queen Victoria’s Foundation and Swiss National Science Foundation (to W.W.).

1 M.J. and L.C. have contributed equally to this article. Back

Abbreviations: GH c, Continuous infusion of GH; PPAR, peroxisome proliferator-activated receptor.

Received June 17, 2002.

Accepted for publication October 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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