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Molecular Endocrinology, doi:10.1210/me.2006-0152
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Molecular Endocrinology 21 (2): 388-400
Copyright © 2007 by The Endocrine Society

A Novel Peroxisome Proliferator-Activated Receptor Responsive Element-Luciferase Reporter Mouse Reveals Gender Specificity of Peroxisome Proliferator-Activated Receptor Activity in Liver

Paolo Ciana1, Andrea Biserni1, Laura Tatangelo, Cecilia Tiveron, Anna Floriana Sciarroni, Luisa Ottobrini and Adriana Maggi

Center of Excellence on Neurodegenerative Diseases, Department of Pharmacological Sciences (P.C., A.B., L.O., A.M.), and Institute of Radiological Sciences (A.B., L.O.), University of Milan, 20133 Milan, Italy; Regina Elena Institute (L.T., C.T.), 00158 Rome, Italy; and Sigma-Tau Industrie Farmaceutiche Riunite S.p.A. (A.F.S.), Department of Endocrinology and Metabolism, 00040 Pomezia, Italy

Address all correspondence and requests for reprints to: Adriana Maggi, Center of Excellence on Neurodegenerative Diseases, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. E-mail: adriana.maggi{at}unimi.it.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
There is a growing interest in peroxisome proliferator-activated receptors (PPARs) as major players in the regulation of lipid and carbohydrate metabolism. Drugs targeting PPARs were in fact shown to have major relevance for the treatment of diseases associated with aging, such as arteriosclerosis and diabetes. However, a variety of toxic effects associated with PPAR ligand administration has been documented, including hepatocarcinogenesis, which may severely limit its therapeutic use. A better comprehension of the multiplicity of PPAR physiological functions is therefore mandatory for the development of novel, safer drugs. We here describe the generation of a novel transgenic mouse for the detection of the generalized activities of PPARs, the PPAR responsive element-Luc reporter mouse. In this model luciferase expression is under the control of a PPAR-inducible promoter in all target organs. By optical imaging and ex vivo analysis, we were able to demonstrate the remarkable gender specificity of the PPAR transcriptional activity in liver. In fact, in the liver of female PPAR responsive element-Luc, the PPAR reporter transgene is more than one order of magnitude less expressed, thus leading to the conclusion that the signaling in females is much less activated than in males. Diet or hormonal manipulations as demonstrated here by treatments with high-fat diet or gonad removal and hormone replacement do not influence this low activation. The extent of the gender difference in PPAR transcriptional activity and the ineffectiveness of hormone treatments or diet to significantly elevate liver PPAR activity in females led us to hypothesize that gender-specific epigenetic events occurring during development may affect PPAR signaling in the liver. This study sets the ground for understanding the differential susceptibility of the two genders to metabolic disorders; furthermore, the model generated provides a novel opportunity for the molecular characterization of PPAR activity in pathophysiological conditions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PEROXISOME PROLIFERATOR-ACTIVATED receptors (PPARs) are of major clinical interest because of their significant role in the etiopathogenesis of metabolic diseases such as diabetes, arteriosclerosis, other cardiovascular pathologies (1), and rare genetic diseases such as X-linked adrenoleukodystrophy (2). PPARs are transcription factors activated by fatty acids and eicosanoids (3, 4, 5). So far, three receptor subtypes have been characterized and named PPAR{alpha}, -{gamma}, and -{delta} (also known as PPARß). To regulate gene transcription, PPARs heterodimerize with the retinoid-X-receptor and bind to PPAR responsive elements [PPREs, also known as direct repeats (DR1)] (6, 7) located in the promoter of target genes. PPAR subtypes have distinct tissue localization and physiological activities: PPAR{alpha} is mainly expressed in liver and heart, where it operates as a major regulator of lipid catabolism (8); PPAR{gamma} is the prevailing subtype in adipocytes where, by controlling lipid remodeling and adipocyte differentiation, it plays a key role in lipid storage and influences, indirectly, carbohydrate metabolism (9); the physiological role of PPAR{delta}, the ubiquitously expressed subtype, is less well characterized, even though a growing number of studies suggests its involvement in lipid combustion in skeletal muscles and heart (10). The finding that drugs that are widely used for the prevention and treatment of arteriosclerosis and diabetes, such as fibrates and thiazolidinediones, target PPARs (11, 12) further stresses the pathophysiological relevance of these receptors and their importance as novel drug targets.

Thus, in view of the importance of PPAR functions, a major effort should be made to generate model systems that would enable the acquirement of an in-depth view of the physiological functions of these receptors and a better evaluation of the systemic actions of their synthetic ligands.

Our laboratory recently developed a reporter mouse technology for the ubiquitous and quantitative measurement of the transcriptional activity of intracellular receptors (13). These mice are generated by genomic integration of a transgene flanked by insulators and carrying a reporter gene under the control of a specific receptor-responsive regulatory element. Previous studies demonstrated that this construct leads to generalized and hormonally controlled expression of the reporter (14). The use of bioluminescent luciferase as a reporter system allows for the quantitative analysis of the activity of a specific receptor class in space and time also in living animals. This mouse technology provides an unprecedented opportunity to gain novel insights into nuclear receptor physiological functions and to identify novel drugs for their selective regulation (15, 16). Indeed, the first prototype of reporter mouse generated, estrogen receptor responsive element-Luc model, originally proposed as a tool to identify selective estrogen receptor modulators (14), significantly facilitated the study of the pharmacological activity of estrogen receptor ligands (15, 17, 18, 19, 20). In addition, this mouse model revealed novel and unsought mechanisms controlling estrogen receptor activity (21, 22, 23).

The aim of the present study was to generate a PPRE-Luc reporter mouse as a model to investigate PPAR physiological activity and as a test system for innovative therapies for metabolic disorders. The characterization of the PPRE-Luc mouse revealed a gender difference in the activity of hepatic PPARs by demonstrating that, in male mice, the PPARs are more transcriptionally active than in female mice. This dimorphism in PPAR activity is restricted to liver and is not observed in other organs. Interestingly, the regulation of liver PPAR activity by physiological stimuli such as fasting maintains the liver PPAR differential activity in the two genders.

The relevance of the study is 2-fold: We demonstrate for the first time the significant difference in liver PPAR activity in the two genders, and we provide a novel model for the study of PPAR activity in living animals.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of the Transgene: The Highest Response to PPARs Is Obtained with Five Tandem Repeats of the PPRE
Multimers of the DR1 (6, 7) sequence spaced by one nucleotide were cloned upstream of a minimal thymidine kinase (tk) promoter, driving the transcription of the firefly luciferase reporter gene (Fig. 1AGo). Matrix attachment regions (MAR) from chicken lysozyme (24) flanked the constructs and were previously described as elements indispensable to obtain the generalized expression of the reporter (14). Next, promoter inducibility by PPARs was tested by transient transfections in COS 7 and HepG2 cells treated with 5 µM Wy-14,643 (PPAR{alpha} agonist) and 0.5 µM Rosiglitazone (a thiazolidinedione, PPAR{gamma} agonist) for 16 h. In these experiments, reporter constructs were cotransfected with expression vectors encoding for PPAR{alpha} or PPAR{gamma} subtypes. Figure 1BGo shows that, in the two cell lines considered, the extent of luciferase accumulation induced by the synthetic ligands was directly proportional to the number of PPREs present in the promoter region of the transgene.


Figure 1
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  		Fig. 1. Selection of the Most Responsive Reporter Transgene

A, Four vectors were generated with a variable number of DR1 sequence in a tandem repeated arrangement (1X, 2X, 3X, 5X) and cloned upstream a minimal tk promoter, driving the expression of the luciferase reporter gene. The constructs were flanked by MAR insulator elements from chicken lysozyme gene. B, Promoter inducibility was tested by transient transfection experiments in COS 7 and HepG2 cell lines after treatment with 5 µM Wy-14,643 (PPAR{alpha} agonist) and 0.5 µM Rosiglitazone (PPAR{gamma} agonist) for 16 h. Bars represent the mean ± SEM of three independent experiments, each done in triplicate. *, P < 0.01 as compared with the control; **, P < 0.005 as compared with the control. P values were calculated with ANOVA followed by Scheffé’s test.

 
Identification of the Mouse Line Best Suited to Be Used as PPAR Reporter
On the basis of the transfection experiments reported above, we selected the construct pMAR-PPRE5X-tk-Luc-MAR carrying five PPRE DR1 for the subsequent injection in fertilized oocytes and generation of the novel reporter mice in the C57Bl/6xDBA/2 F2 strain. Of the 22 individual lines obtained, only 13 were viable and grown to generate an independent colony. To establish the responsiveness of the reporter, we first screened the animals using bioluminescence analysis. To this aim, mice were injected with luciferine, as described in the Materials and Methods section, and photon emission was measured by optical imaging with a charge-coupled device (CCD) camera. Using appropriate software, we could visualize the body regions where luciferase, by acting on the substrate luciferine, generated photons. To select the mouse lines in which the reporter was expressed by the transgene and induced by PPAR agonists, photon emission was measured in four heterozygous females/line before and after 6 h of a sc injection of 50 mg/kg PPAR{alpha} agonist (Wy-14,643) or 10 mg/kg PPAR{gamma} agonist (Rosiglitazone) (data not shown). In five lines, luciferase was below the limit of CCD camera detection, and these lines were discarded; therefore, the study was continued in the remaining eight lines, with which we could prove a generalized expression of the reporter (Table 1Go), measuring luciferase content in different tissues by enzymatic assay. Due to the widespread presence of PPARs, luciferase expression could be measured in most tissues, with sporadic exceptions (e.g. line 16). Interestingly, in all lines a similar pattern of reporter expression was detected with maximal luciferase activity in the brain and intestine; this reproducible pattern is indicative of a minimal influence of position effects on the transgene expression.


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  		Table 1. Luciferase Expression in Females PPRE-Luc Mice

 
Considering luciferase expression before and after treatments with the PPAR{alpha} and PPAR{gamma} agonists, line 18 was selected as the line to be used for further studies and was named PPRE-Luc.

Luciferase Expression in PPRE-Luc Mice after Pharmacological Treatments
To evaluate the extent to which luciferase accumulation was dependent on PPAR transcriptional activity, we investigated the rate of luciferase accumulation after treatment with a selected PPAR agonist and the concentration of the PPAR agonist necessary to induce luciferase expression. The study was done using photon counting. To limit the experimental variability due to hormonal and nutritional effects on PPARs, the study was carried out in male mice fed with a schedule experimentally set up to lower the background receptor activity; thus, male PPRE-Luc mice were fed only at night for the two nights preceding bioluminescence imaging, which was routinely carried out in the morning starting from 0900 h. This experimental design reduced significantly the background luminescence and led to a variability among the different mice below 20% of standard deviation, when experimental groups of five mice were used (data not shown).

First, a time-course analysis was done by measuring luciferase-dependent bioluminescence in mice treated sc with vehicle or with 250 mg/kg of the PPAR{alpha} agonist Wy-14,643. Photon counting in chest, abdomen, and limbs was done at time 0, and after 3, 6, and 24 h (Fig. 2AGo). In the 24-h duration of the experiment, photon emission fluctuated reproducibly in the chest of control (vehicle-treated) mice, but not to a significant extent: these changes were ascribed to variation in the nutritional status and to the circadian fluctuation of hepatic PPAR expression. Maximal luciferase accumulation was observed at 6 h after treatment (3.5-fold above time 0); at 24 h, luciferase activity was as low as in controls. A similar pattern of activation was observed in abdomen and limbs.


Figure 2
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  		Fig. 2. Luciferase Activity in PPRE-Luc Mice after Treatment with Synthetic PPAR Ligands

A, Time course: 3- to 5-month-old male heterozygous transgenic mice were injected sc with vehicle (white bars) or 250 mg/kg Wy-14,643 (W250, blue bars). Photon emission was measured as counts per second (cts/s) (as described in Materials and Methods) at 0, 3, 6, and 24 h after treatment. The photon counting (right panel) was made in the specific body areas shown in A, left panel: 1, chest; 2, abdomen; 3, limbs. B and C, Luciferase expression after treatment with different doses of Wy-14,643 and the PPAR{alpha} antagonist MK-886. Male mice were treated sc with vehicle or Wy-14,643 at 50 and 250 mg/kg (W50, W250). Luciferase activity was measured 6 h after treatment as photon emission (B, right panels) or enzymatic assay (C). The PPAR{alpha} antagonist MK-886 was administered sc 30 min before the agonist at the dose of 250 mg/kg (M250). D, mRNA accumulation of the Abcd2 gene was measured by real-time PCR in animals treated as in B. Bars represent the mean ± SEM of five to 10 mice. *, P < 0.01 as compared with the 3- and 6-h vehicle treatment; **, P < 0.005 as compared with the 6-h vehicle treatment; °, P < 0.01 as compared with the W50 treatment; {Delta}, P < 0.01 as compared with the 6-h vehicle treatment. P values were calculated with ANOVA followed by Scheffé’s test. RLU, Relative light units.

 
Once it was established that 6 h of drug treatment resulted in the highest accumulation of luciferase in the PPRE-Luc mice, we tested for the existence of a direct relation between luciferase accumulation and dosage of administered drug. Groups of a minimum of five male mice were treated sc with 50 or 250 mg/kg of Wy-14,643, and luciferase-dependent photon emission was quantified. In addition, mice were euthanized to measure luciferase activity by enzymatic assay. Figure 2BGo shows that photon emission was directly proportional to the dose of the drug used, particularly in chest. Consistent with this finding, we observed that luciferase activity, as measured by enzymatic assay (Fig. 2CGo), increased with the dose of the drug administered in liver. In heart, luciferase activity was relatively elevated in controls, and we did not observe a significant increase after the drug treatment. To further prove that PPAR{alpha} activation was responsible for the luciferase accumulation observed, a selective PPAR{alpha} antagonist (MK-886) was co-administered with Wy-14,643. Due to solubility problems, we could not administer MK-886 at a concentration sufficient enough to antagonize the highest dose of Wy-14,643; therefore, the experiment was done only using the lower dosage of Wy-14,643 with a ratio antagonist to agonist of 5:1. Despite the relatively low ratio between agonist and antagonist, MK-886 completely prevented Wy-14,643 effects in liver (Fig. 2CGo). Interestingly, in heart, MK-886 alone significantly reduced luciferase activity, indicating that endogenous ligands were responsible for the PPAR{alpha} activation at time 0 and that, possibly, this basal PPAR activity was the cause for the lack of responsiveness to agonist administration previously shown.

To demonstrate finally that quantification of luciferase activity provided a reliable measurement of the activity of PPAR{alpha} on their endogenous target promoters, the content of ATP-binding cassette, subfamily D, member 2 (Abcd2) mRNA in liver was measured by means of semiquantitative real-time PCR. It is well known that Abcd2 is induced by PPAR{alpha} in mouse liver (25). Abcd2 mRNA in liver was induced dose-dependently by treatment with Wy-14,643, and MK-886 completely blocked the effect of the agonist (Fig. 2DGo). It is worth underlining that, after Wy-14,643 treatment, the increase of luciferase content as measured by enzymatic assay (8.7-fold) was higher than photon imaging (2-fold) or Abcd2 mRNA quantification (3.1-fold). These differences have to be ascribed to a lower efficiency of the in vivo optical imaging approach in detecting variation in the photon emission as compared with the ex vivo enzymatic assay and to the fact that the transgene promoter consists of a multimerized PPRE, possibly slightly amplifying the response to activated PPAR when compared with endogenous genes.

To further strengthen the use of luciferase as reporter of PPAR{alpha} activity, we measured luciferase activity and the mRNA levels of other endogenous target genes in parallel after acute (Fig. 3AGo) and repeated (Fig. 3BGo) oral treatment with 250 mg/kg and 100 mg/kg·d of Wy-14,643, respectively. Acyl-CoA oxidase1, palmitoyl (Acox1), Acyl-CoA synthetase long-chain family member 1 (Acsl1), Cytochrome P450 family 4, subfamily a, polypeptide 14 (Cyp4a14), and ATP-binding cassette, subfamily D, member 3 (Abcd3) were selected as well-known target genes for PPAR{alpha} (25, 26, 27, 28). We also tested the expression of Abcd1, not modulated by PPAR{alpha}, as a negative control (29). The acute treatment determined a significant induction of Acox1 and Acsl1 gene expression (4.8-fold and 3.1-fold, respectively), whereas only a trend to induction was seen in the expression of two other PPAR{alpha} targets, Cyp4a14 (1.6-fold) and Abcd3 (1.3-fold) (Fig. 3AGo). A significant induction was found in all PPAR{alpha} target genes after chronic treatment (Acox1, 4.3-fold; Acsl1, 4.5-fold; Cyp4a14, 20.1-fold; and Abcd3, 4.3-fold) (Fig. 3BGo). As expected, Abcd1 gene was not induced in both treatment conditions. Luciferase activity provided significant data showing the effect of the agonist in all pharmacological manipulations (sc, oral, acute, and chronic), thus demonstrating its usefulness in reporting the state of PPAR{alpha} activity in response to a specific agonist and antagonist.


Figure 3
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  		Fig. 3. Comparison of PPAR{alpha}-Induced Expression of Luciferase and Endogenous Genes

Endogenous gene mRNAs were measured by semiquantitative real-time PCR in liver and expressed as fold change (Wy-14,643 treated vs. vehicle treated). Luciferase enzymatic activity was evaluated in parallel in the same tissue specimen used for the extraction of mRNAs. The experiments were carried out using heterozygous PPRE-Luc males 3 to 5 months old. A, Animals were treated with a single oral dose of 250 mg/kg Wy-14,643 (W250) and killed 6 h after treatment. B, Animals were treated for 5 d with Wy-14,643, 100 mg/kg·d (W100) and killed 6 h after the last treatment. Bars represent the mean ± SEM of five mice. The experiment was repeated twice. *, P < 0.01; **, P < 0.005 as compared with the vehicle treatment. P values were calculated with ANOVA followed by Scheffé’s test. RLU, Relative light units.

 
PPRE-Luc Reveals a Remarkable Gender Difference in PPAR Signaling
By comparing the images and enzymatic assay data obtained in experiments with female and male mice, we observed a significant difference in chest luminescence. Indeed, in mice fed ad libitum, the reporter activity was much higher in males than in females (24-fold higher as measured by photon emission and 57-fold higher as enzymatic activity in liver protein extracts) (Fig. 4Go, A and B). A more extended comparative study, where luciferase enzymatic activity was measured in different organs of the two sexes, confirmed that no significant difference was detectable in all organs with the exception of liver. This lack of gender dimorphism was observed in the absence of pharmacological and dietary treatments (data not shown) or after treatment with Wy-14,643 for 6 h (Fig. 4BGo and supplementary Fig. 1Go, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). The liver gender difference was not a peculiarity of line 18 because this was observed in all the C57Bl/6 lines generated (data not shown). In addition, the liver gender difference could not be ascribed to a specific mouse strain (Fig. 4CGo).


Figure 4
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  		Fig. 4. Luciferase Expression in Male and Female PPRE-Luc Mice

A, Optical imaging and photon counting in untreated male and female C57Bl/6 heterozigous PPRE-Luc mice. B, Luciferase enzymatic activity in tissue extracts of male and female (C57Bl/6) PPRE-Luc mice after sc treatment with 50 mg/kg Wy-14,643 for 6 h. C, Different strains of PPRE-Luc mice were generated by backcross, and photon emission was detected in the chest of males and females. Images were taken after treatment with 50 mg/kg Wy-14,643 for 6 h. Bars represent the mean ± SEM of five to 10 mice. **, P < 0.005 as compared with males. P values were calculated with ANOVA followed by Scheffé’s test. RLU, Relative light units.

 
Intriguingly, in chest of male, but not female mice, luciferase activity appeared to fluctuate during the day and in relation to the animal nutritional status (Fig. 2AGo and supplementary Fig. 2Go). To better assess the effect of nutrition on PPAR activity, independently of any circadian rhythm, PPRE-Luc mice of both sexes were fed either during the day or the night for 2 d. Photon emission and luciferase activity were measured in vivo and in liver dissected on the morning of the third day at 0900 h. Figure 5Go shows that this nutritional schedule affects PPAR activity in males, but not in females. In males fed only during the day, liver PPARs were significantly more activated than in those fed during the night, as shown by stronger bioluminescence (Fig. 5AGo) in chest and by higher luciferase enzymatic activity in liver (Fig. 5BGo). No significant changes were observed in the other parts of the body (such as abdomen). In females this feeding program did not appear to perturb luciferase activity, which remained significantly lower than in males. Similarly to what was observed in males, luciferase activity in abdomen did not change significantly in relation to this nutritional schedule.


Figure 5
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  		Fig. 5. Effect of Different Nutritional States on Luciferase Expression in PPRE-Luc Mice

Two groups of four PPRE-Luc male and four female mice each were fed during the day (0900–1800 h) or during the night (1800–0900 h) for 48 h, and luciferase expression was evaluated at the 0900 h of the third day by photon imaging and enzymatic activity. A, Upper panels: male (left) and female (right) representative individuals from the groups fed either at night or during the day. Lower panels: mean ± SEM of chest and abdomen photon emission of groups of four animals. B, Enzymatic luciferase activity in the liver extracts of males (left) and females (right). Bars represent the mean ± SEM of four mice. *, P < 0.01 as compared with the group fed during the night. P values were calculated with Student’s t test. RLU, Relative light units.

 
It is now well recognized that food deprivation (30, 31) causes a rapid transcriptional activation of genes encoding mitochondrial, peroxisomal, and microsomal fatty acid oxidation in liver of healthy individuals. Some of the critical enzymes of these oxidation systems are transcriptionally controlled by PPAR, and the study of knockout mice identified PPAR{alpha} as the subtype that plays a key role in coordinating the synthesis of the enzymes involved in fatty acid oxidation. To verify the extent to which the effects of food deprivation on PPAR activity were affected by gender, PPRE-Luc mice were deprived of food for 2 d, and photon emission was measured at time 0 (0900 h of the first day), and at 6, 24, and 48 h time points (Fig. 6AGo). In the chest of male mice, we observed a significant increase of photon emission at 6 h (2-fold); this increase has to be ascribed to PPAR{alpha} activation because it was completely prevented by treatment with the specific PPAR{alpha} antagonist MK-886 (250 mg/kg) (Fig. 6BGo).


Figure 6
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  		Fig. 6. Luciferase Expression in Male and Female PPRE-Luc Mice during Food Deprivation

A, Imaging of a single representative mouse at time 0 or after 6, 24, and 48 h of food deprivation; the left panels refer to male and the right panels to female mice. Bars represent the mean ± SEM of photon emission of four mice. B, Quantification of photon emission in the chest area of fasted male PPRE-Luc mice injected sc with the specific PPAR{alpha} antagonist MK-886 (250 mg/kg, M250) or vehicle. Bars represent the mean ± SEM of photon emission of four mice. C, Luciferase enzymatic assay on protein extract from the liver of the same group of animals. Bars represent the mean ± SEM of four mice. *, P < 0.01 as compared with 0 h; °, P < 0.01 as compared with the 6-h-fasted, untreated animals. P values were calculated with ANOVA followed by Scheffé test. RLU, Relative light units.

 
At 24 and 48 h, chest bioluminescence and liver luciferase activity decreased significantly to reach levels below time 0 (Fig. 6Go, A and C). The pattern of PPAR activity in chest of females was similar to males, with a tendency to increase at 6 h and to decline to levels below the limits of detection at 24 and 48 h. In both sexes, the pattern of luciferase expression in the abdomen was different than in liver because it increased significantly at 24 and 48 h.

These results indicated that, with food deprivation, PPAR activity is differentially regulated in chest and abdomen. It is possible that this reflects activation of the various PPAR subtypes known to contribute in a different way to metabolism. Interestingly, females and males appeared to have a very similar response to starvation with abdominal PPAR activation after 24–48 h of food deprivation and rapid induction in chest response (6 h). The response in female chest, however, was quite variable in the experimental group of animals, possibly due to the low photon emission. Next, we verified the extent to which dietary lipids affected PPAR activity in PPRE-Luc mice with a long-term exposure to a high-fat diet. Indeed, hepatic PPAR activity is known to be directly induced by dietary lipids in males (26), but it was unknown whether females had a similar response. Groups of five PPRE-Luc mice of both sexes were fed with a standard or a high-fat diet (see Materials and Methods for details). Figure 7AGo shows that the high-fat diet resulted in a significant weight increase in females (+25%), but not in males (+5%); on the other hand, no specific gender difference was observed in PPAR activity in both chest and abdomen (Fig. 7BGo). In the chest of male mice, PPAR activity was significantly elevated after 1 month of high-fat diet. In females, a trend to increase was observed; however, the response in the different animals was quite variable. At month 6, PPAR activity was increased 2.8-fold in males and 4.3-fold in females, indicating a similarity of response in the two sexes. A trend to increase PPAR activity in abdomen was observed in both groups of animals.


Figure 7
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  		Fig. 7. High-Fat Diet Increases the Reporter Expression in the Chest of PPRE-Luc Mice

High-fat diet was administered to two groups of four male (left) and female (right) PPRE-Luc mice for 6 months. A, Mice weight was assessed in the beginning of the treatment (0 months) and after 1 and 6 months. B, Photon imaging of one representative mouse. Bars represent the average ± SEM of four mice. *, P < 0.01; **, P < 0.005 as compared with 0. P values were calculated with ANOVA followed by Scheffé test.

 
Altogether, the data obtained in different nutritional conditions indicated that the PPRE-Luc model is able to report physiological changes in PPAR activation. In addition, our results indicated that, in females, chest PPAR signaling is sensitive to nutritional status, but, consistently, the effect on the transcription of luciferase is more than one order of magnitude lower than in males. Again, no gender-specific differences in PPAR signaling were observed in organs other than liver (data not shown).

Previous reports provided evidence of an effect of sex hormones on PPAR activity in rodents (32). However, in the PPRE-Luc model, neither ovariectomy (Fig. 8AGo) nor treatment with testosterone or dihydrotestosterone (Fig. 8BGo) affected female luciferase expression in PPRE-Luc mice. Moreover, the orchiectomy of male mice did not result in a reduction of the photon counting in the chest as compared with sham operated (supplemental Fig. 3Go, A and B), demonstrating that sex hormones do not have a major influence on the hepatic PPAR state of activity. Because several sex dimorphisms are heightened by the presence of a ligand, we treated gonadectomized mice with Wy-14,643 (250 mg/kg sc) (supplementary Fig. 3CGo). Similarly to what was shown with intact animals, the ligand increased luciferase activity in males but not in females. Thus, these results further support the concept of an impaired PPAR signaling in female liver.


Figure 8
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  		Fig. 8. Ovariectomy and Testosterone Administration Do Not Affect Luciferase Expression in Female PPRE-Luc Mice

A, Two groups of four female mice were ovariectomized (OVX) or sham operated (Sham) and subjected to the in vivo imaging procedure at the basal condition, after 3 d, and 1 and 2 wk after surgery. Pictures are showing the luminescence distribution of one representative individual/group at the indicated time points. Bars of the inserted graphs (right panel) are the quantification of the photon emission expressed as mean values ± SEM of four animals. B, Imaging analysis after hormonal manipulation of the ovariectomized animals. Two weeks after ovariectomy, three groups of four female mice were sc treated with 1 mg/kg testosterone, 1 mg/kg dihydrotestosterone (DHT), or vegetal oil (vehicle). Images show the luminescence of a representative animal. Bars represent mean photon emission values ± SEM of four mice. The experiment was repeated twice.

 
These experiments led us to conclude that the low PPAR activity observed in female PPRE-Luc mice could not be ascribed to the hormonal milieu in adult animals, but to changes occurring earlier in liver differentiation leading to a lower activity of PPARs in adult animals.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The present study shows the validity of a novel PPRE-Luc reporter mouse system for the in vivo study of PPAR activity and sheds new light on the gender specificity of hepatic PPAR signaling. These data might be of major relevance for the study of metabolic diseases and the development of drugs active in these disorders.

We here demonstrated that in adult mice PPAR signaling in liver of females is consistently lower than in males. This is in agreement with previous studies carried out in several laboratories (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). It is in fact well known that PPAR{alpha} is the mediator of the hepatic, gender-specific response to fibrates (33, 34, 35, 36, 37, 38, 39, 40, 41); this receptor subtype is also important for gender-specific lipid and glucose metabolism (43), fat storage (44), and responses to food assumption (42) or inflammatory stimuli (45). It has also been demonstrated that the liver content of PPAR{alpha} mRNA and protein is significantly lower in females (2- and 5-fold lower than in males, respectively) and that testosterone is able to elevate female PPAR{alpha} to male levels. This body of evidence led to the postulation that levels of PPAR{alpha} expression are directly responsible for the above reported sex differences (32). This hypothesis is challenged by the observation here presented revealing that hepatic PPAR activity in females remains significantly lower than in males, despite any hormonal or dietary manipulation. Thus, we conclude that female liver cells have impaired transcriptional activity through DR1 elements. The extent of such an impairment suggests that factors other than receptor content or hormonal influences contribute to the limited activity of PPAR{alpha}. The extent to which these differences have to be ascribed to developmental cues, possibly acting on coregulators or to epigenetic changes, needs to be investigated.

The molecular characterization of this gender-specific transcriptional blockage is of relevance considering the key role of PPAR signaling in the liver physiology and, most importantly, in view of the interest of pharmacological manipulation of liver PPARs for the treatment of metabolic syndrome.

It is worth emphasizing that only the use of the PPRE-Luc reporter mouse could provide a direct demonstration of impaired PPAR signaling in female liver. Before our study, reports on the PPAR{alpha} knockout (41, 42, 43, 44, 45) or on the endogenous PPAR{alpha} target genes (32, 35, 36, 37, 39, 46, 47) provided correlative evidence of altered PPAR functions in female rodents. When using knockout mice, a function is deleted from embryos, a fact that may instigate several compensative mechanisms, masking the real contribution of the deleted function. On the other hand, trying to infer PPAR{alpha} action from the analysis of endogenous target gene expression can be misleading because of multiple signals converging into a complex promoter. Thus, PPAR contribution can be masked by the activity of other transcription factors regulating a given promoter. The novel perspective view, offered by imaging the receptor activity in living mice, allowed to define, for the first time, that in chest and abdomen PPAR activation follows a different kinetic in fasting conditions. For example, our data indicate that in chest PPAR activity is induced quite early upon food withdrawal, whereas it is turned off later on at 24 and 48 h. The specific requirements of hepatic PPAR{alpha} activation obtained 6 h after food deprivation is in accordance with the master regulator role of the main PPAR subtype in the liver during this metabolic process. In fact, PPAR{alpha} activation is considered a key step to turn on a set of genes responsible for change in fuel source from carbohydrates and fats to fats only (30, 31). Moreover, our results indicate that in the abdomen, the PPAR transcriptional activation is detected only at 24 h and is more persistent than in chest, where it is still detectable after 48 h. Although the physiological significance of these tissue-specific differences in PPAR activation has to be firmly elucidated, they likely reflect a differential involvement of PPAR subtypes in fasting. The molecular basis of these differential responses are under investigation and may provide novel clues regarding the physiological role of the PPAR signaling pathway in different tissues.

The present study further highlights the usefulness of reporter animals to investigate receptor activity in vivo. The successful generation of the PPRE-Luc reporter mouse by using the insulator strategy adopted for the estrogen receptor responsive element-Luc model (14) validates the overall strategy for the creation of pharmacologically valuable reporter mice in which the reporter gene is widely expressed and can report the state of activity of nuclear receptors in all possible target organs. In seven of the eight transgenic lines expressing luciferase, the reporter: a) is inducible by PPARs, b) is distributed in all the tissue examined so far (n = 10 tissues shown in Table 1Go and 4 others not shown), and c) has a pattern of expression consistent among different lines (with the highest level in brain and intestine and, in males, liver). These results, together with our previous findings (14), lead to the conclusion that insulators, such as the MAR sequences, are quite efficient in preventing position effects in transgenesis. In addition, the present study confirms the importance of the use of firefly luciferase as a reporter of choice for its possible detection by in vivo imaging technologies and because of its short half-life, which enables a dynamic view of the state of transcriptional activity and of the receptor of interest to be acquired.

The model here generated, particularly if crossed with other mice models in which the ablation of a specific PPAR subtype has been operated, will be of major interest for the study of the numerous disorders where PPARs are implicated (e.g. metabolic syndrome, diabetes, arteriosclerosis, X-linked adrenoleukodystrophy) and for the identification of novel synthetic ligands to be used for the therapy of these disorders.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids Construction
The two oligonucleotides, PPRE sense: 5'-TCGACAGGGGACCAGGACAAAGGTCACGTTCGGGAC-3' and PPRE antisense: 3'-GTCCCCTGGTCCTGTTTCCAGTGCAAGCCCTGAGCT-5', containing the PPRE sequence of the Acyl-CoA oxidase gene (6, 7) and carrying the SalI and XhoI restriction sites at the ends were annealed, ligated (in conditions forming monomer and multimers), and cloned into the SalI site of the ptk-Luc vector, which was previously constructed (14) to generate: pPPRE1x-tk-Luc, pPPRE2x-tk-Luc, pPPRE3x-tk-Luc, and pPPRE5x-tk-Luc. The XhoI fragments containing the promoter of these constructs were then subcloned into the XhoI site of the pMAR vector (14), generating the final constructs used in the transfection experiments: pMAR-PPRE1x-tk-Luc-MAR, pMAR-PPRE2x-tk-Luc-MAR, pMAR-PPRE3x-tk-Luc-MAR, and pMAR-PPRE5x-tk-Luc-MAR.

Cell Cultures and Transfections
Human hepatic carcinoma HepG2 and SV40-transformed African Green Monkey kidney COS 7 cell lines (American Type Culture Collection) were grown in MEM supplemented with 10% fetal bovine serum (FBS) and DMEM supplemented with 10% FBS, respectively (Invitrogen Corp., Carlsbad, CA). Transient transfections of HepG2 and COS 7 cells were performed by DNA-calcium phosphate precipitation as previously described (48). 1 x 106 HepG2 and COS 7 cells per well were seeded in 24-well plates (Corning Inc., Action, MA) in the appropriate medium supplemented with 10% FBS. Six hours before the addition of the CaPO4/DNA mixture, the medium was replaced with medium deprived of phenol red and supplemented with DCC serum. The CaPO4/DNA mixture used for transfection contained 800 ng of pMAR-PPRE(n)-tk-Luc-MAR reporter vector, 200 ng of pCMVlacZ as internal control of transfection efficiency (Promega, Madison, WI), 25 ng of pSG5mPPAR{alpha} or pSG5mPPAR{gamma}, and 225 ng of the filler (salmon sperm DNA). Sixteen hours after transfection, the medium was replaced with media supplemented with 10% FBS. Six hours later, cells were treated with 5 µM Wy-14,643 (ChemSyn Laboratories, Lenexa, KS) or 0.5 µM Rosiglitazone maleate (Axxora Life Sciences Inc., San Diego, CA) for 16 h. Control cells were treated for 16 h with the same percentage (5 ppm) of dimethyl sulfoxide (used to dissolve Wy-14,643 and Rosiglitazone powders). To carry out the enzymatic assay, protein extracts were obtained as previously described (49).

Real-Time PCR Gene Expression Analysis
Real-time PCR experiments were done with total liver RNAs extracted after homogeneization in TRIzol reagent (Invitrogen Corp.) as suggested by the manufacturer’s instructions. For the preparation of cDNA, 1 µg RNA was denatured at 75 C for 5 min in the presence of 1.5 µg of random primers (Promega) in 15 µl final volume. Deoxynucleotide triphosphate (GE Healthcare, Piscataway, NJ) and Moloney murine leukemia virus reverse transcriptase (RT) (Promega) were added at 0.5 mM and 8 U/µl final concentration, respectively, in a final volume of 25 µl. The RT reaction was performed at 37 C for 1 h; the enzyme was inactivated at 75 C for 5 min. Control reactions without addition of the RT enzyme were performed for each sample. Real-time PCR experiments were performed using TaqMan technology. The reaction mix for each sample was made up of 2 µl of cDNA, 12.5 µl of TaqMan 2x Universal PCR Master Mix No AmpErase UNG (Applied Biosystems, Foster City, CA) and 10.5 µl of primers and probes mix: 200 nM Abcd1 forward and reward primers (5'-TCACAGCCACTGGCTATGCA-3', 5'-CATTTCCAAGGCTGCCTTCTT-3'), 150 nM Abcd1 TaqMan MGB probe 5'-6FAM-TCAGACTCAGAAGCCATG-MGB-3'; 300 nM Abcd2 forward and reward primers (5'-TGGTGGCTTCCAGGCTAAAC-3', 5'-GGGACCAGTTATCAAGAGATGCA-3'), 200 nM Abcd2 TaqMan MGB probe 5'-6FAM-TCAAAGTGGAAGAAGGG-MGB-3'; 200 nM Abcd3 forward and reward primers (5'-GTCTCAAGCTTTGGGTCGTATAGTT-3', 5'-GCCGTAAAACCAGCCAATCTAG-3'), 150 nM Abcd3 TaqMan MGB probe 5'-6FAM-CTGGGCGTGAAATG-MGB-3'; pre-made TaqMan Gene Expression assays for the endogenous gene Acox1 (Mm00443579_m1, Applied Biosystems), Acsl1 (Mm00484217_m1, Applied Biosystems), Cyp4a14 (Mm00484132_m1, Applied Biosystems), and as a reference gene assay 18S rRNA VIC–MGB-PDAR (Applied Biosystems). The reaction was carried out according to the manufacturer’s protocol using Applied Biosystems 7000 Sequence Detection System device with the following thermal profile: 2 min at 50 C; 10 min 95 C; 40 cycles (15 sec 95 C, 1 min at 60 C), and data were analyzed using the ABI Prism 7000 SDS Software and the 2 –{Delta}{Delta}Ct method (50). The analysis of each sample was repeated six times.

Transgenic Mice
For oocyte microinjection, linearized pMAR-PPRE5x-tk-Luc-MAR constructs depleted of plasmid sequences were obtained with BsshII restriction enzyme (Roche, Basel, Switzerland) digestion and purification of the 10-kb transgene fragment from agarose gel. Transgenic mice were generated by pronuclear DNA injection of zygotes C57Bl/6xDBA/2 F2 generation using standard procedures (51). Microinjected zygotes were reimplanted into pseudopregnant C57Bl/6xDBA/2 F2 foster mothers to complete their development. For genotyping genomic DNA was extracted as previously described (52) from tail biopsies. Briefly, tissues were lysed by addition of 1% sodium dodecyl sulfate, 50 mM Tris-HCl, (pH 8), and 200 mg/ml Proteinase K and incubation overnight at 37 C. DNA was then purified by phenol extraction and ethanol precipitation. DNAs from the founders and their littermates were screened by PCR analysis. PCR amplification was carried out in a buffer containing 10 mM Tris-HCl, pH 8.0; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM deoxynucleotide triphosphates (GE Healthcare); 0.25 mM of each primer; and 2 U of Taq Vent DNA polymerase (New England Biolabs, Inc., Ipswich, MA) for 10 ng genomic DNA template. The primers used were 5'-GGCAGAAGCTATGAAACGAT-3' and 5'-CGACTGAAATCCCTGGTAAT-3'. After 40 cycles (30 sec at 96 C, 30 sec at 55 C, and 30 sec at 72 C), the products were analyzed on 3% agarose gels stained with ethidium bromide. At 3 wk of age, all the potential founders obtained from microinjection were screened by PCR. Twenty-two individuals were identified as positives for the presence of the transgene. Unless otherwise specified, all experiments were carried out with heterozygous mice obtained by mating our founders with C57Bl/6 (Charles River, Wilmington, MA) wild-type mice.

Dietary and Pharmacological Treatments
All the experiments were done with 3- to 5-month-old heterozygous mice fed only at night for 2 d before experimentation with standard diet. The animals were fed ad libitum before experimentation only in the day-night feeding experiments. The high-fat diet experiments were performed by giving a diet composed of 35% crude fat, 18% crude protein, 6% crude fiber, and 7% ash for 6 months; the diet included also vitamins integration (Mucedola, Settimo Milanese, Italy). Fasting experiments were performed keeping male and female PPRE-Luc mice in metabolic cages for 48 h in the absence of food and water ad libitum. In male mice, sc injection of MK-886 (250 mg/kg) or vehicle was done at 0900 h for 3 consecutive days; the third morning after the injection, animals were subjected to fasting.

For the in vivo pharmacological studies with PPARs agonist and antagonists, 3- to 5-month-old heterozygous male and female mice were treated by sc injections of the different compounds dissolved in vegetal oil: 50 and 250 mg/kg Wy-14,643 (ChemSyn Laboratories) and 250 mg/kg MK-886 (Biomol International, Plymouth Meeting, PA). MK-886 was given 30 min before the injections of the corresponding agonists. Photon emission analysis of gonadectomized male and female mice was performed at the same time (1700 h) just before gonadectomy, after 3, 7, and 14 d. Gonadectomized mice were treated with vehicle or 1 mg/kg dihydrotestosterone (Sigma, St. Louis, MO), 1 mg/Kg testosterone (Sigma), and 250 mg/kg Wy-14,643 sc.

In oral treatment Wy-14,643 was dissolved in a water solution of 0.05 M D(–)-N-methylglucamine (Merk) and then administered for 6 h (250 mg/kg, acute treatment) or 5 d (100 mg/kg, chronic treatment).

Experimental Animals
Experiments performed in this study were conducted according to the Guidelines for Care and Use of Experimental Animals. Use of experimental animals was approved by the Italian Ministry of Research and University and controlled by the panel of experts of the Department of Pharmacological Sciences, University of Milan.

Bioluminescence Reporter Imaging
Mice were visualized with a Night Owl imaging unit (Berthold Technologies, Bad Wildbad, Germany) consisting of a Peltier cooled CCD slow-scan camera equipped with a 25 mm/0.95 lens. The images were generated by a Night Owl LB981 image processor and transferred via video cable to a peripheral component interconnect frame grabber using WinLight (32) software (Berthold Technologies). For the detection of bioluminescence, mice were anesthetized and received ip injection of an aqueous solution of luciferin (beetle luciferin potassium salt; Promega; 25 mg/kg), 20 min before bioluminescence quantification, to obtain a uniform biodistribution of the substrate. We administered the substrate luciferine after a described procedure (53, 54). Mice were placed in the light-tight chamber, and a gray-scale image of the animals was first taken with dimmed light. Photon emission was then integrated over a period of 5 min. Luminescence measurements are expressed as the integration of the average brightness/pixel unit expressed as photon counts per second. For colocalization of the bioluminescent photon emission on the animal body, gray-scale and pseudocolor images were merged using the WinLight (32) software. Because layers of tissue may limit photon emission from inner organs, to better identify the original source of luminescence from the chest, we killed several mice after the in vivo analysis and identified the liver as the main photon-emitting source in the chest area by a direct evaluation of the emission from this organ. After sacrifice, the tissues were rapidly dissected and immediately frozen on dry ice.

Luciferase Enzymatic Assay
Tissue extracts of mice killed at 1700 h were prepared by homogenization in 200 µl of 100 mM KPO4 lysis buffer (pH 7.8, containing 1 mM dithiothreitol, 4 mM EGTA, 4 mM EDTA, and 0.7 mM phenylmethylsulfonyl fluoride), three cycles of freezing-thawing, and 30 min of minifuge centrifugation (Eppendorf, Hamburg, Germany) at maximum speed. Supernatants containing luciferase were collected, and protein concentrations were determined by Bradford’s assay (55). Luciferase enzymatic activity in tissue extracts was measured by a commercial kit (Luciferase assay system, Promega) according to the instructions of the supplier. The light intensity was measured with a luminometer (Lumat LB 9501/16, Berthold) in 10-sec time periods and expressed as relative light units per µg protein.


   ACKNOWLEDGMENTS
 
We thank Cristina Martelli, Monica Rebecchi, Clara Meda, Paolo Sparaciari, and Samanta Oldoni for their technical and vet assistance. We thank Prof. Giovanni Lucignani for his continuous support and the numerous and helpful scientific exchanges we had during the preparation of the manuscript.

The study was supported by Sigma-Tau, Telethon GGP02336, National Institutes of Health (NIH) RO1AG027713-01, and the European Community (Network of Excellence Diagnostic Molecular Imaging LSHB-CT-2005-512146 and European Molecular Imaging Laboratories LSHC-CT-2004-503569).


   FOOTNOTES
 
Disclosure Summary: A.M. and P.C. are inventors of the "Transgenic Mouse for Screening and for Studies of the Pharmacodynamics and Pharmacokinetics of Ligands Acting on Intracellular Receptors, and Method for the Preparation Thereof" (European Patent no. 1298988, PTC extension international publication no. WO 02/01949 A2) and are cofounders of TOP S.r.l. A.B., L.O., L.T., and C.T. have nothing to declare. A.F.S. is employed by Sigma-Tau Industrie Farmaceutiche Riunite S.p.A.

First Published Online December 7, 2006

1 P.C. and A.B. have contributed equally to the work. Back

Abbreviations: Abcd2, ATP-binding cassette, subfamily D, member 2; CCD, charge-coupled device; DR1, direct repeats; FBS, fetal bovine serum; MAR, matrix attachment regions; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR responsive element; RT, reverse transcriptase; tk, thymidine kinase.

Received for publication April 6, 2006. Accepted for publication November 28, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Berger JP, Akiyama TE, Meinke PT 2005 PPARs: therapeutic targets for metabolic disease. Trends Pharmacol Sci 26:244–251[CrossRef][Medline]
  2. Kemp S, Wei HM, Lu JF, Braiterman IT, McGuinness MC, Moser AB, Watkins PA, Smith KD 1998 Gene redundancy and pharmacological gene therapy: implications for x-linked adrenoleukodystrophy. Nat Med 4:1261–1268[CrossRef][Medline]
  3. Gottlicher M, Widmark E, Li Q, Gustafsson JA 1992 Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor. Proc Natl Acad Sci USA 89:4653–4657[Abstract/Free Full Text]
  4. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM 1995 15-Deoxy-{delta} 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR {gamma}. Cell 83:803–812[CrossRef][Medline]
  5. Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehmann JM 1995 A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor {gamma} and promotes adipocyte differentiation. Cell 83:813–819[CrossRef][Medline]
  6. Kliewer SA, Umesono K, Noonan DJ, Heyman RA, Evans RM 1992 Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature 358:771–774[CrossRef][Medline]
  7. Tugwood JD, Issemann I, Anderson RG, Bundell KR, McPheat WL, Green S 1992 The mouse peroxisome proliferator activated receptor recognizes a response element in the 5' flanking sequence of the rat acyl coa oxidase gene. EMBO J 11:433–439[Medline]
  8. Fruchart JC, Duriez P, Staels B 1999 Peroxisome proliferator-activated receptor-{alpha} activators regulate genes governing lipoprotein metabolism, vascular inflammation and atherosclerosis. Curr Opin Lipidol 10:245–257[CrossRef][Medline]
  9. Rosen ED, Walkey CJ, Puigserver P, Spiegelman BM 2000 Transcriptional regulation of adipogenesis. Genes Dev 14:1293–1307[Free Full Text]
  10. Fredenrich A, Grimaldi PA 2005 PPAR {delta}: an uncompletely known nuclear receptor. Diabetes Metab 31:23–27[Medline]
  11. Issemann I, Green S 1990 Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 347:645–650[CrossRef][Medline]
  12. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA 1995 An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor {gamma} (PPAR {gamma}). J Biol Chem 270:12953–12956[Abstract/Free Full Text]
  13. Maggi A, Ottobrini L, Biserni A, Lucignani G, Ciana P 2004 Techniques: reporter mice—a new way to look at drug action. Trends Pharmacol Sci 25:337–342[CrossRef][Medline]
  14. Ciana P, Di Luccio G, Belcredito S, Pollio G, Vegeto E, Tatangelo L, Tiveron C, Maggi A 2001 Engineering of a mouse for the in vivo profiling of estrogen receptor activity. Mol Endocrinol 15:1104–1113[Abstract/Free Full Text]
  15. Ciana P, Mussi P, Raviscioni M, Biserni A, Ottobrini L, Vegeto E, Maggi A 2004 The ERE-Luc reporter mouse. Ernst Schering Res Found Workshop:151–168
  16. Maggi A, Ciana P 2005 Reporter mice and drug discovery and development. Nat Rev Drug Discov 4:249–255[CrossRef][Medline]
  17. Di Lorenzo D, Villa R, Biasiotto G, Belloli S, Ruggeri G, Albertini A, Apostoli P, Raviscioni M, Ciana P, Maggi A 2002 Isomer-specific activity of dichlorodyphenyltrichloroethane with estrogen receptor in adult and suckling estrogen reporter mice. Endocrinology 143:4544–4551[Abstract/Free Full Text]
  18. Penza M, Bonetti E, Villa R, Ganzerla S, Bergonzi R, Biasiotto G, Caimi L, Apostoli P, Ciana P, Maggi A, Di Lorenzo D 2004 Whole body action of xenoestrogens with different chemical structures in estrogen reporter male mice. Toxicology 205:65–73[CrossRef][Medline]
  19. Villa R, Bonetti E, Penza ML, Iacobello C, Bugari G, Bailo M, Parolini O, Apostoli P, Caimi L, Ciana P, Maggi A, Di Lorenzo D 2004 Target-specific action of organochlorine compounds in reproductive and nonreproductive tissues of estrogen-reporter male mice. Toxicol Appl Pharmacol 201:137–148[CrossRef][Medline]
  20. Ciana P, Raviscioni M, Mussi P, Vegeto E, Que I, Parker MG, Lowik C, Maggi A 2003 In vivo imaging of transcriptionally active estrogen receptors. Nat Med 9:82–86[CrossRef][Medline]
  21. Ciana P, Brena A, Sparaciari P, Bonetti E, Di Lorenzo D, Maggi A 2005 Estrogenic activities in rodent estrogen-free diets. Endocrinology 146:5144–5150[Abstract/Free Full Text]
  22. Klotz DM, Hewitt SC, Ciana P, Raviscioni M, Lindzey JK, Foley J, Maggi A, Diaugustine RP, Korach KS 2002 Requirement of estrogen receptor-{alpha} in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross-talk. J Biol Chem 277:8531–8537[Abstract/Free Full Text]
  23. Patrone C, Cassel TN, Pettersson K, Piao YS, Cheng G, Ciana P, Maggi A, Warner M, Gustafsson JA, Nord M 2003 Regulation of postnatal lung development and homeostasis by estrogen receptor ß. Mol Cell Biol 23:8542–8552[Abstract/Free Full Text]
  24. Stief A, Winter DM, Stratling WH, Sippel AE 1989 A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature 341:343–345[CrossRef][Medline]
  25. Fourcade S, Savary S, Albet S, Gauthe D, Gondcaille C, Pineau T, Bellenger J, Bentejac M, Holzinger A, Berger J, Bugaut M 2001 Fibrate induction of the adrenoleukodystrophy-related gene (ABCD2): promoter analysis and role of the peroxisome proliferator-activated receptor PPAR{alpha}. Eur J Biochem 268:3490–3500[Medline]
  26. Patsouris D, Reddy JK, Muller M, Kersten S 2006 Peroxisome proliferator-activated receptor {{alpha}} mediates the effects of high-fat diet on hepatic gene expression. Endocrinology 147:1508–1516[Abstract/Free Full Text]
  27. Stauber AJ, Brown-Borg H, Liu J, Waalkes MP, Laughter A, Staben RA, Coley JC, Swanson C, Voss KA, Kopchick JJ, Corton JC 2005 Constitutive expression of peroxisome proliferator-activated receptor {alpha}-regulated genes in dwarf mice. Mol Pharmacol 67:681–694[Abstract/Free Full Text]
  28. Harano Y, Yasui K, Toyama T, Nakajima T, Mitsuyoshi H, Mimani M, Hirasawa T, Itoh Y, Okanoue T 2006 Fenofibrate, a peroxisome proliferator-activated receptor {alpha} agonist, reduces hepatic steatosis and lipid peroxidation in fatty liver Shionogi mice with hereditary fatty liver. Liver Int 26:613–620[CrossRef][Medline]
  29. Berger J, Albet S, Bentejac M, Netik A, Holzinger A, Roscher AA, Bugaut M, Forss-Petter S 1999 The four murine peroxisomal ABC-transporter genes differ in constitutive, inducible and developmental expression. Eur J Biochem 265:719–27[Medline]
  30. Kersten S, Seydoux J, Peters JM, Gonzalez FJ, Desvergne B, Wahli W 1999 Peroxisome proliferator-activated receptor {alpha} mediates the adaptive response to fasting. J Clin Invest 103:1489–1498[Medline]
  31. Leone TC, Weinheimer CJ, Kelly DP 1999 A critical role for the peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) in the cellular fasting response: the PPAR{alpha}-null mouse as a model of fatty acid oxidation disorders. Proc Natl Acad Sci USA 96:7473–7478[Abstract/Free Full Text]
  32. Jalouli M, Carlsson L, Ameen C, Linden D, Ljungberg A, Michalik L, Eden S, Wahli W, Oscarsson J 2003 Sex difference in hepatic peroxisome proliferator-activated receptor {alpha} expression: influence of pituitary and gonadal hormones. Endocrinology 144:101–109[Abstract/Free Full Text]
  33. Svoboda D, Azarnoff D, Reddy J 1969 Microbodies in experimentally altered cells. II. The relationship of microbody proliferation to endocrine glands. J Cell Biol 40:734–746[Abstract/Free Full Text]
  34. Gray RH, De La Iglesia FA 1984 Quantitative microscopy comparison of peroxisome proliferation by the lipid-regulating agent gemfibrozil in several species. Hepatology 4:520–530
  35. Hawkins JM, Jones WE, Bonner FW, Gibson GG 1987 The effect of peroxisome proliferators on microsomal, peroxisomal, and mitochondrial enzyme activities in the liver and kidney. Drug Metab Rev 18:441–515[Medline]
  36. Kawashima Y, Uy-Yu N, Kozuka H 1989 Sex-related differences in the enhancing effects of perfluoro-octanoic acid on stearoyl-Coa desaturase and its influence on the acyl composition of phospholipid in rat liver. Comparison with clofibric acid and tiadenol. Biochem J 263:897–904[Medline]
  37. Kawashima Y, Uy-Yu N, Kozuka H 1989 Sex-related difference in the inductions by perfluoro-octanoic acid of peroxisomal ß-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-Coa hydrolase in rat liver. Biochem J 261:595–600[Medline]
  38. Yamada J, Sakuma M, Ikeda T, Fukuda K, Suga T 1991 Characteristics of dehydroepiandrosterone as a peroxisome proliferator. Biochim Biophys Acta 1092:233–243[Medline]
  39. Sundseth SS, Waxman DJ 1992 Sex-dependent expression and clofibrate inducibility of cytochrome P450 4A fatty acid o-hydroxylases. Male specificity of liver and kidney CYP4A2 mRNA and tissue-specific regulation by growth hormone and testosterone. J Biol Chem 267:3915–3921[Abstract/Free Full Text]
  40. Sugiyama H, Yamada J, Suga T 1994 Effects of testosterone, hypophysectomy and growth hormone treatment on clofibrate induction of peroxisomal ß-oxidation in female rat liver. Biochem Pharmacol 47:918–921[CrossRef][Medline]
  41. Nakajima T, Kamijo Y, Usuda N, Liang Y, Fukushima Y, Kametani K, Gonzalez FJ, Aoyama T 2000 Sex-dependent regulation of hepatic peroxisome proliferation in mice by trichloroethylene via peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}). Carcinogenesis 21:677–682[Abstract/Free Full Text]
  42. Lewitt MS, Brismar K 2002 Gender difference in the leptin response to feeding in peroxisome-proliferator-activated receptor-{alpha} knockout mice. Int J Obes Relat Metab Disord 26:1296–1300[CrossRef][Medline]
  43. Djouadi F, Weinheimer CJ, Saffitz JE, Pitchford C, Bastin J, Gonzalez FJ, Kelly DP 1998 A gender-related defect in lipid metabolism and glucose homeostasis in peroxisome proliferator-activated receptor {alpha}-deficient mice. J Clin Invest 102:1083–1091[Medline]
  44. Costet P, Legendre C, More J, Edgar A, Galtier P, Pineau T 1998 Peroxisome proliferator-activated receptor {alpha}-isoform deficiency leads to progressive dyslipidemia with sexually dimorphic obesity and steatosis. J Biol Chem 273:29577–29585[Abstract/Free Full Text]
  45. Tai ES, Bin Ali A, Zhang Q, Loh LM, Tan CE, Retnam L, El Oakley RM, Lim SK 2003 Hepatic expression of PPAR{alpha}, a molecular target of fibrates, is regulated during inflammation in a gender-specific manner. FEBS Lett 546:237–240[CrossRef][Medline]
  46. Yang X, Schadt EE, Wang S, Wang H, Arnold AP, Ingram-Drake L, Drake TA, Lusis AJ 2006 Tissue-specific expression and regulation of sexually dimorphic genes in mice. Genome Res 16:995–1004[Abstract/Free Full Text]
  47. Clodfelter KH, Holloway MG, Hodor P, Park SH, Ray WJ, Waxman DJ 2006 Sex-dependent liver gene expression is extensive and largely dependent upon signal transducer and activator of transcription 5b (STAT5b): STAT5b-dependent activation of male genes and repression of female genes revealed by microarray analysis. Mol Endocrinol 20:1333–1351[Abstract/Free Full Text]
  48. Ma ZQ, Spreafico E, Pollio G, Santagati S, Conti E, Cattaneo E, Maggi A 1993 Activated estrogen receptor mediates growth arrest and differentiation of a neuroblastoma cell line. Proc Natl Acad Sci USA 90:3740–3744[Abstract/Free Full Text]
  49. Patrone C, Gianazza E, Santagati S, Agrati P, Maggi A 1998 Divergent pathways regulate ligand-independent activation of ER {alpha} in SK-N-BE neuroblastoma and COS-1 renal carcinoma cells. Mol Endocrinol 12:835–841[Abstract/Free Full Text]
  50. Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(–{Delta}{Delta}Ct) method. Methods 25:402–408[CrossRef][Medline]
  51. Hogan B BR, Costantini F, Lacy E 1994 Manipulating the mouse embryo. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press
  52. Ciana P, Braliou GG, Demay FG, Von Lindern M, Barettino D, Beug H, Stunnenberg HG 1998 Leukemic transformation by the v-ErbA oncoprotein entails constitutive binding to and repression of an erythroid enhancer in vivo. EMBO J 17:7382–7394[CrossRef][Medline]
  53. Contag CH, Spilman SD, Contag PR, Oshiro M, Eames B, Dennery P, Stevenson DK, Benaron DA 1997 Visualizing gene expression in living mammals using a bioluminescent reporter. Photochem Photobiol 66:523–531[Medline]
  54. Honigman A, Zeira E, Ohana P, Abramovitz R, Tavor E, Bar I, Zilberman Y, Rabinovsky R, Gazit D, Joseph A, Panet A, Shai E, Palmon A, Laster M, Galun E 2001 Imaging transgene expression in live animals. Mol Ther 4:239–249[CrossRef][Medline]
  55. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]

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Nuclear Receptors:   PPARα  |  PPARδ  |  PPARγ
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