MCB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Peters, J. M.
Right arrow Articles by Gonzalez, F. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Peters, J. M.
Right arrow Articles by Gonzalez, F. J.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, July 2000, p. 5119-5128, Vol. 20, No. 14
0270-7306/00/$04.00+0

Growth, Adipose, Brain, and Skin Alterations Resulting from Targeted Disruption of the Mouse Peroxisome Proliferator-Activated Receptor beta (delta )

Jeffrey M. Peters,1,* Susanna S. T. Lee,1,† Wen Li,2,‡ Jerrold M. Ward,3 Oksana Gavrilova,4 Carrie Everett,4 Marc L. Reitman,4 Lynn D. Hudson,2 and Frank J. Gonzalez1

Laboratory of Metabolism, National Cancer Institute,1 Laboratory of Developmental Neurogenetics, National Institute of Neurological Disorders and Stroke,2 and Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases,4 National Institutes of Health, Bethesda, Maryland 20892, and Veterinary and Tumor Pathology Section, Office of Laboratory Animal Resources, National Cancer Institute, Frederick, Maryland 217023

Received 18 January 2000/Returned for modification 28 February 2000/Accepted 13 April 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

To determine the physiological roles of peroxisome proliferator-activated receptor beta  (PPARbeta ), null mice were constructed by targeted disruption of the ligand binding domain of the murine PPARbeta gene. Homozygous PPARbeta -null term fetuses were smaller than controls, and this phenotype persisted postnatally. Gonadal adipose stores were smaller, and constitutive mRNA levels of CD36 were higher, in PPARbeta -null mice than in controls. In the brain, myelination of the corpus callosum was altered in PPARbeta -null mice. PPARbeta was not required for induction of mRNAs involved in epidermal differentiation induced by O-tetradecanoylphorbol-13-acetate (TPA). The hyperplastic response observed in the epidermis after TPA application was significantly greater in the PPARbeta -null mice than in controls. Inflammation induced by TPA in the skin was lower in wild-type mice fed sulindac than in similarly treated PPARbeta -null mice. These results are the first to provide in vivo evidence of significant roles for PPARbeta in development, myelination of the corpus callosum, lipid metabolism, and epidermal cell proliferation.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In the past 10 years, specific roles for peroxisome proliferator-activated receptor alpha  (PPARalpha ) and PPARgamma have emerged while information defining PPARbeta -dependent processes is lacking. PPARs are members of the nuclear receptor superfamily (34). The three PPARs exhibit unique tissue distribution, are encoded by separate genes in all species examined to date, and are designated by the subtypes alpha , beta  (delta , NUC1), and gamma  (14, 18, 34, 47, 48). Acting as regulatory transcription factors, the PPARs heterodimerize with retinoid X receptors and modulate gene expression in target genes containing peroxisome proliferator-responsive elements (PPREs) in response to ligand activation.

The three PPARs have related but distinct activities. Activation of PPARalpha can occur as a result of cold shock (19), food restriction (26), dietary fatty acids (44), and treatment with the hypolipidemic fibrate class of drugs (31). Peroxisomal and mitochondrial beta -oxidizing enzymes, microsomal omega -oxidizing enzymes, hepatic fatty acid binding protein, carnitine palmitoyltransferases, and a number of apolipoproteins are all regulated by PPARalpha ligands/activators (3, 26, 31, 38, 41, 44). These data, obtained in part from the PPARalpha -null mouse, provide strong in vivo evidence that PPARalpha regulates lipid metabolism by regulating gene expression of numerous proteins which are clinically relevant for a number of diseases including diabetes, obesity, and atherosclerosis.

Another PPAR isoform, PPARgamma , is required for adipocyte differentiation and regulation of adipocyte-specific genes such as the gene for adipocyte fatty acid binding protein aP2 (47). Similar to PPARalpha , PPARgamma is activated by specific ligands, most notably the thiazolidinedione drugs used for type 2 diabetes therapy (32). The phenotype of a PPARgamma -null mouse line is embryo lethal due in part to disrupted placental function (4). Tetraploid rescue experiments to bypass the placental defect confirmed an in vivo role for the receptor in adipogenesis (4). Analysis of heterozygotes and chimeras also established a role for PPARgamma in adipocyte function and glucose homeostasis (29, 45). Thus, it is clear from null mouse studies that there are distinct metabolic roles for PPARalpha and PPARgamma .

The function of PPARbeta has remained elusive. While PPARbeta is ubiquitously expressed, some tissues express relatively higher levels of the mRNA including the brain, adipose tissue, and skin (2, 8). Expression of PPARbeta is considerably higher in the developing neural tube and the epidermis during rat development (9). No target genes that are controlled only by PPARbeta have been identified, but activators for PPARbeta including fatty acids (27), bezafibrate (28), and a furan-conjugated linoleic acid metabolite (39) are reported to activate reporter gene constructs containing PPREs through PPARbeta . Despite the lack of a specific PPARbeta ligand to induce activation, there are several reports suggesting roles for PPARbeta in adipocyte differentiation (5), brain function (51), epidermal differentiation (37), uterine implantation (33), and colon cancer (20). In large part, these studies are correlative associations; definitive proof for PPARbeta function requires the use of a null mouse model. In the present study, a PPARbeta -null mouse was generated and characterized to identify physiological functions dependent on PPARbeta .


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Construction of the targeting vector. Genomic clones corresponding to mouse PPARbeta (mPPARbeta ) were obtained by screening an amplified Sv/129 genomic mouse liver library (Stratagene, La Jolla, Calif.) with a partial (nucleotides [nt] 140 to 1039, 900 bp) mPPARbeta cDNA (2) obtained by reverse transcription-PCR (RT-PCR) of RNA from the 3T3 adipocyte cell line. Since there is significant homology between the other PPARs, these clones were subsequently screened with mPPARgamma and mPPARalpha cDNA probes. The PPARbeta genomic clones did not hybridize with the two cDNAs. Restriction mapping and sequencing analysis of these clones resulted in the identification of one 9.5-kb genomic clone that contained the last exon and intron of the mPPARbeta gene and that was used for constructing the targeting vector. To disrupt the mPPARbeta gene, the 1.14-kb phosphoribosyltransferase II gene conferring neomycin resistance (NEO; derived from plasmid pMC1NeoPolyA; Stratagene) was inserted into the XbaI site of the last exon in the same direction of transcription of the genomic clone. The targeting vector contained 1.8 kb of homologous sequence 5', and 3.5 kb of homologous sequence 3', of the NEO cassette. A herpes simplex virus thymidine kinase gene inserted at the 5' end of the construct allowed negative selection.

Electroporation and selection of recombinant ES cells. Conditions for embryonic stem (ES) cell culture and electroporation have been previously described (31). Twenty-five micrograms of XhoI-linearized targeting vector was used to electroporate Sv/129 ES cells (Genome Systems, St. Louis, Mo.). Of the 56 ES clones that were picked up after positive and negative selection, four were positive for recombination as verified by Southern analysis using both the genomic and NEO-specific probes.

Generation of chimeras. One of the positive ES clones (JP31) was used for microinjections into recipient C57BL/6N blastocysts as previously described (31). Five chimeras with >60% agouti coat color were used to breed with C57BL/6N females, and one of these produced agouti offspring. The genotype of the F1 agouti litter was determined by Southern blot analysis of BamHI-digested tail DNA isolated from 3-week-old pups. Mice heterozygous for the disrupted PPARbeta gene were mated, and homozygous PPARbeta -null mice were identified by Southern blot analysis. Since F2 offspring did not exhibit Mendelian distributions of genotypes, F1 heterozygotes were bred with wild-type C57BL/6N mice to obtain F2 heterozygotes. The heterozygous F2 offspring from these matings were subsequently used to establish a colony of homozygous mice, and normal Mendelian distributions were obtained in the F3 generation. The genetic background of mice produced from this colony was on average 75% C57BL/6N, and the mice were used for all experiments unless otherwise noted.

Southern blot analysis. DNA was isolated from ES cells and mouse tails (30), digested with BamHI, electrophoresed, blotted to nylon membranes, and fixed as previously described (31). The blot was hybridized with 3'-flanking probe A, a 650-bp XhoI-AflII fragment. Probe A hybridizes to a 9.5-kb BamHI restriction fragment from wild-type genomes (see Fig. 1A and B). When one allele of the mPPARbeta gene is replaced with the targeting vector sequence by homologous recombination, probe A hybridizes with a 6.4-kb BamHI restriction fragment (see Fig. 1A and B). An internal NEO probe was used to hybridize with DNA digested with BamHI to demonstrate single-copy insertion of the targeting vector by a homologous recombination event (Fig. 1B).

Northern blot analysis. Total RNA was isolated from gonadal adipose samples (adipose tissue from one or two mice) after disruption of cells in guanidine thiocyanate. Total RNA from corpus callosum and skin samples was isolated using Trizol reagent and the manufacturer's recommended procedures (GIBCO-BRL, Grand Island, N.Y.). Five to 10 µg of total RNA was electrophoresed on a 1.0% agarose gel containing 0.22 M formaldehyde, transferred to a nylon membrane, and baked in a vacuum oven to fix the RNA. Membranes were hybridized in ULTRAhyb hybridization buffer (Ambion, Austin, Tex.) with one of the following previously described cDNA probes: mPPARalpha (22), mPPARbeta (delta ) (2), mPPARgamma (27), mouse myelin basic protein (MBP) (23), mouse proteolipid protein (PLP) (21), rat transglutaminase I (TG-I) (42), rat involucrin (12), small proline-rich (SPR) proteins SPR1A (25) and SPR2H (46), mouse cyclin B1 (10), mouse cyclin-dependent kinase-1 (CDK-1) (49), mouse CDK-4 (36), mouse proliferating cellular nuclear antigen (PCNA) (40), or mouse beta -actin (31). Mouse cDNA fragments for ornithine decarboxylase (ODC), CD36, and acyl coenzyme A synthases (ACS) ACS-2 and ACS-3 were obtained by cloning as described below.

RT-PCR cloning of mouse cDNAs. Mouse cDNA clones for CD36, ACS-2, ACS-3, and ODC were obtained by RT-PCR from 0.5 µg of total RNA isolated from adipose tissue, whole brain, or O-tetradecanoylphorbol-13-acetate (TPA)-treated skin. The PCR primers selected were based on the published cDNA sequences of mouse CD36 (13), rat ACS-2 (16), rat ACS-3 (15), and mouse ODC (24). The second-strand cDNA was amplified by subsequent PCR with designed primers for each gene. The amplified cDNA fragment for mouse CD36 was 1,102 bp, corresponding to nt 229 to 1330. The amplified cDNA fragment for mouse ACS-2 was 942 bp, corresponding to nt 61 to 1002. The mouse cDNA fragment for ACS-2 was 95% homologous with the rat sequence. The amplified cDNA fragment for mouse ACS-3 was 810 bp, corresponding to nt 56 to 865. The mouse cDNA fragment for ACS-3 was 94.2% homologous with the rat sequence. The amplified cDNA fragment for mouse ODC was 1,009 bp, corresponding to nt 855 to 1863. The identity of each clone was confirmed by sequencing. The BLASTN software, version 2.1.10 (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.), was used to show that all four of these cloned RT-PCR products (CD36, ODC, ACS-2, and ACS-3) were only significantly homologous with the respective mRNA of interest (1).

Western blot analysis. Nuclear extracts were obtained from skin and liver samples by grinding tissue submerged in liquid nitrogen with a mortar and pestle. After centrifugation, nuclei were resuspended in a lysis buffer (20 mM HEPES, 0.4 M sodium chloride, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate). The protein concentration was quantified (BCA kit; Pierce, Rockford, Ill.), and 50 µg of protein was separated on a 10% gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After being blocked overnight in Tris-buffered saline plus Tween 20 (TBST)-5% milk at 4°C, the membrane was incubated at room temperature with an anti-PPARbeta antibody raised against an amino terminus peptide (Santa Cruz Biotechnology, Santa Cruz, Calif.) for 2 h. After being washed with TBST, the membrane was incubated with horseradish peroxidase-conjugated donkey anti-goat antibody, followed by a washing with TBST. Detection of PPARbeta protein was performed using a chemiluminescence kit (ECL; Amersham Life Science, Cleveland, Ohio). A detergent extract of transfected COS cells expressing PPARbeta was used as a positive control (kindly provided by John Woods and David Moller, Merck Pharmaceuticals).

Animal studies. To assess body weight gain, male and female wild-type and PPARbeta -null mice were weighed on postnatal day 3, week 4, week 8, week 10, and weeks 48 to 54. Mice were fed stock rodent chow and were provided water ad libitum.

The effect of 24-h food restriction on body temperature, physical activity, and adipocyte lipid metabolism was determined for male and female wild-type and PPARbeta -null mice. Biotelemetry chips (Mini Mitter, Sunriver, Oreg.) were implanted into the abdomens of mice under anesthesia to monitor body temperature and motor activity (17). After surgery, mice were allowed to recover before baseline activity levels were determined. Values obtained after 1 week of monitoring showed a typical diurnal variation. After this period, food was removed from each mouse and body temperature and motor activity were measured during the next 24-h fasting period. Daily food intakes were also measured over a 12-day period using mice that were individually housed.

Two separate cohorts of animals were used to determine the effect of 24-h fasting on adipose mRNA levels. The first group of male and female wild-type and PPARbeta -null mice were euthanized, and gonadal adipose tissue was weighed and snap frozen for future RNA analysis. The second group of mice were fasted for 24 h and adipose tissue was collected as before for RNA analysis.

To assess the role of PPARbeta in brain, male and female wild-type and PPARbeta -null mice, 12 or 36 weeks of age, were euthanized. Brains were removed and assessed for myelination as described below. To analyze RNA expression in specific regions of the brain, the corpus callosum, cerebellum, and brain stem were dissected and RNA was isolated from these samples as described above.

To assess the role of PPARbeta in the epidermal response to TPA, female wild-type and PPARbeta -null mice, 8 weeks of age, were shaved to remove back hair. Twenty-four hours later, either 5 µg of TPA (Sigma Chemical Co., St. Louis, Mo.) dissolved in 200 µl of acetone or 200 µl of acetone was applied to the shaved area. Eight or 48 h after TPA application, mice were euthanized and the skin was removed and snap frozen in liquid nitrogen. Total RNA was isolated as described above. Another section of skin was also removed and placed in 10% phosphate-buffered formalin for further histological analysis of epidermal cell proliferation.

To assess the effect of the nonsteroidal anti-inflammatory drug (NSAID) sulindac on TPA-induced inflammation, female wild-type and PPARbeta -null mice, 8 weeks of age, were fed a diet containing 0.32 g of sulindac/kg for 10 days. Mice were then shaved to remove back hair and 24 h later were treated topically with either acetone or 5 µg of TPA dissolved in acetone. Eight hours after TPA treatment, skin sections were obtained from euthanized mice and histological analysis for inflammation and hyperplasia was performed.

Skin histology. Tissues were fixed in 10% neutral buffered formalin (Fisher Scientific, Fair Lawn, N.J.) and embedded in paraffin, and 4- to 6-µm-thick sections were prepared. Sections were stained with hematoxylin and eosin, and the epidermis was evaluated for hyperplastic growth.

Brain histology. Brains were removed immediately after euthanasia and frozen on dry ice. Sagittal sections (10 µm) were cut with a cryostat. Sections were stored at -70°C until use. A Luxol fast blue (LFB) solution was prepared by dissolving 0.2 g of LFB (Sigma Chemical Company) in 200 ml of 95% ethanol and adding 1 ml of 10% acetic acid. After removal from the freezer and equilibration at room temperature, brain sections were fixed in 4% paraformaldehyde for 15 min. Following two rinses in distilled water, sections were dehydrated with successive immersion in 75, 95, and 100% ethanol. Sections were immersed in LFB solution overnight at 60°C in tightly sealed staining jars. Removal of excess LFB with 95% ethanol rinses was followed by rinsing with distilled water and then immersing for 30 s in 0.05% lithium carbonate (Sigma Chemical Company). The sections were subjected to several changes of 70% ethanol until the grey and white matter were clearly distinguished. Thereafter, the sections were washed thoroughly in distilled water and counterstained with 1% methyl green (Fisher Scientific) for 5 min and rinsed with tap water. Sections were then destained with successive incubations in 80, 90, and 100% ethanol, cleared with a 5-min incubation in xylene, and mounted. Sections were examined under a Zeiss microscope.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

PPARbeta -null mice are smaller with reduced adipose stores. Targeted disruption of the ligand binding domain of the mPPARbeta gene was performed by inserting a phosphoribosyltransferase II expression cassette into the last exon of the gene (Fig. 1A and B). Successful integration of the targeting vector into the mouse genome was confirmed by Southern blot analysis (Fig. 1B). Northern blot analysis of RNA from selected tissues demonstrated successful disruption of the PPARbeta gene. In the brain, an mRNA fragment ~1 kb larger than the wild-type mRNA was detected in null mice at a substantially lower level than in wild-type mice (Fig. 1C). In adipose tissue, both the larger mRNA and a truncated form were also detected in null mice and the levels of these transcripts were substantially lower than levels of wild-type PPARbeta mRNA expression (Fig. 1C). Both of these mRNA transcripts were also detected in liver mRNA from null mice (data not shown). However, despite the presence of these mRNAs, expression of the PPARbeta protein was not detected in hepatic nuclear extracts from PPARbeta -null mice (Fig. 1D). Neither the larger nor the truncated mRNA was detected in the skin of PPARbeta -null mice (Fig. 1C). Western blot analysis of nuclear extracts from skin of wild-type mice demonstrated an increase in PPARbeta protein levels as a result of TPA, while expression of the PPARbeta protein was not detected in either control or TPA-treated skin samples from PPARbeta -null mice (Fig. 1D).


View larger version (29K):
[in this window]
[in a new window]
  		FIG. 1.   Targeted disruption of the mPPARbeta gene. (A) Strategy for the mPPARbeta knockout. (I) Partial map of a mouse genomic fragment containing the second-to-last and last exons encoding the mPPARbeta ligand binding domain. Restriction enzymes: B, BamHI; Xh, XhoI; Xb, XbaI; A, AflII. The wild-type 9.5-kb BamHI fragment detected by probe A, a 0.65-kb XhoI-AflII fragment from the 3' end of the mPPARbeta genomic DNA, is indicated. (II) Targeting vector with a total of 5.3 kb of homologous sequence contained in the XhoI fragment of the genomic clone. The 1.14-kb NEO gene in the same orientation relative to mPPARbeta transcription was inserted into the XbaI site indicated. The NEO cassette introduces a new BamHI restriction site used for genotyping by Southern blot analysis. A pMCITK expression cassette (herpes simplex virus thymidine kinase [HSV-TK]) was added at the 3' end of the construct for negative selection. DX, disrupted XhoI site. (III) The expected homologous recombination event of mPPARbeta . When one allele of the mPPARbeta gene was replaced with the targeting vector sequences by homologous recombination, a 6.4-kb restriction fragment appeared when the gene was analyzed with probe A. (B) Genomic Southern blots of ES cell DNA (top) and Southern blot of tail DNA from wild-type (+/+), heterozygous (+/-), and homozygous mutant (-/-) mice (bottom). (C) Northern analysis of PPARbeta mRNA in selected tissues from wild-type (+/+) and PPARbeta -null (-/-) mice. (D) Western blot analysis of skin and liver from wild-type (+/+) and PPARbeta -null (-/-) mice. Samples from skin of TPA-treated mice were also analyzed. +, positive control. Con, control.

Breeding mixed-genetic-background (C57BL/6N × Sv/129) heterozygous offspring resulted in fewer null mice than expected (Table 1). An analysis of embryos on gestation day 10 (GD10) and fetuses on GD18 revealed that the absence of PPARbeta was not lethal to embryo or fetal development since relatively normal distributions of genotypes were found and the conceptus morphology appeared grossly normal (Table 2; data not shown). However, PPARbeta -null fetuses on GD18 had significantly smaller crown-to-rump lengths and weighed less than controls (Table 3). F2 offspring were subsequently backcrossed one generation with C57BL/6N mice, and the heterozygous mice from these matings were used to establish homozygous wild-type and PPARbeta -null colonies. The colony of PPARbeta -null mice reproduced successfully, and normal Mendelian genotype distributions were found from subsequent heterozygous matings (Table 1).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Genotype of litters from matings of heterozygous PPARbeta  mice


                              
View this table:
[in this window]
[in a new window]
  		TABLE 2.   Genotypes of embryos and fetuses from heterozygous PPARbeta mice on a mixed genetic background (C57BL/6N × Sv/129)


                              
View this table:
[in this window]
[in a new window]
  		TABLE 3.   Developmental assessment of GD18 fetuses from C57BL/6N × Sv/129 micea

Postnatal development of PPARbeta -null mice between 3 days and 48 weeks appeared grossly normal except that they were significantly smaller than controls (Table 4). This effect was more pronounced in female mice than in males. Contributing to the smaller body weights were smaller gonadal fat stores in the PPARbeta -null mice than in the wild-type mice (Table 5). The difference in gonadal adipose stores was not found in older mice aged 48 to 54 weeks (data not shown). Food consumption normalized for body weight indicated that the male PPARbeta -null mice consumed more energy than wild-type controls (Table 6). Total oxygen consumption rates corrected for body weight for wild-type males, PPARbeta -null males, wild-type females, and PPARbeta -null females were 10.2 ± 0.2, 11.5 ± 0.6, 11.4 ± 0.4, and 12.2 ± 0.3 ml/(g of body weight)0.75/h, respectively (n = 5 mice per group). While oxygen consumption tended to be higher in PPARbeta -null mice than in controls, this was not significantly different between genotypes.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 4.   Body weights in wild-type (+/+) and PPARbeta -null (-/-) mice


                              
View this table:
[in this window]
[in a new window]
  		TABLE 5.   Gonadal adipose stores in wild-type (+/+) and PPARbeta -null (-/-) micea


                              
View this table:
[in this window]
[in a new window]
  		TABLE 6.   Food consumption in wild-type (+/+) and PPARbeta -null (-/-) micea

PPARbeta -null mice respond similarly to wild-type mice after fasting. Body temperatures and basal activity levels of wild-type and PPARbeta -null mice were similar and showed a normal circadian rhythm (increased body temperature and activity during the dark cycle). Fasting for 24 h caused similar decreases in body temperature in both genotypes (Fig. 2). Levels of weight loss during a 24-h fast were not different in the two genotypes and ranged from 7 to 9% of total body weight. Serum analysis revealed no consistent differences between genotypes. Typical changes in serum chemistry associated with fasting were detected in both genotypes including increased free fatty acids and beta -hydroxybutyrate and decreased triglycerides, and no change in blood urea nitrogen was detected (data not shown). Since fatty acid transporters can be regulated by PPARs, levels of CD36 (also known as FAT) were quantified in adipose RNA. Constitutive expression of adipocyte CD36 mRNA was higher in PPARbeta -null mice than in controls, while levels of PPARgamma mRNA in adipose tissue of the PPARbeta -null mice and controls were similar (Fig. 3). Fasting had no effect on either mRNA, as similar expression patterns were observed after fasting (Fig. 3).


View larger version (22K):
[in this window]
[in a new window]
  		FIG. 2.   Body temperature and activity in wild-type (+/+) and PPARbeta -null (-/-) mice. Daytime (10 a.m. to 5 p.m.) and nighttime (10 p.m. to 5 a.m.) measurements were made continuously for 14-week-old mice. The body temperature during a 24-hour fast is reported both as the mean of hours 15 to 24 and the minimum during the fast. Values are means ± standard errors of the means (n = 5/group).


View larger version (48K):
[in this window]
[in a new window]
  		FIG. 3.   Effect of 24-h fasting on gonadal adipose mRNA of PPARgamma and CD36/FAT in wild-type (+/+) and PPARbeta -null (-/-) mice. Male and female +/+ and -/- mice (8 weeks of age) were used. For expression of CD36 and PPARgamma mRNA 5 µg of total RNA was subjected to Northern analysis. Values for the respective hybridization signals normalized to beta -actin are means ± standard deviations. *, significantly different from wild-type control (P < 0.05). Con, control.

PPARbeta -null mice have altered myelination in the central nervous system. Since expression of PPARbeta mRNA is reported to be high in the developing neural tubes of embryos and fetuses as well as the adult rodent brain (8, 9, 11, 51), brains from PPARbeta -null mice were examined. The diameters of the brains of PPARbeta -null mice were significantly smaller than those of wild-type mice, which is likely due to the relatively smaller size of the PPARbeta -null mice (data not shown). Histological examination revealed alterations in the extent of myelination in the corpus callosum compared to controls (Fig. 4). This difference was found more often in female mice than in males (three of five females; two of seven males). No consistent differences in myelination of other brain regions including the cerebellum and brain stem between genotypes were found. Levels of mRNA encoding proteins that are important in the myelination process, such as MBP and PLP, were similar in the corpus callosums from both genotypes (Fig. 5). Since two ACS are expressed in the developing rodent brain and have important roles in fatty acid utilization (15, 43, 50) and since recent data suggest that PPARbeta regulates ACS-2 (6), expression of the mRNAs for these enzymes were also analyzed. As shown in Fig. 5, mRNA levels for ACS-2 were similar between genotypes. The levels of RNA encoding ACS-3 were also similar between genotypes (data not shown).


View larger version (113K):
[in this window]
[in a new window]
  		FIG. 4.   Altered myelination of corpus callosum in PPARbeta -null mice. Sagittal sections (10 µm) were cut and stained with LFB as described in Materials and Methods. Magnification, ×170. Arrows, regions of altered myelination. (A) Twelve-week-old females; (B) 36-week-old females; (C) 12-week-old males; (D) 36-week-old males.


View larger version (75K):
[in this window]
[in a new window]
  		FIG. 5.   Northern analysis of selected mRNAs from corpora callosa from wild-type (+/+) and PPARbeta -null (-/-) mice. The corpus callosum was dissected, RNA was isolated, and 5 µg of total RNA was subjected to Northern analysis. Shown are MBP, PLP, and brain ACS-2. Values for the respective hybridization signals normalized to beta -actin are means ± standard deviations.

PPARbeta deficiency results in accentuated TPA-induced hyperplasia. Since induction of PPARbeta mRNA is coincident with increased expression of mRNAs encoding TG-I and SPR proteins in keratinocytes cultured in the presence of TPA (37), the epidermal response to TPA in PPARbeta -null mice was assessed. Topical application of TPA to wild-type mice caused an increase in the mRNA encoding PPARbeta , involucrin, ODC, TG-I, and SPR proteins SPR1A and SPR2H 8 h after TPA treatment (Fig. 6). However, induction of mRNAs encoding proteins associated with differentiation of the epidermis was also found in TPA-treated PPARbeta -null mice despite the absence of PPARbeta mRNA (Fig. 6). Expression of PPARalpha and PPARgamma mRNA was not detectable in any of the skin RNA samples (data not shown). Interestingly, the hyperplastic response typically observed in the epidermis 48 h after TPA treatment was greater in the PPARbeta -null mice than in controls (Fig. 7A), and this effect was found at both low and high doses (2.5 and 10 µg, respectively; data not shown). Associated with the enhanced hyperplastic response observed in the PPARbeta -null mice were higher levels of mRNA encoding proteins involved in cell cycle regulation including CDK-1, CDK-4, cyclin B1, and PCNA (Fig. 7B).


View larger version (71K):
[in this window]
[in a new window]
  		FIG. 6.   Northern analysis of skin mRNAs in wild-type (+/+) and PPARbeta -null (-/-) mice 8 h after TPA. Ten micrograms of total RNA was analyzed from representative skin from two mice. mRNAs associated with epidermal differentiation and cell proliferation were measured. Con, control.



View larger version (181K):
[in this window]
[in a new window]
  		FIG. 7.   Analysis of TPA-induced hyperplasia in skin of wild-type (+/+) and PPARbeta -null (-/-) mice 48 h posttreatment. (A) Histological examination of representative skin topically treated with 5 µg of TPA 48 h posttreatment. Note the enhanced hyperplasia of the epidermis in the -/- skin compared to similarly treated +/+ skin. Magnification, ×267. (B) Northern analysis of skin RNA 48 h after TPA. Total RNA from skin was isolated and analyzed for mRNAs of genes involved in cell proliferation as described in Materials and Methods. mRNAs for the indicated proteins were measured. Values for the respective hybridization signals normalized to beta -actin are means ± standard deviations. *, significantly different from wild-type control (P < 0.05). Con, control.

PPARbeta -null mice are refractory to the anti-inflammatory drug sulindac. Since it was recently suggested that the PPARbeta may influence the effect of the NSAID sulindac (20), the effect of this drug on the inflammatory response induced by TPA was examined. Wild-type mice fed a sulindac-containing diet and then treated with TPA showed no signs of epidermal hyperplasia and mild to moderate inflammation (Fig. 8). In contrast, inflammation was more severe in TPA-treated PPARbeta -null mice than in controls (Fig. 8). Further, early hyperplasia was also detected in TPA-treated PPARbeta -null mice but was not observed in TPA-treated wild-type mice (Fig. 8).


View larger version (69K):
[in this window]
[in a new window]
  		FIG. 8.   TPA-induced inflammation in skin of representative wild-type (+/+) and PPARbeta -null (-/-) mice fed a sulindac diet, 8 h after TPA treatment. Mice were fed 0.32 g of sulindac/kg of diet for 10 days and treated topically with 5 µg of TPA as described in Materials and Methods. Magnification, ×132. Note that there is less inflammation (blue cells) in dermis and subcutaneous tissue in the wild-type section and more infiltration of inflammatory cells in the dermis and subcutaneous tissue in the PPARbeta -null section. The epidermis of the PPARbeta -null mouse is approximately twice the size of the wild-type mouse epidermis.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Developmental role of PPARbeta . The phenotype of a PPARbeta -null mouse line offers clues to the function of this receptor. Since the number of homozygous null offspring was less than expected from heterozygote breedings of the original mixed-genetic-background mice, PPARbeta may have a role in embryonic, fetal, and/or postnatal development. However, the normal distribution of genotypes and gross morphology of PPARbeta -null conceptuses on GD10 and -18 do not support the hypothesis that PPARbeta is required for implantation (33). Nonetheless, these results do provide evidence that, in the absence of PPARbeta , development is impaired since both the weights of GD18 fetuses and the postnatal weights of PPARbeta -null mice are significantly lower than those of wild-type mice, in particular of female null mice.

The role of the PPARbeta in adipocyte function. The phenotype of the PPARbeta -null mouse also indicates that the receptor is involved in adipocyte function. Indeed, overexpression of PPARbeta in fibroblasts promotes induction of adipocyte differentiation (5). In the absence of PPARbeta , adipose stores are smaller and constitutive expression of CD36/FAT mRNA is higher than those for wild-type mice. Thus the smaller adipose tissue may be due to alterations in fatty acid transport. However, the influence of fasting on measures of lipolysis was not different for the different genotypes, indicating that the role of PPARbeta in adipose metabolism may be complex. While it is known that the CD36 gene is responsive to PPAR activators in a tissue-specific manner (35, 38), these data do not address whether PPARbeta is required for inducible expression of this gene.

PPARbeta and brain development. The alteration in myelination of the corpus callosum is another unique phenotype of the PPARbeta -null mouse. There are a number of possible mechanisms that could explain this effect. PPARbeta may be required during postnatal development of the brain, functioning as a regulator of genes involved in this process. However, three likely candidate genes were unaffected in the PPARbeta -null mouse corpus callosum including genes for MBP, PLP, and two brain-specific ACS (ACS-2 and ACS-3). For PLP, there is a reported PPRE in the promoter of the gene (7), and thus it is surprising that the level of its RNA is unaffected since PPARbeta is the more predominant PPAR expressed in the brain. Reduced fatty acid utilization resulting from reduced acyl coenzyme A derivatives is also not likely to contribute to altered myelination since no difference in ACS-2 expression was found. Despite recent in vitro evidence that PPARbeta regulates ACS-2 mRNA upon activation (6), these data demonstrate that constitutive expression of this gene is not influenced by the absence of PPARbeta . Lastly, MBP RNA was not different for the different genotypes. Combined, these results suggest that the alteration in myelination observed in the PPARbeta -null mouse corpus callosum is the result of events that are mediated by PPARbeta during development but that were not detectable at the age we analyzed. Further analysis of this process is needed, but these data do provide strong evidence that PPARbeta is required for brain development, possibly for regulation of genes that have not been identified. It is noteworthy that preliminary behavioral assessments of older mice using a rotorod revealed no differences between PPARbeta -null and wild-type mice. While further behavioral studies are warranted, the physiological and behavioral consequences of the altered myelination remain a mystery.

The role of PPARbeta in skin. The PPARbeta gene is one of the genes involved in epidermal cell proliferation and differentiation induced by TPA (37). It was hypothesized that PPARbeta is required for induction of other genes involved in epidermal differentiation since PPARbeta mRNA is increased coincidently with TG-I and SPR1A in vitro (37). Data provided from PPARbeta -null mice demonstrate that PPARbeta is not required for this effect, since mRNAs for TG-I, involucrin, ODC, SPR1A, and SPR2H were all induced to similar levels in the skin of wild-type and PPARbeta -null mice treated with TPA. Since PPARbeta mRNA is increased, the role of this receptor in the TPA response is of great interest and may provide a useful model to identify more-specific roles for PPARbeta . The finding that the hyperplastic response to TPA is enhanced in the PPARbeta -null mice suggests the possibility that this receptor is involved in cell cycle control. Support for a putative role of PPARbeta in cell cycle control is provided by the recent report that colon carcinomas have elevated levels of PPARbeta (20).

Interestingly, PPARbeta -null mice fed the NSAID sulindac were more sensitive to the inflammatory response induced by TPA. These data indicate that the sulindac-mediated anti-inflammatory response is dependent on PPARbeta . These data support recent observations that sulindac inhibits PPARbeta from binding to recognition sites of unidentified target genes (20). Further support for a role for PPARbeta in cell cycle control is also provided by the observation that an early hyperplastic response not found in wild-type mice was observed in TPA-treated PPARbeta -null mice. The precise mechanisms for these effects are unknown, but these data clearly demonstrate that PPARbeta can influence the effects of sulindac. Further studies are necessary to delineate the mechanisms underlying the PPARbeta influence on cell cycle regulation in both tumor promotion and tumor formation.

The PPARbeta -null mouse model. In summary, this is the first report that provides in vivo evidence for the roles of PPARbeta in development, lipid metabolism, myelination of the corpus callosum, and epidermal cell proliferation. These results support previous reports suggesting a role for this receptor in adipose tissue and brain, with a consistent theme of lipid metabolism being demonstrated for all three PPAR subtypes. In addition, these studies significantly extend our understanding of other important physiological functions that are likely regulated by PPARbeta by showing that development and cell proliferation are also likely targets of this nuclear receptor.


    ACKNOWLEDGMENTS

We gratefully acknowledge Karen Chandross for dissection of specific brain regions for RNA isolation, Colin Stewart for his analysis of GD10 embryos, Debra Wolgemuth for providing the mouse CDK-1, CDK-4, and cyclin B1 cDNA plasmids, Robert Rice for providing the rat TG-I and involucrin cDNA plasmids, and Tonja Kartasova for providing the mouse SPR1A and SPR2H cDNA plasmids.


    FOOTNOTES

* Corresponding author. Mailing address: Center for Molecular Toxicology, Department of Veterinary Science, The Pennsylvania State University, 226 Fenske Laboratory, University Park, PA 16802-4401. Phone: (814) 863-1387. Fax: (814) 863-1696. E-mail: jmp21{at}psu.edu.

dagger Present address: Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.

Dagger Present address: Neurotoxicology Laboratory, Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402[Abstract/Free Full Text].
2. Amri, E. Z., F. Bonino, G. Ailhaud, N. A. Abumrad, and P. A. Grimaldi. 1995. Cloning of a protein that mediates transcriptional effects of fatty acids in preadipocytes. Homology to peroxisome proliferator-activated receptors. J. Biol. Chem. 270:2367-2371[Abstract/Free Full Text].
3. Aoyama, A., J. M. Peters, N. Iritani, T. Nasu-Nakajima, K. Furihata, T. Hashimoto, and F. J. Gonzalez. 1998. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha  (PPARalpha ). J. Biol. Chem. 273:5678-5684[Abstract/Free Full Text].
4. Barak, Y., M. C. Nelson, E. S. Ong, Y. Z. Jones, P. Ruiz-Lozano, K. R. Chien, A. Koder, and R. E. Evans. 1999. PPARgamma is required for placental, cardiac, and adipose tissue development. Mol. Cell 4:585-595[CrossRef][Medline].
5. Bastie, C., D. Holst, D. Gaillard, C. Jehl-Pietri, and P. A. Grimaldi. 1999. Expression of peroxisome proliferator-activated receptor PPARdelta promotes induction of PPARgamma and adipocyte differentiation in 3T3C2 fibroblasts. J. Biol. Chem. 274:21920-21925[Abstract/Free Full Text].
6. Basu-Modak, S., O. Braissant, P. Escher, B. Desvergne, P. Honegger, and W. Wahli. 1999. Peroxisome proliferator-activated receptor beta  regulates acyl-CoA synthetase 2 in reaggregated rat brain cell cultures. J. Biol. Chem. 274:35881-35888[Abstract/Free Full Text].
7. Bogazzi, F., L. D. Hudson, and V. M. Nikodem. 1994. A novel heterodimerization partner for thyroid hormone receptor. Peroxisome proliferator-activated receptor. J. Biol. Chem. 269:11683-11686[Abstract/Free Full Text].
8. Braissant, O., F. Foufelle, C. Scotto, M. Dauca, and W. Wahli. 1996. Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha , -beta , and -gamma in the adult rat. Endocrinology 137:354-366[Abstract].
9. Braissant, O., and W. Wahli. 1998. Differential expression of peroxisome proliferator-activated receptor-alpha , -beta , and -gamma during rat embryonic development. Endocrinology 139:2748-2754[Abstract/Free Full Text].
10. Chapman, D. L., and D. J. Wolgemuth. 1992. Identification of a mouse B-type cyclin which exhibits developmentally regulated expression in the germ line. Mol. Reprod. Dev. 33:259-269[CrossRef][Medline].
11. Cullingford, T. E., K. Bhakoo, S. Peuchen, C. T. Dolphin, R. Patel, and J. B. Clark. 1998. Distribution of mRNAs encoding the peroxisome proliferator-activated receptor alpha , beta , and gamma  and the retinoid X receptor alpha , beta , and gamma  in rat central nervous system. J. Neurochem. 70:1366-1375[Medline].
12. Djian, P., M. Phillips, K. Easley, E. Huang, M. Simon, R. H. Rice, and H. Green. 1993. The involucrin genes of the mouse and the rat: study of their shared repeats. Mol. Biol. Evol. 10:1136-1149[Abstract].
13. Endemann, G., L. W. Stanton, K. S. Madden, C. M. Bryant, R. T. White, and A. A. Protter. 1993. CD36 is a receptor for oxidized low density lipoprotein. J. Biol. Chem. 268:11811-11816[Abstract/Free Full Text].
14. Fruchart, J. C., P. Duriez, and B. Staels. 1999. Peroxisome proliferator-activated receptor-alpha activators regulate genes governing lipoprotein metabolism, vascular inflammation and atherosclerosis. Curr. Opin. Lipidol. 10:245-257[CrossRef][Medline].
15. Fujino, T., M. J. Kang, H. Suzuki, H. Iijima, and T. Yamamoto. 1996. Molecular characterization and expression of rat acyl-CoA synthetase 3. J. Biol. Chem. 271:16748-16752[Abstract/Free Full Text].
16. Fujino, T., and T. Yamamoto. 1992. Cloning and functional expression of a novel long-chain acyl-CoA synthetase expressed in brain. J. Biochem. (Tokyo) 111:197-203[Abstract/Free Full Text].
17. Gavrilova, O., L. R. Leon, B. Marcus-Samuels, M. M. Mason, A. L. Castle, S. Refetoff, C. Vinson, and M. L. Reitman. 1999. Torpor in mice is induced by both leptin-dependent and -independent mechanisms. Proc. Natl. Acad. Sci. USA 96:14623-14628[Abstract/Free Full Text].
18. Gelman, L., and J. Auwerx. 1999. Peroxisome proliferator-activated receptors: mediators of a fast food impact on gene regulation. Curr. Opin. Clin. Nutr. Metab. Care 2:307-312[CrossRef][Medline].
19. Guardiola-Diaz, H. M., S. Rehnmark, N. Usuda, T. Albrektsen, D. Feltkamp, J. A. Gustafsson, and S. E. Alexson. 1999. Rat peroxisome proliferator-activated receptors and brown adipose tissue function during cold acclimatization. J. Biol. Chem. 274:23368-23377[Abstract/Free Full Text].
20. He, T. C., T. A. Chan, B. Vogelstein, and K. W. Kinzler. 1999. PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 99:335-345[CrossRef][Medline].
21. Hudson, L. D., J. A. Berndt, C. Puckett, C. A. Kozak, and R. A. Lazzarini. 1987. Aberrant splicing of proteolipid protein mRNA in the dysmyelinating jimpy mutant mouse. Proc. Natl. Acad. Sci. USA 84:1454-1458[Abstract/Free Full Text].
22. Issemann, I., and S. Green. 1990. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 347:645-650[CrossRef][Medline].
23. Jordan, C. A., V. L. Friedrich, Jr., C. Godfraind, C. B. Cardellechio, K. V. Holmes, and M. Dubois-Dalcq. 1989. Expression of viral and myelin gene transcripts in a murine CNS demyelinating disease caused by a coronavirus. Glia 2:318-329[CrossRef][Medline].
24. Kahana, C., and D. Nathans. 1985. Nucleotide sequence of murine ornithine decarboxylase mRNA. Proc. Natl. Acad. Sci. USA 82:1673-1677[Abstract/Free Full Text].
25. Kartasova, T., N. Darwiche, Y. Kohno, H. Koizumi, S. Osada, N. Huh, U. Lichti, P. M. Steinert, and T. Kuroki. 1996. Sequence and expression patterns of mouse SPR1: correlation of expression with epithelial function. J. Investig. Dermatol. 106:294-304[CrossRef][Medline].
26. Kersten, S., J. Seydoux, J. M. Peters, F. J. Gonzalez, B. Desvergne, and W. Wahli. 1999. Peroxisome proliferator-activated receptor alpha  mediates the adaptive response to fasting. J. Clin. Investig. 103:1489-1498[Medline].
27. Kliewer, S. A., B. M. Forman, B. Blumberg, E. S. Ong, U. Borgmeyer, D. J. Mangelsdorf, K. Umesono, and R. M. Evans. 1994. Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc. Natl. Acad. Sci. USA 91:7355-7359[Abstract/Free Full Text].
28. Krey, G., O. Braissant, F. L'Horset, E. Kalkhoven, M. Perroud, M. G. Parker, and W. Wahli. 1997. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay. Mol. Endocrinol. 11:779-791[Abstract/Free Full Text].
29. Kubota, N., Y. Terauchi, H. Miki, H. Tamemoto, T. Yamauchi, K. Komeda, S. Satoh, R. Nakano, C. Ishii, T. Sugiyama, K. Eto, Y. Tsubamoto, A. Okuno, K. Murakami, H. Sekihara, G. Hasegawa, M. Naito, Y. Toyoshima, S. Tanaka, K. Shiota, T. Kitamura, T. Fujita, O. Ezaki, S. Aizawa, R. Nagai, K. Tobe, S. Kimura, and T. Kadowaki. 1999. PPARgamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol. Cell 4:597-609[CrossRef][Medline].
30. Laird, P. W., A. Zijderveld, K. Linders, M. A. Rudnicki, R. Jaenisch, and A. Berns. 1991. Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 19:4293[Free Full Text].
31. Lee, S. S., T. Pineau, J. Drago, E. J. Lee, J. W. Owens, D. L. Kroetz, P. M. Fernandez-Salguero, H. Westphal, and F. J. Gonzalez. 1995. Targeted disruption of the alpha  isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol. Cell. Biol. 15:3012-3022[Abstract].
32. Lehmann, J. M., L. B. Moore, T. A. Smith-Oliver, W. O. Wilkison, T. M. Willson, and S. A. Kliewer. 1995. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma  (PPARgamma ). J. Biol. Chem. 270:12953-12956[Abstract/Free Full Text].
33. Lim, H., R. A. Gupta, W. G. Ma, B. C. Paria, D. E. Moller, J. D. Morrow, R. N. DuBois, J. M. Trzaskos, and S. K. Dey. 1999. Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPARdelta . Genes Dev. 13:1561-1574[Abstract/Free Full Text].
34. Mangelsdorf, D. J., and R. M. Evans. 1995. The RXR heterodimers and orphan receptors. Cell 83:841-850[CrossRef][Medline].
35. Martin, G., K. Schoonjans, A. M. Lefebvre, B. Staels, and J. Auwerx. 1997. Coordinate regulation of the expression of the fatty acid transport protein and acyl-CoA synthetase genes by PPARalpha and PPARgamma activators. J. Biol. Chem. 272:28210-28217[Abstract/Free Full Text].
36. Matsushime, H., M. E. Ewen, D. K. Strom, J. Y. Kato, S. K. Hanks, M. F. Roussel, and C. J. Sherr. 1992. Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins. Cell 71:323-334[CrossRef][Medline].
37. Matsuura, H., H. Adachi, R. C. Smart, X. Xu, J. Arata, and A. M. Jetten. 1999. Correlation between expression of peroxisome proliferator-activated receptor beta  and squamous differentiation in epidermal and tracheobronchial epithelial cells. Mol. Cell. Endocrinol. 147:85-92[CrossRef][Medline].
38. Motojima, K., P. Passilly, J. M. Peters, F. J. Gonzalez, and N. Latruffe. 1998. Expression of putative fatty acid transporter genes are regulated by peroxisome proliferator-activated receptor alpha  and gamma  activators in a tissue- and inducer-specific manner. J. Biol. Chem. 273:16710-16714[Abstract/Free Full Text].
39. Moya-Camarena, S. Y., J. P. van den Heuvel, and M. A. Belury. 1999. Conjugated linoleic acid activates peroxisome proliferator-activated receptor alpha  and beta  subtypes but does not induce hepatic peroxisome proliferation in Sprague-Dawley rats. Biochim. Biophys. Acta 1436:331-342[Medline].
40. Peters, J. M., T. Aoyama, R. C. Cattley, U. Nobumitsu, T. Hashimoto, and F. J. Gonzalez. 1998. Role of peroxisome proliferator-activated receptor alpha  in altered cell cycle regulation in mouse liver. Carcinogenesis 19:1989-1994[Abstract/Free Full Text].
41. Peters, J. M., N. Hennuyer, B. Staels, J. C. Fruchart, C. Fievet, F. J. Gonzalez, and J. Auwerx. 1997. Alterations in lipoprotein metabolism in peroxisome proliferator-activated receptor alpha -deficient mice. J. Biol. Chem. 272:27307-27312[Abstract/Free Full Text].
42. Phillips, M. A., B. E. Stewart, Q. Qin, R. Chakravarty, E. E. Floyd, A. M. Jetten, and R. H. Rice. 1990. Primary structure of keratinocyte transglutaminase. Proc. Natl. Acad. Sci. USA 87:9333-9337[Abstract/Free Full Text].
43. Reddy, T. S., and N. G. Bazan. 1985. Long-chain acyl CoA synthetase in microsomes from rat brain gray matter and white matter. Neurochem. Res. 10:377-386[CrossRef][Medline].
44. Ren, B., A. P. Thelen, J. M. Peters, F. J. Gonzalez, and D. B. Jump. 1997. Polyunsaturated fatty acid suppression of hepatic fatty acid synthase and S14 gene expression does not require peroxisome proliferator-activated receptor alpha . J. Biol. Chem. 272:26827-26832[Abstract/Free Full Text].
45. Rosen, E. D., P. Sarraf, A. E. Troy, G. Bradwin, K. Moore, D. S. Milstone, B. M. Spiegelman, and R. M. Mortensen. 1999. PPARgamma is required for the differentiatio