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Previous Article | Next Article 
Molecular and Cellular Biology, November 2003, p. 7947-7956, Vol. 23, No. 22
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.22.7947-7956.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Molecular Oncology Group, Department of Medicine, McGill University Health Centre, Montréal, Québec, Canada H3A 1A1,1 Département d'Anatomie et Physiologie, Faculté de Médecine, Université Laval, Québec, Canada G1K 7P42
Received 31 March 2003/ Returned for modification 9 May 2003/ Accepted 30 July 2003
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ABSTRACT |
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(ERR) is an orphan member of the superfamily of nuclear hormone receptors expressed in tissues that preferentially metabolize fatty acids. Despite the molecular characterization of ERR and identification of target genes, determination of its physiological function has been hampered by the lack of a natural ligand. To further understand the in vivo function of ERR, we generated and analyzed Estrra-null (ERR-/-) mutant mice. Here we show that ERR-/- mice are viable, fertile and display no gross anatomical alterations, with the exception of reduced body weight and peripheral fat deposits. No significant changes in food consumption and energy expenditure or serum biochemistry parameters were observed in the mutant animals. However, the mutant animals are resistant to a high-fat diet-induced obesity. Importantly, DNA microarray analysis of gene expression in adipose tissue demonstrates altered regulation of several enzymes involved in lipid, eicosanoid, and steroid synthesis, suggesting that the loss of ERR might interfere with other nuclear receptor signaling pathways. In addition, the microarray study shows alteration in the expression of genes regulating adipogenesis as well as energy metabolism. In agreement with these findings, metabolic studies showed reduced lipogenesis in adipose tissues. This study suggests that ERR functions as a metabolic regulator and that the ERR-/- mice provide a novel model for the investigation of metabolic regulation by nuclear receptors. |
INTRODUCTION |
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The estrogen-related receptor (ERR) (NR3B1) was the first orphan nuclear receptor identified more than a decade ago on the basis of its close homology to the classic estrogen receptor (ER) (NR3A1) (14). ERR was also identified as a repressor of the simian virus 40 major late promoter, revealing that ERR had the ability to regulate gene expression in the absence of exogenous stimuli (67). Two other orphan nuclear receptors, ERRß (NR3B2) (6, 14) and ERR
(NR3B3) (9, 16, 18), also belong to the same subfamily.
ERR expression begins at day 9.5 post coitum in extraembryonic tissues and is later detected in the heart, intestine, brain, spinal cord, brown fat, and bone (4, 50, 62). ERR is expressed in a nearly ubiquitous fashion in adult tissues but most prominently in tissues demonstrating a high capacity for fatty acid ß-oxidation or activation, suggesting that ERR may play a role in regulating cellular energy balance (14, 21, 50). This is further supported by the observations that ERR is a direct regulator of the medium-chain acyl coenzyme A dehydrogenase gene (MCAD) (50, 64) and that PGC-1, a coactivator regulating transcription in response to signals relaying metabolic needs, is a potent regulator of ERR activity (19, 21, 49).
Recent experiments have also shown that ERR is functionally closer to the ERs than originally anticipated (reviewed in reference 13). Like the ERs, the ERRs recognize the ERE as a homodimer, suggesting that these receptors may control overlapping regulatory pathways (32, 45, 73). In addition, ERR binds to extended half-sites with the consensus sequence TCAAGGTCA referred to as an ERRE (22, 50, 64, 67, 71). ER has also been shown to recognize a subset of ERREs and stimulate the transcriptional activity of promoters containing such sites, reinforcing the concept that the two classes of related receptors share common target genes (63). It has also been discovered that the synthetic estrogen diethylstilbestrol and the partial estrogen antagonist 4-hydroxytamoxifen can act as selective inverse agonists on the three ERR isoforms, leading to the dissociation of coactivator proteins and loss of transcriptional activation function (8, 58, 59). In addition, we have recently demonstrated that the ERR isoforms can regulate the transcriptional activity of the human breast cancer marker gene pS2 and, unexpectedly, that the full transcriptional activity of the ERs and the ERRs on the pS2 gene is dependent on the presence of a previously unidentified ERRE within its promoter (32). Taken together, these results suggest that ERR may also influence classic endocrine estrogenic pathways. However, in spite of these studies, the exact physiological roles of ERR remain unknown.
To explore the biological functions of ERR in vivo, we used gene targeting technology to generate ERR deficient mice (ERR-/- mice). These ERR-/- mice are viable and fertile and show no gross anatomical abnormalities. However, ERR-/- mice display reduced fat mass and are resistant to high-fat diet-induced obesity, and their adipose tissue shows alterations in the expression of several genes directly linked to lipid metabolism and adipogenesis. ERR-/- mice thus provide a new model to investigate nuclear receptor-regulated metabolic pathways and associated physiology and pathology.
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MATERIALS AND METHODS |
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and production of chimeric mice.
Three overlapping 
clones containing the mouse Estrra locus were isolated from a 129Sv genomic library (a gift from A. Joyner, Skirball Institute, New York, N.Y.) and characterized by restriction mapping and direct sequencing of the exon boundaries. The knockout construct was created by using pNT (60) and contained 6.73 kb of genomic DNA flanking the second exon of Estrra. An endfilled 4.36-kb BamHI/NotI fragment, lying upstream of the second exon, was cloned into the XhoI site of pNT, whereas a 2.37-kb HindIII fragment was cloned between the neor and thymidine kinase cassettes to provide the 3' arm of the construct. Correct targeting of the Estrra locus replaces the second exon of the receptor, which encodes the amino-terminal region and an essential component of its DNA-binding domain, with a neo cassette. The linearized construct was electroporated into R1 embryonic stem (ES) cells (41), which were selected with G418 (150 µg/ml) and ganciclovir (2 µM). Two ES cell clones were isolated and injected into C57BL/6 blastocysts to generate chimeras, and three chimeras transmitted the mutation to their offspring. Heterozygous mice, generated by mating the chimeric animals with 129SvJ mice were mated with C57BL/6 animals to generate hybrid F1 animals: physiologic studies were performed by using the F2-null mutant and wild-type offspring obtained by mating the F1 hybrid heterozygotes, with the exception of the study on high-fat diet-induced obesity, which was performed with ERR-null mice in the C57BL/6 background. Complete disruption of the Estrra allele was verified by performing Northern and Western blots with RNA and proteins obtained from kidney and intestine, respectively, as previously described (50). Studies of Estrra-/- mice. Mice were housed in an specific-pathogen-free facility with a daily 12-h light cycle (7:00 a.m. to 7:00 p.m.) and with free access to food and water. Between two and four mice were housed in each cage. Growth curves were obtained by weighing mice of defined ages between 10:00 and 12:00 a.m. Fasting serum and biochemical studies were performed between 10:00 and 12:00 a.m. with animals that had been deprived of food for 18 h. Body composition was determined by desiccating mouse carcasses from which the intestines had been removed (29). After desiccation, the carcass was homogenized and a 1-g aliquot was saponified by using potassium hydroxide and extracted with petroleum ether. After complete evaporation of the ether, the residue was weighed to determine the fat content. Statistical comparisons of the body composition data was performed by using the Mann-Whitney U test. Baseline biochemical studies were performed with serum samples obtained from tail bleeds of restrained animals at between 20 and 28 weeks of age. Enzymatic assays were used to determine serum triglycerides (GPO-PAP; Boehringer Mannheim) and glycerol (TC Glycerin; Boehringer Mannheim), glucose (Glucose Oxidase-Trinder; Sigma), free fatty acids (GPO-PAP Half Micro Test; Boehringer Mannheim), and ß-hydroxybutyrate (TC ß-hydroxybutyrate; Boehringer Mannheim). Energy balance and body composition measurements were performed as previously described (46). For high-fat feeding, a diet containing 45% (wt/wt) fat content (D12451; Research Diets, Inc., New Brunswick, N.J.) was used.
DNA microarrays and quantitative PCR.
Microarray analysis was performed with pooled adipose tissue obtained from two wild-type and two ERR-/- adult male mice. The animals were fasted overnight and euthanized by using Avertin. Total RNA was prepared from homogenized adipose tissue by using Trizol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.). Probes for microarray analysis were prepared by using 10 µg of total RNA and hybridized to Affymetrix (Santa Clara, Calif.) Mu74Av2 GeneChips. Detailed protocols for the probe synthesis and hybridization reactions, as well as for the posthybridization washing and staining, have been previously described (43). The hybridized arrays were scanned and raw data extracted by using Microarray Analysis Suite 5.0 (Affymetrix). Expression profiles obtained from both knockout animals were compared individually to those obtained from each wild-type mouse by using Microarray Analysis Suite 5.0, which examines the hybridization intensities of individual probe pairs in order to estimate the significance of observed changes in gene expression: in the present study, the default range for the change P value (lower cutoff, 0.0025; upper cutoff, 0.9975) was used to identify genes whose expression changed in each comparison. Genes whose expression levels differed in three of four comparisons were considered to show significantly different levels of expression between the wild-type and knockout animals. For quantitative PCR, total RNA was extracted from white and brown fat tissues of wild-type and knockout animals by using Trizol reagent. cDNA was prepared by reverse transcription of 1 µg of total RNA with Superscript II enzyme and oligo(dT) primer. The resulting cDNAs were amplified by using the LightCycler FastStart DNA Master SYBR Green I kit and a LightCycler instrument according to the manufacturer's instructions (Roche Diagnostics, Indianapolis, Ind.). All mRNA expression data was normalized to hypoxanthine phosphoribosyltransferase expression in the corresponding sample.
Lipogenesis rate. Mice were studied at 10:00 a.m. after free access to food overnight. The animals were conditioned by sham intraperitoneal injections of water. On the day of the experiment, the animals were injected intraperitoneally with 3H2O (0.5 mCi per 100 g of body weight) and sacrificed by cervical dislocation 30 min later. Adipose tissue samples were harvested and stored at -80°C. The tissues were homogenized and heated in ethanolic KOH. The resulting tissue extracts, which contained saponified lipids, were acidified with concentrated sulfuric acid and extracted by using petroleum ether. The extracts were dried by evaporation, and 3H incorporation in the lipid fraction was determined by scintillation counting.
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RESULTS |
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+/- ES cell lines (Fig. 1B) confirmed that the locus had been targeted as expected.
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-/- mice are viable and fertile.
Heterozygotes for the disrupted ERR allele were apparently normal and were crossed to generate homozygous mutant mice. Figure 1C shows a representative Southern blot of 4-week-old offspring from crosses of heterozygous mice. Homozygous ERR-/- mice were obtained at the expected Mendelian frequency and did not differ from their wild-type littermates in overall health, fertility (including interbreeding between ERR-/- mice), and longevity. Histological examinations detected no obvious abnormalities in the brain, spinal cord, pituitary, heart, kidneys, adrenal, testis, ovary, intestine, and liver (data not shown). Northern blot analysis with total mRNA extracted from kidneys failed to detect ERR transcripts in ERR-/- mice, whereas approximately half the amount of transcripts was observed in ERR+/- mice (Fig. 1D). Interestingly, the levels of the ERRß transcript were found to be downregulated in both heterozygous and ERR knockout animals, suggesting that ERR may regulate ERRß expression in the kidneys. Finally, no ERR protein could be detected by Western blot analysis of proteins extracted from the intestine of knockout animals (Fig. 1E).
ERR-/- mice have reduced fat mass.
To verify the general well-being of ERR-/- mice and explore the possibility that ERR may play a role in maintaining homeostasis in the adult animal, their growth rates relative to those of wild-type littermate controls were measured in terms of total body weight (Fig. 2A and B). During the first postnatal week, the mean body weight of ERR-/- mice was slightly decreased, although it was not statistically different from wild-type littermates. However, beginning at postnatal week 2, ERR-/- mice displayed a significant decrease in body weight. At postnatal week 10, for example, both male and female ERR-/- mice weighed ca. 15% less than their wild-type littermates (P < 0.01). No significant reduction in body weight was observed in heterozygous ERR+/- mice (Table 1 and data not shown). The body length (nose to anus) of 10-week-old male and female mice showed that linear growth was not impaired in ERR-/- mice (Fig. 2C). In addition, the weight of the heart did not differ in ERR-/- mice (Fig. 2D). However, ERR-/- had a 50 to 60% reduction in the weight of the inguinal, epididymal and peritoneal fat pads (Fig. 2E to G). Biochemical characterization of carcass composition showed that the ERR-null mice had, in comparison with control littermates, 80.6% fat, 105% lean mass, and 106.4% water (Table 1). The histology of fat tissue shows an apparent normal number of adipocytes with decreased adipocyte size (Fig. 3).
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-/- mice.
To further investigate the biological basis of this phenotype, a series of physiological tests were performed on the ERR-/- mice. Fasting glucose levels, as well as serum free fatty acids and triglyceride levels, were not significantly affected by the absence of ERR (data not shown).
Food consumption and energy expenditure.
To examine whether the reduction in fat mass observed in ERR-/- mice was caused by decreased food intake or increased energy expenditure, both parameters were measured over a 4-day period. We observed no statistically significant change in either parameter (Fig. 4 and data not shown).
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-/- mice are resistant to high-fat diet-induced obesity.
We next challenged 10-week-old mice with a high-fat diet containing 45% (wt/wt) fat for 5 weeks to stimulate weight gain. As shown in Fig. 5A, weight gain was significantly reduced in the ERR-/- mice relative to the control littermates. A statistical difference between the two groups was observed in the third week of feeding. By the end of the experiment, the control littermates had become mildly obese (Fig. 5B), whereas the ERR-/- mice weighed only slightly more than wild-type mice fed a normal diet.
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ablation causes changes in gene expression in adipose tissue (Table 2). Pooled adipose tissue was harvested from fasted adult male mice and studied by using Affymetrix GeneChips. The expression studies show downregulation of ERR and changes in the expression of two known ERR target genes: lactotransferrin and MCAD. The expression studies also demonstrate altered regulation of several enzymes involved in lipid metabolism (fatty acid synthase [gene Fasn], stearoyl-coenzyme A desaturase 2 [Scd2], fatty acid coenzyme A ligase long-chain 5, [Facl5], acetyl-coenzyme A dehydrogenase medium chain [Acadm], and acetyl-coenzyme A dehydrogenase long chain [Acadl]) and energy metabolism (cytochrome c somatic [Cycs], carnitine acetyltransferase [Crat], creatine kinase mitochondrial 2 [Ckmt2], and uncoupling protein 1 [Ucp1]). These changes create an imbalance between enzymes involved in fat synthesis and fat catabolism that may result in decreased levels of de novo triglyceride synthesis in the knockout animals. The alterations in transcript expression observed in the microarray studies may include genes that are direct ERR targets, as well as genes that are regulated by other transcription factors or cell signaling pathways. In particular, the microarray studies demonstrate alterations in sterol regulatory element binding factor 1 (Srebf1) and CCAAT/enhancer binding protein gamma (Cebpg) expression, transcription factors that have been implicated in the regulation of adipogenesis (reviewed in reference 11). In addition, decreased expression of prostaglandin D2 synthase (Ptgds) and hydroxysteroid dehydrogenase-4 
5
-3-ß (Hsd3b4) and hydroxysteroid dehydrogenase-6 5-3-ß (Hsd3b6) isoforms may alter peroxisome proliferator-activated receptor (PPAR) activity by regulating levels of their putative endogenous ligands, which in turn may explain prostaglandin-endoperoxide synthase 2 (Ptgs2) upregulation (25, 72). Finally, adipose tissue from ERR knockout animals also expresses increased levels of adrenergic receptor ß3, a known regulator of murine adiposity (54). The quality of the array data was further validated by quantitative PCR analysis of 15 selected genes indicated in bold in Table 1. Of these genes, changes in the expression of 11 of 15 (74%) were validated by quantitative PCR, including Esrra, whose expression could not be detected as expected. The levels of expression for two genes, Ckmt21 and Hdac6, in white adipose tissue (WAT) were also too low to obtain accurate measurements. For two other genes, Ptgds and Ucp1, we observed upregulation (
30- and 2.7-fold, respectively) by quantitative PCR, while the microarray data indicated that the expression of these genes was downregulated. Thus, we are confident that, for more than 75% of genes identified as differentially expressed, the results of our microarray gene screening method is accurate. The changes in the expression of the validated genes in the WAT of ERR-/- mice compared to wild-type mice, as obtained by quantitative PCR, are shown in Fig. 6A. Of note, true fold changes are generally greater than the averaged microarray data, leading us to include genes whose level of expression was as low as 1.2-fold (Table 2). Since brown fat expressed high levels of ERR, we also measured changes in expression levels of the selected genes in this tissue (Fig. 6B). In most cases, changes in gene expression observed in brown fat parallel those seen in white fat, with the exception of Elovl3 and Srebf1.
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-/- mice with 3H2O: the amount of radioactive label incorporated into triacylglycerol can be measured by saponification and ether extraction of adipose tissues and other organs (12). ERR-/- mice demonstrated significantly decreased lipogenesis in comparison to littermate controls: knockout animals show a 30 to 55% decrease in 3H incorporation into adipose tissue lipids (Fig. 7). This finding demonstrates that adipose tissues of knockout mice possess a defect in triglyceride synthesis, which may result from decreased adipocyte glycolytic activity, decreased fatty acid synthesis, or decreased esterification.
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DISCUSSION |
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-deficient mice through homologous recombination in ES cells. No ERR expression was detectable in these mice by Northern and Western blot analyses and quantitative PCR. The mice were viable and fertile and have a normal life span. Although ERR-deficient mice weighed less compared to their wild-type littermates, histological analysis of adult tissues failed to show morphological abnormalities. Therefore, we conclude that there is no absolute requirement for ERR function in development, sexual maturation, and homeostasis. This finding is in sharp contrast with the phenotype observed with the ERRß-null mice who die in utero due to the complete absence of the diploid trophoblast layers of the placenta (36). This finding is also surprising in view of the very high level of ERR expression in brown adipose tissue (50) and the proposed role for ERR in bone formation (3, 4).
Although ERR deficiency by itself may be largely asymptomatic, if present with other genetic defects such as null mutations in other members of the ERR or ER families, it could conceivably give rise to an overtly abnormal developmental, metabolic, or endocrine phenotype. In fact, deletion of genes encoding nuclear receptors does not always produce an apparent phenotype. Perhaps the most dramatic example is that of the genes encoding the three retinoic acid receptors (RAR, -ß, and -). Mice devoid of either RAR (30, 34), RARß (35, 40), or RAR (31) display only subtle developmental phenotypes. An extensive functional redundancy between RARs accounts for this observation as severe developmental defects results from the lack any combinations of two RAR genes (reviewed in reference 38). Disruption of other nuclear receptor genes produce a phenotype only when animals are physiologically or pharmacologically challenged, such as with a high cholesterol diet in LXR mutant mice (44) or xenobiotic agents in PXR/SXR-null mice (52, 68, 69). Therefore, a more evident phenotype may be observed only in ERR double mutants or in response to a specific physiological stress.
Although the ERR-/- mice appear normal, they display reduced body weight and diminished fat mass (Fig. 2). We were unable to detect statistically significant changes in eating behavior or energy expenditure between the wild-type and ERR-null littermates. One possible explanation for these results is that the putative increase in energy expenditure is small and significant only over a long period of time. How the absence of ERR leads to this phenotype at the molecular level is currently unknown. However, gene expression profiling experiments in adipose tissue of wild-type versus knockout mice are providing interesting avenues of investigation. First, the expression studies confirmed a role of ERR in MCAD (Acadm) gene regulation, predominantly as a repressor, since MCAD expression is upregulated in the adipose tissue of the knockout mice. This result indicates that ERR can indeed act as a direct regulator of metabolic genes. Second, the loss of ERR function leads to alterations in the expression of a wide variety of genes encoding transcriptional regulators, two of them (Srebf1 and Cebpg) directly implicated in the regulation of adipogenesis. The absence of ERR might therefore induce significant changes in a regulatory cascade that normally controls the expression of genes involved in fat and sterol metabolism. We have also noted significant alterations in the expression of genes involved in sterol, steroid, and prostaglandin synthesis, suggesting that the loss of ERR might indirectly influence the activity of other nuclear receptor-based signaling pathways, many of which are known to regulate lipid metabolism an adipogenesis (1, 11, 33, 47). Third, the gene microarray analysis detected changes in the expression of metabolic enzymes directly involved in lipid synthesis, an observation correlated with a marked decrease in lipogenesis rate in white fat tissues. Finally, the observed upregulation of the gene encoding the ß3 adrenergic receptor suggests that lipolysis in white adipocytes could also be affected (7), although more specific studies will be required to validate this hypothesis. It is, however, interesting that the Adrb3 promoter has been shown to encode a putative binding site for ERR (28). Although the physiological significance of the alteration in expression of each specific gene remains to be fully investigated, this ensemble is in agreement with the observed reduced fat mass in the ERR-/- mice.
As introduced above, ERR has been shown to possess the ability to interfere with estrogen signaling (reviewed in reference 13). Estrogen is known to play an important role in WAT regulation: in rodents, ovariectomy increases the amount of WAT, whereas estrogen replacement decreases the amount of WAT (65). Similar observations were made in postmenopausal women subjected to hormone replacement therapy (56). More recently, increased adipose tissue in male and female ER-null mice was observed, demonstrating conclusively a role for estrogen and ER in WAT functions (17). The ERR-null mice displays the opposite phenotype, an observation consistent with the fact that ERR can act as a potent transcriptional repressor in specific biological settings and compete with the ERs for binding sites and coactivators (27, 50, 57, 67, 73). The upregulation of Fos, a direct target of ER signaling (20), in the white and brown adipose tissues of the ERR-null mice is in agreement with the proposed cross talk between the two nuclear receptor-based regulatory pathways. In addition, the inhibition of estrogen production in aromatase-deficient mice results in increased adipose tissue mass and hepatic steatosis (23), which is associated with reduced rates of hepatic ß-oxidation and decreased expression of AOX, VLACS, and MCAD (42). It will be interesting to test whether the combined loss of ER and ERR in mice "resets" their metabolism toward a more wild-type-like state.
In light of the well-known caveat that mouse development and life under controlled laboratory conditions do not reproduce the environment that mice find themselves in the wild, it is now evident that ERR is not absolutely required for normal mouse development and growth. However, it is also evident that the ERR-null mice display metabolic abnormalities that warrant further investigation. The development of ERR specific ligands and the current availability of high-throughput gene expression profiling technology coupled with future ERR-/--based mouse models will provide further insight into the role played by ERR in metabolic control and other biological processes.
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ACKNOWLEDGMENTS |
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V.G. is a Senior Scientist of the Canadian Institutes of Health Research (CIHR). This work was supported by operating grants from the CIHR to V.G.
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FOOTNOTES |
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