Reduced fat mass in mice lacking orphan nuclear receptor estrogen-related receptor alpha

Abstract

The estrogen-related receptor alpha (ERRalpha) is an orphan member of the superfamily of nuclear hormone receptors expressed in tissues that preferentially metabolize fatty acids. Despite the molecular characterization of ERRalpha 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 ERRalpha, we generated and analyzed Estrra-null (ERRalpha-/-) mutant mice. Here we show that ERRalpha-/- 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 ERRalpha 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 ERRalpha functions as a metabolic regulator and that the ERRalpha-/- mice provide a novel model for the investigation of metabolic regulation by nuclear receptors.

FIG. 1.

Targeted disruption of the murine Estrra gene in ES cells and mice. (A) Schematic representation of the Estrra locus and targeting vector. Black boxes in the wild-type allele schematics represent exons of the Estrra gene. The open boxes in the targeting vector schematics correspond to the pgk-neo and hsv-tk selectable marker genes. The position of the 3′ flanking and neomycin probes used in Southern blot analysis are indicated by filled boxes. (B) Homologous recombination of the targeting vector in ES cells was verified by Southern blot analysis, digesting genomic DNA with BamHI, and hybridization with a 3′-flanking probe (upper panel) and a neomycin probe (lower panel). The wild-type allele generated a 10.9-kb band, while the mutant allele produced a 4.2-kb band due to the introduction of a BamHI site in the targeting vector. The neomycin probe recognized a 6.0-kb band in the targeted locus. (C) Southern blot analysis of 4-week-old offspring from heterozygote intercrosses with the 3′-flanking probe. (D) Northern blot analysis of ERRα expression. Total RNA isolated from adult wild-type, heterozygous, and homozygous kidneys. Twenty micrograms of total RNA were used in each lane. ERRα and ERRβ transcripts were detected by using mouse cDNA probes (51). A mouse β-actin cDNA probe was used as a loading control (lower panel). (E) Western analysis of ERRα knockout mice. Intestine lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blot was probed with antibodies against mouse ERRα, which were made by using the amino-terminal domain of the ERRα protein.

FIG. 2.

Comparison of total weight, length, and fat pad weight of wild-type and ERRα-null mice. (A) Growth of female ERRα-null mice. ERRα^−/− F₂ mice (▪; n = 15) and littermate wild-type controls (○; n = 16) were weighed, and the data were binned by using 1- to 4-week intervals. The data are means ± the standard error of the mean (sem). (B) Growth of male ERRα-null mice. ERRα^−/− F₂ mice (▪; n = 13) and littermate wild-type controls (○; n = 10) were weighed, and the data were binned by using 1- to 4-week intervals. The data are mean ± the SEM. (C to F) Anatomical characteristics of wild-type plus heterozygote (n = 9) and ERRα-null (n = 7) mice at 26 weeks of age. The P values (✽) were as follows: inguinal, 0.008; epididymal, 0.002; and peritoneal, 0.005.

FIG. 3.

Histology of adipose tissue. Hematoxylin-and-eosin-stained wild-type (WT) and ERRα^−/− epididymal WATs are shown. Magnification, ×150.

FIG. 4.

Energy consumption in wild-type (n = 4) and ERRα^−/− (n = 8) male mice over a 4-day period.

FIG. 5.

ERRα^−/− mice are resistant to high-fat diet-induced obesity. (A) Ten-week-old male mice (n = 7 for each group) were fed a high-fat diet. The increases in body weights were calculated based on the initial body weight at day 0 of high-fat feeding. The average initial body weight was 10% smaller (P < 0.05) for the ERRα^−/− mice compared to the control littermates. (B) Representative male ERRα^−/− mouse and control littermate after 5 weeks of feeding with a high fat diet.

FIG. 6.

The absence of ERRα^−/− influences the expression of genes involved in lipid metabolism in both white (A) and brown (B) adipose tissues. Quantitative PCR data shows the difference in expression of both upregulated (□) and downregulated (▪) genes in these tissues. The data displayed represent the means of at least two experiments.

FIG. 7.

ERRα^−/− mice demonstrate decreased lipogenesis in comparison to littermate controls. After intraperitoneal injection of with ³H₂O, ERRα^−/− mice incorporate 30 to 55% less ³H into adipose tissue lipids. IF, inguinal fat; EF, epididymal fat; PF, perirenal fat. ✽, P < 0.05; ✽✽, P < 0.01.