The nuclear orphan receptor COUP-TFII plays an essential role in adipogenesis, glucose homeostasis, and energy metabolism

Abstract

Adipose tissue development and function play a central role in the pathogenesis and pathophysiology of metabolic syndromes. Here, we show that chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) plays a pivotal role in adipogenesis and energy homeostasis. COUP-TFII is expressed in the early stages of white adipocyte development. COUP-TFII heterozygous mice (COUP-TFII(+/-)) have much less white adipose tissue (WAT) than wild-type mice (COUP-TFII(+/+)). COUP-TFII(+/-) mice display a decreased expression of key regulators for WAT development. Knockdown COUP-TFII in 3T3-L1 cells resulted in an increased expression of Wnt10b, while chromatin immunoprecipitation analysis revealed that Wnt10b is a direct target of COUP-TFII. Moreover, COUP-TFII(+/-) mice have increased mitochondrial biogenesis in WAT, and COUP-TFII(+/-) mice have improved glucose homeostasis and increased energy expenditure. Thus, COUP-TFII regulates adipogenesis by regulating the key molecules in adipocyte development and can serve as a target for regulating energy metabolism.

Figure 1. COUP-TFII^+/− mice display reduced adiposity

(A). The photograph depicts the appearance of the 3-month-old COUP-TFII^+/+ and COUP-TFII^+/− mice (top), and gross morphology of BAT and epididymal WAT (EWAT) (bottom). (B). Body weight of 3-month-old COUP-TFII^+/+ (black box, n=7) and COUP-TFII^+/− (gray box, n=7) mice fed a normal chow. (C). H&E staining of paraffin-embedded EWAT and BAT sections. The upper scale bar shown is equal to 20 μm and applicable to the top four sections; the lower scale bar shown is equal to 100 μm and applicable to the bottom two sections. (D). One side of whole mount EWAT were collected from COUP-TFII^+/+ and COUP-TFII^+/− mice respectively, and were completely homogenized in a 0.5 ml RIPA buffer. Picture was taken after the samples were centrifuged for 15 min at 12,000 rpm. COUP-TFII^+/− mice had much less lipid accumulation in EWAT than the control (top layer). (E). COUP-TFII^+/− mice have similar food intake (gray box, n=7) in comparison with COUP-TFII^+/+ mice (black box, n=7). (F). MRI analysis revealed that COUP-TFII^+/− mice (gray box, n=7) have less fat mass and are leaner than COUP-TFII^+/+ mice (black box, n=7). (G). Real-time PCR analysis of cyclophilin A gene number in EWAT genomic DNA from both COUP-TFII^+/+ (black box, n=7) and COUP-TFII^+/− (gray box, n=7) mice. Data in (B) and (E-G) represent mean ± SEM. * p<0.05; † p<0.005.

Figure 2. COUP-TFII is required for adipocyte differentiation in vivo

(A). Real-time PCR analysis of the mRNA expression of COUP-TFII and the adipogenic transcription factors PPARγ and C/EBPα in EWAT collected from COUP-TFII^+/+ mice at different developmental stages. Expression levels of each gene were normalized to the levels of the 18S rRNA (n=6). (B). X-gal staining of histological sections of EWAT taken from different ages of COUP-TFII-LacZ knock-in mice. The scale bar shown equals 50 μm, and is applicable to all sections. (C). Oil-red-O staining of frozen sections of EWAT from COUP-TFII^+/+ and COUP-TFII^+/− mice at different ages. The scale bar shown equals 50 μm, and is applicable to all sections. (D). Real-time PCR analysis of adipocyte marker genes and transcription factors. RNAs were isolated from EWAT of COUP-TFII^+/+ (black box) and COUP-TFII^+/− (gray box) mice. Expression levels of each gene were normalized to the levels of the 18S rRNA (n=7). Data in (A) and (D) indicate mean ± SEM. * p<0.05; ** p<0.01; ‡ p<0.001; § p<0.0005. (E). ChIP analysis of COUP-TFII binding to a PGC-1α promoter at the promoter region of 2 kb upstream of transcriptional initiation site (top row) and at the second intron region (bottom row) which served as a control.

Figure 3. COUP-TFII is necessary for adipocyte differentiation from 3T3-L1 cells and embryonic fibroblasts

(A). Western blot analysis of COUP-TFII and PPARγ expression during differentiation of 3T3-L1 cells. Cells were harvested at the indicated times. Arrowhead indicates positions of bands corresponding to PPARγ. (B). 3T3-L1 cells were transfected with COUP-TFII-siRNA (COUP-TFII) or control siRNA (Ctrl), expression of COUP-TFII and PGC-1α in cells 8 hours and 1 day after induction were analyzed by western blot (left). siRNA knock down 3T3-L1 cells were induced to differentiate. Cells were stained with Oil-Red-O on day 8 (right). H in (A-B) stands for hours, d stands for days. (C). 3T3-L1 cells transfected with a control plasmid (Ctrl) or one expressing COUP-TFII (OE). Expression of COUP-TFII in cells was analyzed by western blot (left). COUP-TFII overexpression in cells was subsequently induced to differentiate. Cells were stained with Oil-Red-O on day 8 (right). (D). Impaired adipocyte differentiation in COUP-TFII null embryonic fibroblasts (MEFs). Immunoblot analysis of COUP-TFII in MEFs prepared from COUP-TFII ^flox/flox and ROSA26^CRE-ERT2/+, COUP-TFII ^flox/flox embryos. Cells were harvested 2 days after having been cultured in DMEM with or without 1 μM 4-hydroxy-tamoxifen (4OH-TM) (left). MEFs were induced to differentiate and cells were staining with Oil-Red-O on day 8 (right). (E). COUP-TFII negatively regulates Wnt10b expression. Real-time PCR analysis of COUP-TFII and Wnt10b expressions in COUP-TFII knock down 3T3-L1 cells (left). Expression levels of each gene were normalized to the levels of the 18S rRNA (n=3). Western blot analysis of molecules in Wnt signaling pathway (middle). ChIP analysis of COUP-TFII binding to Wnt10b promoter at the promoter region 1.8 kb upstream of the transcriptional initiation site (top row) and at the second intron region (bottom row) which was served as a control (right). Data indicates mean ± SEM. ** p<0.01, § p<0.0005.

Figure 4. COUP-TFII ^+/- mice have improved glucose homeostasis

(A). Plasma glucose, insulin, adiponectin, cholesterol, NEFA and triglyceride levels in 3-month-old COUP-TFII^+/+ (black box, n=11) and COUP-TFII^+/− (gray box, n=11) mice in the fed and fasted overnight states. * p<0.05; † p<0.005; § p<0.0005. (B). Oil-red-O staining of histological sections of liver taken from 3-month-old COUP-TFII^+/+ and COUP-TFII^+/− mice fed with normal chow. The red staining indicates neutral lipids. The scale bar shown equals 20 μm, and is applicable to all sections. (C). Glucose tolerance test and insulin tolerance test on 3-month-old COUP-TFII^+/+ (filled circle, n=7) and COUP-TFII^+/− (opened circle, n=7) mice. * p<0.05; ** p<0.01; † p<0.005. (D). Following an overnight fast, 3-month-old COUP-TFII^+/+ (black box, n=4) and COUP-TFII^+/− (gray box, n=5) mice were subjected to a conscious euglycemic-hyperinsulinemic clamp. Basal glucose production (left); whole-body glucose disposal rate during hyperinsulinemic-euglycemic clamp (middle); muscle (soleus and gastrocnemius) and white adipose tissue glucose uptake during the hyperinsulinemic-euglycemic clamp (right). * p<0.05. Data in (A), (C) and (D) represent mean ± SEM.

Figure 5. COUP-TFII^+/− mice are resistant to high-fat diet induced obesity

(A). Body weight gain of COUP-TFII^+/+ (filled circle, n=9) and COUP-TFII^+/− (opened circle, n=8) mice fed a high-fat diet. 8-week-old mice were fed a high-fat diet for 12 weeks. Body weight was measured weekly. * p<0.05. (B). MRI analysis of body fat and lean content in COUP-TFII^+/+ (black box, n=9) and COUP-TFII^+/− (gray box, n=8) mice after 12 weeks of high-fat feeding. ‡ p<0.001. (C). Glucose tolerance test and insulin tolerance test on COUP-TFII^+/+ (filled circle, n=9) and COUP-TFII^+/− (opened circle, n=8) mice after 12 weeks of high-fat feeding. * p<0.05; † p<0.005; § p<0.0005. (D). The photograph depicts the appearance of COUP-TFII^+/+ and COUP-TFII^+/− mice after 12 weeks of high-fat feeding (top left); exposed ventral view of the mice revealed much less WAT mass in COUP-TFII^+/− mice (bottom left); much less BAT and EWAT in COUP-TFII^+/− mice (top right), and gross morphology of the liver (bottom right). Paler BAT and liver color of COUP-TFII^+/+ mice indicated higher lipid accumulation in these organs. (E). Oil-red-O staining of the liver collected from COUP-TFII^+/+ and COUP-TFII^+/− mice. The scale bar shown equals 50 μm, and is applicable to all sections. (F). Locomotion activity was measured in COUP-TFII^+/+ (black box, n=6) and COUP-TFII^+/− (gray box, n=6) mice under a high-fat diet with the VersaMax animal activity monitoring system. Shown is the average movement during the monitoring period. ** p<0.01. Data in (A-C and F) represent mean ± SEM.

Figure 6. COUP-TFII^+/− mice have increased energy expenditure

(A). Oxygen consumption (VO₂) and carbon dioxide generation (VCO₂) analyzed by indirect calorimetry in COUP-TFII^+/+ (filled circle, n=8) and COUP-TFII^+/− (open circle, n=8) mice. ** p<0.01; † p<0.005; ‡ p<0.001; § p<0.0005. (B). Heat generation of COUP-TFII^+/+ (black box, n=8) and COUP-TFII^+/− (gray box, n=8) mice. ‡ p<0.001. (C). Respiratory exchange ratio of COUP-TFII^+/+ (black box, n=8) and COUP-TFII^+/− (gray box, n=8) mice. ** p<0.01. (D). Immunoblots of EWAT (in triplicate) extracts for COUP-TFII, PGC-1α, UCP1 and other structural mitochondrial markers. Tissues were collected from COUP-TFII^+/+ and COUP-TFII^+/− mice. (E). Transmission electron microscopy revealed mitochondria in the WAT (bottom) and BAT (top) from COUP-TFII^+/+ and COUP-TFII^+/− mice. Scale bar for BAT equals 1 μm; scale bar for the WAT equals 500 nm. (F). Comparison of mitochondrial volume densities from white adipocyte of COUP-TFII^+/+ (black box) and COUP-TFII^+/− (gray box) depicted in (E) (n=20 micrographs per group). § p<0.0005. (G). Real-time PCR analysis PRDM16 and CIDEa expression in EWAT collected from COUP-TFII^+/+ (black box, n=5) and COUP-TFII^+/− (gray box, n=5) mice that have been treated with CL316243 for 6 days. Data in (A-C, F and G) represent mean ± SEM.