Higher core body temperature and expression of Ucp1 in BAT and WAT of LXRα^−/− mice. (A) Core body temperature in WT (○), LXRα^−/− (•), and LXRα^−/− LXRβ^−/− (▾) mice before and after cold (5°C) challenge. *, P < 0.05 versus WT (two-tailed t test). (B to E) RT-PCR analysis. Ucp1 gene in BAT (B) and WAT (C) of WT and LXRα^−/− mice at 25°C and 5°C. Symbols: ***, P < 0.001 versus WT at 25°C; †††, P < 0.001 versus LXRα^−/− at 25°C; ‡‡‡, P < 0.001versus WT at 5°C (one-way ANOVA). PGC-1α mRNA in BAT (D) and WAT (E) of WT and LXRα^−/− mice at 25°C and 5°C. Symbols: *, P < 0.05; ***, P < 0.001 versus WT at 25°C; ‡, P < 0.05; ‡‡‡, P < 0.001 versus WT at 5°C (one-way ANOVA). All results are means ± SEM for five mice for each genotype.

Increased expression of mitochondrial and brown fat markers in BAT and WAT of LXRα^−/− mice. RT-PCR analysis of genes in BAT (A) and WAT (C) of WT and LXRα^−/− mice at 25°C. Data are expressed relative to those for the WT mice. Symbols: *, P < 0.05; **, P < 0.001; ***, P < 0.0001 versus corresponding gene in WT (two-tailed t test). Immunohistochemical staining for UCP1 in BAT (B) and gonadal WAT (D) from WT and LXRα^−/− mice. All results are means ± SEM for five mice for each genotype.

Increased mitochondrial mass, respiration, and brown adipocyte markers in LXRα^−/− adipocytes. Expression of adipogenic marker genes aP2 (A) and β₃AR (B) in undifferentiated (UnD) and differentiated (Diff) WT and LXRα^−/− MEFs. Symbols: ***, P < 0.001 versus WT (UnD); †††, P < 0.001 versus LXRα^−/− (UnD); ‡‡‡, P < 0.001 versus WT (Diff) (one-way ANOVA; n = 6). (C) Expression levels of genes related to mitochondrial biogenesis and brown fat in adipocytes derived from WT and LXRα^−/− MEFs. ***, P < 0.001 versus the corresponding gene in the WT (two-tailed t test; n = 6). (D) Percent increase in mitochondrial mass upon differentiation of WT and LXRα^−/− MEFs measured by MitoTracker green and flow cytometry. *, P < 0.05 versus WT (two-tailed t test; n = 2). (E) Oxygen consumption in adipocytes derived from WT and LXRα^−/− MEFs by using a Clark-type oxygen electrode under basal conditions or following ATP synthetase inhibitor oligomycin (2 μg/ml; uncoupled respiration) or FCCP (8 μM; maximum respiration). Symbols: *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus corresponding treated WT; †, P < 0.01; †††, P < 0.001 versus basal untreated WT (one-way ANOVA; n = 3). All data are means ± SEM.

LXR agonists selectively suppress cAMP-stimulated Ucp1 gene expression in brown adipocytes. Expression levels of Ucp1(A) and SREBP1 (B) in differentiated HIB-1B cells treated for 6 h with vehicle (DMSO), 10 μM FSK, 1 μM 22R-OH, and 5 μM T09 as indicated. Transcripts were measured by RT-PCR using TaqMan probes. Results are means ± SEM (n = 4) relative to values for vehicle-treated cells. (C to F) Ucp1 and PGC-1α transcripts were measured by RT-PCR by TaqMan probes in differentiated primary brown adipocytes (C and D) and HIB-1B cells (E and F). Cells were treated for 6 h with vehicle (DMSO), 10 μM FSK, 10 μM Iso, and 5 μM T09 as indicated. Results from three experiments were performed in duplicate and are presented as means ± SEM relative to values for vehicle-treated cells. Symbols: **, P < 0.01; ***, P < 0.001 versus vehicle; †††, P < 0.001 versus FSK; ‡‡, P < 0.01, ‡‡‡; P < 0.001 versus Iso (one-way ANOVA).

Suppression of cAMP-stimulated Ucp1 expression is absent from LXRα^−/− adipocytes. Differentiated MEFs were treated for 6 h with vehicle (DMSO), 5 μM T09, 10 μM FSK, and FSK plus T09 as indicated. RT-PCR analysis of Ucp1(A) and PGC-1α (B) gene expression in WT and LXRα^−/− adipocytes. Results are means ± SEM relative to vehicle-treated cells for each genotype. Symbols: ***, P < 0.001 versus vehicle-treated control; †††, P < 0.001 versus FSK; ‡‡‡, P < 0.001 versus Iso (one-way ANOVA; n = 4 to 6).

Activated LXRα suppresses cAMP-stimulated Ucp1 enhancer activity through a DR-4 element. (A) Alignment of DR-4, DR-1, and CRE2 in the enhancer regions of the Ucp1 genes from five species. (B) Design of synthetic oligonucleotides containing LXRE from mGlut4 (13) and DR-4 from the mouse Ucp1 enhancer. (C) Gel shift assays using nuclear extracts (5 μg) from HIB-1B cells incubated with radiolabeled LXRE or DR-4 in the absence or presence of increasing molar amounts (10×, 100×) of the unlabeled WT and mutant competitor oligonucleotides shown in panel B. For supershift analysis, anti-LXRα antiserum (1 μl, 2 μl) was added to the binding reaction mixture. Lanes 1 and 6 contain no nuclear protein. The black arrowhead indicates the protein-DNA complex. The white arrowhead indicates the supershifted complex. (D and E) Transient transfection of HIB-1B cells with tk-CAT reporter genes containing the 220-bp enhancer element from the mouse Ucp1 gene with WT or mutant (dDR-4) DR-4 sequences shown in panel B plus β-actin-luc as an internal control, without (D) or with (E) cotransfected LXRα/RXRα. Results presented are means ± SEM. Symbols: **, P < 0.01; ***, P < 0.001 versus basal untreated WT-DR-4; ††, P < 0.01; †††, P < 0.001 versus FSK-treated WT-DR-4 (one-way ANOVA; n = 3).

Dismissal of PPARγ from the Ucp1 enhancer by activated LXRα and role of RIP140 in suppressing of Ucp1 gene transcription. Differentiated HIB-1B cells (A, B, D, and E) and adipocytes derived from LXR^−/− (C) or RIP140^−/− (F) MEFs were treated with FSK (10 μM) or FSK plus T09 (5 μM) for 6 h and collected for immunoprecipitations with anti-RIP140, anti-LXRα, and anti-PPARγ sera as illustrated and as described in Materials and Methods. Occupancy of the Ucp1 enhancer by LXRα (A and B) and PPARγ (A, B, C, and F) and PGC-1α and RIP140 (D and E) was detected by ChIP. (G and H) HIB-1B cells were transfected with siRNA targeting RIP140 as described in Materials and Methods and subsequently treated (G) or not (H) with delivery of vector-containing Ucp1 enhancer (Ucp1-En-tk-CAT) and β-actin-luc as an internal control. Six hours prior to collection for assays, cells were treated with the following: T09 (5 μM), FSK (10 μM), or FSK plus T09. Ucp1 enhancer activity and transcripts were measured by CAT and luciferase enzyme activity assay and by RT-PCR using TaqMan probes with GAPDH as an internal control, respectively. Immunoprecipitated DNA was analyzed by agarose gel imaging (A and D) and quantitative PCR using specific primers for Ucp1 enhancer (B, C, E, and F). Bar graphs are presented as means ± SEM. V, vehicle; MW, molecular weight. Symbols: ***, P < 0.001 versus vehicle or vehicle of scrambled sequence; †††, P < 0.001 versus FSK of scrambled sequence (one-way ANOVA).