Nr4a1 is required for fasting-induced down-regulation of Pparγ2 in white adipose tissue

Abstract

Expression of the nuclear receptor gene, Nur77 (Nr4a1), is induced in white adipose tissue (WAT) in response to β-adrenergic stimulation and fasting. Recently, Nur77 has been shown to play a gene regulatory role in the fasting response of several other major metabolic tissues. Here we investigated the effects of Nur77 on the WAT transcriptome after fasting. For this purpose, we performed gene expression profiling of WAT from wild-type and Nur77(-/-) mice submitted to prolonged fasting. Results revealed Nur77-dependent changes in expression profiles of 135 transcripts, many involved in insulin signaling, lipid and fatty acid metabolism, and glucose metabolism. Network analysis identified the deregulated genes Pparγ2 and Nur77 as central hubs and closely connected in the network, indicating overlapping biological function. We further assayed the expression level of Pparγ2 in a bigger cohort of fasted mice and found a significant Nur77-dependent down-regulation of Pparγ2 in the wild-type mice (P = 0.021, n = 10). Consistently, the expression of several known Pparγ2 targets, found among the Nur77-regulated genes (i.e. G0s2, Grp81, Fabp4, and Adipoq), were up-regulated in WAT of fasted Nur77(-/-) mice. Finally, we show with chromatin immunoprecipitation and luciferase assays that the Pparγ2 promoter is a direct target of Nurr-related 77-kDa protein (Nur77)-dependent repressive regulation and that the N-terminal domain of Nur77 is required for this regulation. In conclusion, we present data implicating Nur77 as a mediator of fasting-induced Pparγ2 regulation in WAT.

Fig. 1.

Expression of Nr4as in WAT from fasted mice. A, qPCR-based Nur77 expression analysis of WAT mRNA from wild-type (Wt) mice, either fasted or overnight fasted followed by 8 h refeeding (n = 3). *, P < 0.05. B, qPCR-based analysis of NOR1 and Nurr1 expression in WAT mRNA from fasted wild-type and Nur77^−/− mice (n = 6). *, P < 0.05. C, Western blot showing expression of Nur77 in fasted wild-type (Wt) but not Nur77-knockout (KO) mice WAT.

Fig. 2.

Expression of Nur77 and Pparγ2 in white adipocytes. Expression (mRNA) levels (qPCR) of Nur77 and Pparγ2 were determined for the following: A, WAT harvested from wild-type (Wt) mice, either fasted or fasted followed by 8 h refeeding (n =3); B, Mature 3T3-L1 adipocytes were stimulated with isoprenaline (1 μm at 0 h); C, WAT of 24-h fasted Nur77^−/− [knockout (KO)] mice and their Wt littermates kept on either regular chow (n = 7) or HFD (n = 5); D, left panel, WAT of Wt mice overnight fasted and refed for 2 and 8 h (n ≥ 4); right panel, WAT of Wt and Nur77^−/− mice (KO) fasted overnight and refed for 2 and 8 h (n ≥ 3). The Wt 2-h refeeding was set to 1. *, P < 0.05; **, P < 0.01. E, Western blot showing protein analyses in protein extracts from 3T3-L1 adipocytes with (Sh-Nur77) or without (Sh-control) silencing of Nur77. Sh, Short hairpin silencing construct. Protein was harvested at the indicated time points after isoprenaline induction. β-Actin was included as loading control. The images shown are representative of three biological replicate experiments. F, Western blot showing protein analyses in protein extracts from WAT from fasted or fed Wt or Nur77^−/− (KO) mice. Three mice are shown for each condition. G, Expression (mRNA) levels (qPCR) of Nur77, Pparγ2, and Pparγ1 were determined for the cohort of mice used for Western blot (fasted mice, n ≥ 10; fed mice, n ≥ 3).

Fig. 3.

Nuclear Nur77 localization imaging. Fluorescent images of mature adipocytes [with Nur77 silencing (Nur77-sh) or without (control)] stained with the nuclear stain DAPI and with anti-Nur77 antibody and visualized with Alexa dye-coupled secondary antibody at the indicated time points after isoprenaline induction.

Fig. 4.

Nur77-dependent changes in the expression of Pparγ2 target genes. A, WAT was harvested from 24-h fasted Nur77^−/− and wild-type mice. Expression of G0s2, GPR81, Ap2, Adipoq, and Lep was assayed with qPCR (n = 7). *, P < 0.05; ***, P < 0.01.

Fig. 5.

Nur77 binds to a highly conserved region of the Pparγ2 promoter, containing predicted Nur77 binding sites (NBREs). The mouse Pparγ2 promoter sequence, encompassing −4000/+500 bp relative to the TSS, was extracted from the CSHL promoter database. A, Cross-species conservation analysis (ENSEMBL, GERP score), showing a highly conserved region in the proximal part of the promoter. B, MatInspector analysis (Genomatix), performing a NBRE position weight matrix scan of the Pparg2 promoter resulting in five predicted NBREs located at position −600, −906, −1688, −2181, and −3490 bp relative to the TSS, respectively. C, ChIP-qPCR of Nur77-bound chromatin from 3T3-L1 cells with constitutive Nur77 expression. Enrichment was evaluated for various amplicons within the Pparγ2 promoter (the bars are aligned with the corresponding part of the promoter, shown above in B). The qPCR results were normalized to a non-NBRE-containing part of the S18 gene (for load normalization). Enrichment is shown relative to a negative control (non-NBRE) of the Pai-1 promoter. A previously experimentally confirmed a Nur77 binding site of the Pai-1 promoter (NBRE) was added as positive control. The figure represents triplicate analysis of three independent experiments. D, Schematic presentation of the Nur77 NBRE position weight matrix (Genomatix V$NBRE/NBRE.01) applied for the predictions in B. Below, the sequences of the predicted NBREs are shown.

Fig. 6.

Nur77 represses the Pparγ2 promoter activity. A, fragments of the Pparγ2 promoter were cloned into luciferase reporter vectors and used for luciferase assays in NIH-3T3 cells. Predicted NBREs are indicated relative to the TSS. B, luciferase assays were carried out after transfection with increasing amounts of a Nur77 expression vector and luciferase reporter vectors containing different fragments of the Pparγ2 promoter (A-luc, B-luc, and C-luc). B, Cotransfection of a Cebpα expression vector with the A-luc luciferase reporter vector. D, Luciferase assay of cells transfected with the reporter vector, A-luc, and 75 ng of a Nur77 expression vector. After transfection, increasing amounts of the Nur77 ligand cytosporone B (Cyt B) were added as indicated. E, Luciferase assays of cells transfected with the reporter vector, A-luc, and increasing amounts of a Nur77DN expression vector. For all assays, a renilla expression vector was employed as a transfection efficiency control. Stippled lines indicate the base activity of the luciferase reporter vector without promoter inserts. RLU, Relative light units. *, P < 0.05; n ≥ 3.