Interaction of ApoA-IV with NR4A1 and NR1D1 Represses G6Pase and PEPCK Transcription: Nuclear Receptor-Mediated Downregulation of Hepatic Gluconeogenesis in Mice and a Human Hepatocyte Cell Line

Abstract

We have previously shown that the nuclear receptor, NR1D1, is a cofactor in ApoA-IV-mediated downregulation of gluconeogenesis. Nuclear receptor, NR4A1, is involved in the transcriptional regulation of various genes involved in inflammation, apoptosis, and glucose metabolism. We investigated whether NR4A1 influences the effect of ApoA-IV on hepatic glucose metabolism. Our in situ proximity ligation assays and coimmunoprecipitation experiments indicated that ApoA-IV colocalized with NR4A1 in human liver (HepG2) and kidney (HEK-293) cell lines. The chromatin immunoprecipitation experiments and luciferase reporter assays indicated that the ApoA-IV and NR4A1 colocalized at the RORα response element of the human G6Pase promoter, reducing its transcriptional activity. Our RNA interference experiments showed that knocking down the expression of NR4A1 in primary mouse hepatocytes treated with ApoA-IV increased the expression of NR1D1, G6Pase, and PEPCK, and that knocking down NR1D1 expression increased the level of NR4A1. We also found that ApoA-IV induced the expression of endogenous NR4A1 in both cultured primary mouse hepatocytes and in the mouse liver, and decreased glucose production in primary mouse hepatocytes. Our findings showed that ApoA-IV colocalizes with NR4A1, which suppresses G6Pase and PEPCK gene expression at the transcriptional level, reducing hepatic glucose output and lowering blood glucose. The ApoA-IV-induced increase in NR4A1 expression in hepatocytes mediates further repression of gluconeogenesis. Our findings suggest that NR1D1 and NR4A1 serve similar or complementary functions in the ApoA-IV-mediated regulation of gluconeogenesis.

Fig 1. Colocalization of apoA-IV and NR4A1.

(A) Subcellular colocalization of apoA-IV and NR4A1 was detected by immunofluorescence. HepG2 cells were transfected with the human NR4A1 expression plasmid for 48 h, and treated with r-h-apoA-IV-GFP (20 μg/mL) or GFP protein (negative control) for 2 h. The cells were probed with rabbit anti-human NR4A1 and mouse anti-GFP primary antibodies, followed by Alex Flour-594-conjugated goat anti-rabbit (red) and FITC-conjugated goat anti-mouse secondary antibodies, respectively. The cells were examined using a Zeiss LSM-510 confocal fluorescence microscope. (B and C) Colocalization of apoA-IV and NR4A1 was confirmed by PLA (Materials and Methods). (B) HepG2 cells and (C) HEK-293 cells were transfected as described above for immunofluorescence. The PLA was performed using anti-NR4A1 and anti-GFP primary antibodies, generating fluorescent red foci when examined using a Zeiss Axiovert 200 fluorescence microscope, which indicated the presence of both NR4A1 and r-h-apoA-IV-GFP in close proximity. The cells were counterstained with DAPI (blue) to visualize the nucleus.

Fig 2. Effect of NR4A1 on the ApoA-IV-mediated regulation of G6Pase promoter activity.

(A) HepG2 cells were treated with r-h-apoA-IV-GFP or GFP (control) for 6 h. Nuclear proteins were extracted from the cells, and immunoprecipitation was performed using an anti-GFP antibody. The precipitates were analyzed for the presence of ApoA-IV-GFP and NR4A1 by western blotting. (B) The colocalization of exogenous ApoA-IV and endogenous NR4A1 at the human G6Pase promoter was detected using ChIP. HepG2 cells were treated as described above for coimmunoprecipitation. Immunoprecipitation was performed using anti-NR4A1 and anti-apoA-IV antibodies. Primers were used to amplify the RORE sequence in the human G6Pase promoter or the GAPDH promoter (control) by PCR. The mean ± SE of three samples is shown (*P < 0.05 vs. vehicle control). (C) The effect of NR4A1 on G6Pase transcription in HEK-293 cells was examined using a luciferase reporter assay. HEK-293 cells were transfected with the G6Pase-luciferase reporter plasmid, human NR4A1 plasmid, renilla luciferase control reporter plasmid, and siNR4A1, control siRNA, or an equivalent volume of solvent for 24 h. Cells were also transfected with the G6Pase-luciferase and renilla luciferase reporter plasmids and the pcDNA3.1 plasmid as a control. The transfected cells were treated with recombinant human ApoA-IV protein or vehicle control for 24 h, and relative luciferase activity was measured. Relative luciferase activities (right) are presented as the mean ± SE of at least three samples from three independent experiments (***P < 0.001 vs. pcDNA or siC controls). (D) Western blotting of cells transfected with the pcDNA plasmid and no siRNA (pcDNA), cells transfected with the NR4A1 expression plasmid and no siRNA (NR4A1), cells cotransfected with the control siRNA (siC), and cells cotransfected with the siNR4A1 (siNR4A1).

Fig 3. Effect of NR4A1 on ApoA-IV-mediated regulation of G6Pase and PEPCK expression and glucose production.

Primary mouse hepatocytes were transfected with siNR4A1 or control siRNA (siC) for 48 h, and treated with 20 μg/mL r-m-apoA-IV or an equivalent volume of PBS (vehicle control) for 6 h or overnight. The levels of the (A) PEPCK and (B) G6Pase mRNAs were measured by qRT-PCR at 6 h posttreatment. (C) The levels of the NR4A1 and GAPDH (control) proteins were assessed by western blotting to demonstrate the inhibition of NR4A1 protein expression at 6 h posttreatment. (D) The level of glucose in the culture medium was also measured at 24 h posttreatment. Data are presented as the mean ± SE of at least three samples from three independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001 vs. vehicle or siC control).

Fig 4. Mutual regulation of NR4A1 and NR1D1 expression in mouse hepatocytes.

(A) Primary mouse hepatocytes were transiently transfected with siNR4A1, siNR1D1, or control siRNA (siC) for 48 h. (Lower panel) The levels of the NR1D1 and NR4A1 mRNAs were measured, relative to the cyclophilin control, using qRT-PCR. (Upper panel) The levels of the NR1D1 and NR4A1 proteins were measured, relative to the GAPDH control, by western blotting. Data are presented as the mean ± SE of at least three samples from three independent experiments (*P < 0.05, and **P < 0.01 vs. vehicle or siC control). (B) Diagram depicting the roles of ApoA-IV, NR4A1, and NR1D1 in the downregulation of hepatic glucose production. The ApoA-IV-induced expression of NR4A1 and NR1D1 represses the transcription of G6Pase and PEPCK in hepatocytes, which in turn reduces glucose output. The expression of NR4A1 and NR1D1 is also regulated by bidirectional feedback, in which each NR represses the expression of the other.

Fig 5. Effect of ApoA-IV treatment on NR4A1 gene expression in cultured cells in vitro and in vivo.

Primary mouse hepatocytes were treated with 20 μg/mL r-m-ApoA-IV or an equivalent volume of PBS (vehicle control). The level of NR4A1 (A) mRNA and (B) protein expression were measured relative to the cyclophilin control using qRT-PCR and western blotting, respectively. The data from three independent experiments are presented as the mean ± SE of at least three samples from three independent experiments (*P < 0.05 and **P < 0.01 vs vehicle). Mice (n = 5 each group) were provided food ad libitum (AL) or fasted for 5 h before receiving an intraperitoneal injection of 1 μg/g r-m-ApoA-IV protein or saline (vehicle control). At 2 h postinjection, the levels of NR1D1 and NR4A1 (C) mRNA and (D) protein expression were measured, relative to the cyclophilin or GAPDH control, a using qRT-PCR and western blotting, respectively (*P < 0.05 and **P < 0.01 vs saline controls).