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

November 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 Xiaoming Li 0 1 Min Xu 0 1 Fei Wang 0 1 Yong Ji 0 1 W. Sean DavidsoN 0 1 Zongfang Li 0 1 Patrick Tso 0 1 0 1 National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, The Second Affiliated Hospital of Medical College, Xi'an Jiaotong University , 157 W 5th Rd, Xincheng, Xi'an, Shaanxi, 710004, China , 2 Department of Pathology and Laboratory Medicine, Metabolic Diseases Institute, University of Cincinnati , 2180 E. Galbraith Road, Cincinnati, 45237-0507 , United States of America 1 Editor: Yanqiao Zhang, Northeast Ohio Medical University , UNITED STATES 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. - Funding: This research was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health awarded to Dr. Tso (grant nos. DK 59630, DK 92138, and DK 76928) and The Cincinnati MMPC (grant no. U24 DK059630). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Metabolic diseases, such as obesity and type 2 diabetes, are a major cause of morbidity and mortality in industrialized nations worldwide. Previous studies have shown that the nuclear receptors (NRs), NR4A1 (also known as Nur77) and NR1D1, contribute to the pathology of metabolic disease through their role in ligand-dependent regulation of cellular glucose metabolism [1]. The NR1D1 and NR4A1 proteins belong to a superfamily of structurally related ligand-dependent transcription factors that have been designated as orphan NRs because no endogenous ligands have been shown to mediate their effects on glucose homeostasis. The transient expression of NR4A1 is rapidly induced by a diverse range of stimuli, including mechanical stress [2], exercise [3], cAMP activation [4], protein kinase A, protein kinase C [5], G protein-coupled receptor [6], mitogen-activated protein kinase [7], and tyrosine kinase signaling pathways[8]. Members of the NR4A subgroup of NRs are involved in various physiological processes, including cardiovascular disease [9], steroidogenesis [10], inflammation [11], type 2 diabetes [12], and metabolic syndrome [13]. After feeding on a high-fat diet, NR4A1-null mice display major metabolic changes, including greater weight gain, lower energy, increased insulin resistance, and a slower blood glucose clearance rate, compared with healthy mice [14], and these types of molecular and cellular events have been shown to be associated with reduced NR4A1 expression both in vivo and in vitro. Synthesized and secreted primarily by enterocytes of the small intestine [15], ApoA-IV is involved in the intestinal absorption of lipids and lipid-soluble vitamins [16], free cholesterol efflux [17], and the inhibition of lipoprotein peroxidation [18]. The ApoA-IV protein is a major component of high-density lipoprotein (HDL) and chylomicrons. During the postprandial state, ApoA-IV in chylomicrons rapidly equilibrates with its lipid-free form in plasma [19]. In our previous study, we showed that elevated ApoA-IV express (...truncated)