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B-Cell Translocation Gene 2 Regulates Hepatic Glucose Homeostasis Via Induction of Orphan Nuclear Receptor Nur77 in Diabetic Mouse Model

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B-Cell Translocation Gene 2 Regulates Hepatic Glucose Homeostasis Via Induction of Orphan Nuclear Receptor Nur77 in Diabetic Mouse Model 1870 Diabetes Volume 63, June 2014 Yong Deuk Kim,1 Sun-Gyun Kim,2 Seung-Lark Hwang,3 Hueng-Sik Choi,4 Jae-Hoon Bae,1 Dae-Kyu Song,1 and Seung-Soon Im1 B-Cell Translocation Gene 2 Regulates Hepatic Glucose Homeostasis via Induction of Orphan Nuclear Receptor Nur77 in Diabetic Mouse Model Diabetes 2014;63:1870–1880 | DOI: 10.2337/db13-1368 B-cell translocation gene 2 (BTG2) is a member of an might serve as a potential therapeutic target for com- emerging gene family that is involved in cellular func- bating metabolic dysfunction. tions. In this study, we demonstrate that BTG2 regu- lates glucose homeostasis via upregulation of Nur77 in diabetic mice. Hepatic BTG2 gene expression was The liver plays a crucial role in glucose homeostasis by elevated by fasting and forskolin. Overexpression of maintaining a balance between uptake and storage of Btg2 increased the expression of hepatic gluconeogenic glucose via glycogenesis and in the release of glucose by genes and blood glucose output and subsequently im- both glycogenolysis and gluconeogenesis (1,2). Gluconeo- paired glucose and insulin tolerance. Upregulation METABOLISM genesis is commonly triggered by key hormones including of the transcriptional activity of Nur77, gluconeogenic insulin, glucagon, and glucocorticoid, which contribute to genes, and glucose production by forskolin was ob- the expression of key metabolic enzymes, such as phospho- served by Btg2 transduction, but not in Btg2 knockdown. enolpyruvate carboxykinase (Pck1), fructose biphosphatase BTG2-stimulated glucose production and glucose-6- (Fbp) 1, and glucose-6-phosphatase (G6pc) (3). A variety phosphatase promoter activity were attenuated by of transcriptional factors and cofactors regulated by dominant-negative Nur77. Coimmunoprecipitation and the cAMP signaling pathway such as cAMP-responsive chromatin immunoprecipitation assays showed that – BTG2 induced Nur77 occupancy on the glucose-6- element binding protein (CREB), FoxA 2 (also known as phosphatase promoter via a physical interaction. hepatocyte nuclear factor [HNF]-3b), HNF-4a, glucocor- Btg2 gene expression was increased in streptozotocin- ticoid receptor (GR), forkhead box protein O1, peroxi- treated and db/db mice. Finally, impairment of glucose some proliferator-activated receptor g coativator-1a homeostasis, such as the increase of blood glucose, (PGC-1a), and transducer of regulated CREB activity 2 con- glucose intolerance, and insulin intolerance, was ele- trol the expression of hepatic gluconeogenic genes (4–9). Es- vated in diabetic mice, whereas this phenomenon was pecially CREB acts as a critical transcriptional checkpoint abolished in knockdown of Btg2. Together, these data for the upregulation of gluconeogenesis by binding cAMP- suggest that BTG2 participates in the regulation of he- response element. Multiple genes involved in the gluco- patic glucose homeostasis, which means that BTG2 neogenic genes including Pck1, G6pc, and Pgc-1a were 1Department of Physiology, Keimyung University School of Medicine, Daegu, Corresponding authors: Seung-Soon Im, [email protected], and Dae-Kyu Song, Republic of Korea [email protected]. 2 Neuroscience Section, Department of Pediatrics, Papé Family Pediatric Research Received 5 September 2013 and accepted 5 February 2014. Institute, Oregon Health and Science University, Portland, OR © 2014 by the American Diabetes Association. See http://creativecommons.org 3College of Pharmacy, Yeungnam University, Gyeongsan, Republic of Korea /licenses/by-nc-nd/3.0/ for details. 4School of Biological Sciences and Technology, National Creative Research Ini- tiatives Center for Nuclear Receptor Signals, Hormone Research Center, Chon- nam National University, Gwangju, Republic of Korea diabetes.diabetesjournals.org Kim and Associates 1871 shown to be increased by recruiting CREB to the pro- mice for quantitative reverse transcription-PCR (qPCR) moters of these genes (4,10,11). analysis. Before the isolation of the liver, the blood The nuclear receptor Nur77/NR4A1 is a member of glucose levels of all mice were measured with Glucostix a family of the NR4A receptor, which has highly Accu-Check (Roche Diagnostics, Mannheim, Germany). conserved DNA- and ligand-binding domains. Nur77 binds All animal experiments were performed in accordance to its cognate DNA sequence, called NGFI-B (Nur77)- with the rules and regulations of the Institutional Animal binding response elements (NBREs) or Nur response Use and Care Committee at Keimyung University School elements (NurREs), and induces transcription of target of Medicine. genes involved in the gluconeogenesis and steroidogen- Metabolic Parameters esis (12,13). In addition, hepatic expression of NR4A Plasma glucose was measured using a glucometer (Roche receptors induced by cAMP in response to glucagon Diagnostics) in blood collected from the tail of mice as and fasting is increased in diabetic mice with elevated previously described (19). For the glucose tolerance test, gluconeogenesis, and ectopic overexpression of the a single dose of 2.5 mg/kg glucose was injected intraper- Nur77 gene induces upregulation of hepatic gluconeogenic itoneally after a 14-h fast. For the insulin tolerance test, genes, glucose production, and increased blood glucose mice fasted for 4 h were administered intraperitoneally level (12). Consistent with these results, Nur77-null mice with insulin (0.5 units/kg), and glucose concentrations on a high-fat diet also show decrease of hepatic glucose production and insulin resistance in the liver (14). were measured at 0, 30, 60, 90, and 120 min. Blood glucose was analyzed under the same conditions. B-cell translocation gene 2 (BTG2) is a member of the BTG/TOBgenefamilyofantiproliferativegenes,which Construction of Plasmids and DNA are highly conserved among various species (15). BTG2 is The reporter plasmids NBRE-Luc, NurRE-Luc, Pck1-Luc, reported to be the initial gene induced by growth factors and G6pc-Luc were described previously (13,18). Expres- and tumor promoters in several cell types (16,17). Re- sion vectors of HA-Nur77, dominant-negative (DN)- cently, we demonstrated that BTG2 acts as a crucial coac- Nur77, Hnf4-a, Pgc-1a, GR, and Flag-Btg2 were described tivator of CREB to regulate hepatic gluconeogenesis, and previously (13,18,20–23). The small interfering RNAs for the increase of BTG2 by glucagon–CREB signaling Btg2 were prepared as described previously (18). The resulted in the elevation of glucose production from hepa- point mutant form of human G6PC-Luc or mouse G6pc- tocytes (18). However, the interrelationship between BTG2 Luc was constructed using a site-directed mutagenesis kit and Nur77 in terms of the regulation of hepatic gluconeo- (Stratagene, La Jolla, CA) and the following primers: hu- genesis has not been addressed yet. man G6PC (hG6PC)-Luc forward, 59-CTGTAACTTTAATTT In the current study, we identified that transcrip- CATTAAATG-39 and reverse, 59-CATTTAATGAAATTAAA tional coregulator BTG2 acts as a novel coactivator GTTACAG-39 and mouse G6pc (mG6pc)-Luc forward, 59- of hepatic gluconeogenesis and elucidated a molecular TAACTT-AAAATTTCACTTCCG-39 and reverse, 59-CGGAA mechanism that links to Nur77 in the regulation of GTGAAATTTTAAGTTA-39. All plasmids were confirmed glucose homeostasis in the livers of fasted and diabetic by sequencing analysis. mice. Cell Culture and Transient Transfection Assays RESEARCH DESIGN AND METHODS AML-12 immortalized mouse hepatocytes were cultured Reagents in DMEM/F-12 medium (Gibco-Brl) supplemented with 10% FBS, insulin-transferrin-selenium (Gibco-Brl), dexa- Forskolin and streptozotocin were purchased from Sigma- methasone (40 ng/mL; Sigma-Aldrich), and antibiotics in Aldrich (St. Louis, MO). Cell-culture media were pur- a humidified atmosphere containing 5% CO at 37°C (24). chased from Gibco-Brl (Grand Island, NY). 2 Transient transfections were carried out as previously de- Animal Studies scribed (18,24). Male C57BL6 mice (8 weeks of age; Samtako, Osan, Isolation and Culture of Primary Mouse Hepatocytes Republic of Korea) were used in the experiments. For studies Mouse primary hepatocytes were isolated from the livers in the fasting and feeding conditions, mice were fed and of 8-week-old male mice (Samtako). The protocol for fasted for 12 h. Diabetes was induced in wild-type (WT) and isolation of hepatocytes was described previously (8). db/db mice by intraperitoneal injections of streptozotocin (80 mg/kg body weight). For overexpression of BTG2, Preparation of Recombinant Ad WT mice were infected with adenoviral vector expressing Ads encoding full-length Btg2 and green fluorescence Btg2 (13 109 plaque-forming units) by tail vein injection. protein (GFP) have been described previously (18,24). To disruption BTG2, db/db mice were intravenously Recombinant lentiviral delivery of Btg2-targeted short injected with lentivirus short hairpin Btg2 (shBtg2; a single hairpin RNA (shRNA) was purchased from Dharmacon dose of 13 109 transducing units/mL). Total RNA was (Lafayette, CO). Ad-expressing DN-Nur77 was gifted from prepared from the livers of streptozotocin-treated dia- Dr. In-Kyu Lee at Kyungpook National University School betic mice and adenovirus (Ad)- or lentivirus-infected of Medicine. 1872 BTG2 Regulates Hepatic Glucose Homeostasis Diabetes Volume 63, June 2014 Measurement of mRNA determined using the Student t test, and multiple com- Total RNA was isolated from liver tissue and used for parisons were analyzed using one-way ANOVA under qPCR as previously described (25,26). The expression of treatment
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