EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 37, No. 2, 101-110, April 2005 Identification and characterization of peroxisome proliferator response element in the mouse GLUT2 promoter Seung-Soon Im1,2,4, Jae-Woo Kim1,3, Introduction Tae-Hyun Kim1,2,4, Xian-Li Song1,2,4, 1,2,4 1 Recently, peroxisome proliferator-activated receptor So-Youn Kim , Ha-il Kim and (PPAR) family draws much attention in the treatment 1,2,4,5 Yong-Ho Ahn of type 2 diabetes. Three isoforms, encoded by sepa- rate genes, have been identified so far: PPAR-α, 1Department of Biochemistry and Molecular Biology PPAR-β(δ), and PPAR-γ (Sher et al., 1993). PPAR-α 2Center for Chronic Metabolic Disease Research is highly expressed in hepatocytes, enterocytes, pro- 3 ximal tubular epithelium of kidney, and cardiac mus- Institute of Genetic Science 4 cle, and the expression pattern parallels, to a certain Brain Korea 21 Project for Medical Science, Korea extent, the sensitivity of various tissues to the β-oxi- Yonsei University College of Medicine dation induction by synthetic peroxisome proliferators 134 Sinchon-dong, Seodaemun-gu (Spiegelman, 1998; Guillemain et al., 2000). PPAR-γ Seoul 120-752, Korea is known to be involved in adipogenesis, and main- 5Corresponding author: Tel, 82-2-361-5183; tenance of differentiation is important in breast, colon, Fax, 82-2-312-5041; E-mail, [email protected] and urinary bladder function. PPAR-γ is a member of the nuclear hormone receptors that contains the li- gand-dependent activation domain (AF-2) (Picard et Accepted 16 March 2005 al., 2002). Upon ligand binding, PPAR-γ heterodimeri- zed with retinoid X receptor (RXR)-α binds to the Abbreviations: ChIP assay, chromatin immunoprecipitation assay; PPAR response element (PPRE) and activates target EMSA, electrophoretic mobility shift assay; GLUT2, glucose trans- gene transcription. Thiazolidinediones (TZDs) are a porter type 2; PPAR, peroxisome proliferator-activated receptor; class of antidiabetic agents that improve insulin sensi- PPRE, peroxisome proliferator response element; RXR, retinoid X tivity in various animal models of diabetes (Cavaghan receptor et al., 1997; Rahimian et al., 2001). Patients with a dominant-negative mutation in the PPAR-γ gene show severe hyperglycemia, which provides a genetic link between PPAR-γ and type 2 diabetes (Barroso et al., Abstract 1999). So far, a mechanism of antihyperglycemic ef- fect of TZDs on the liver is considered to be rather In the present study, we show that the expres- indirect (Jiang et al., 2002), partly because hepatic sion of type 2 glucose transporter isoform (GLUT2) expression of PPAR-γ is rather low, and adipocytes could be regulated by PPAR-γ in the liver. Rosi- specific deletion of the PPAR-γ gene affects the blood glitazone, PPAR-γ agonist, activated the GLUT2 glucose level (He et al., 2003). However, in the patho- mRNA level in the primary cultured hepatocytes logical state, as in obesity and type 2 diabetes pa- tients, the expression of PPAR-γ is known to be in- and Alexander cells, when these cells were trans- creased (Rahimian et al., 2001). Thus, the direct role fected with PPAR-γ/RXR-α. We have localized the of PPAR-γ ligand on the genes involved in hepatic peroxisome proliferator response element in the glucose metabolism cannot be ruled out. Previously, mouse GLUT2 promoter by serial deletion studies we have characterized the peroxisom proliferator-ac- and site-directed mutagenesis. Chromatin immu- tivator response element (PPRE) in the promoter noprecipitation assay using ob/ob mice also regions of rat GLUT2 (Kim et al., 2000) and gluco- showed that PPAR- rather than PPAR- binds to kinase (Kim et al., 2002) gene and reported their phy- γ α siological implications in the insulin secretion in re- the -197/-184 region of GLUT2 promoter. Taken sponse to changing blood glucose level in the pan- together, liver GLUT2 may be a direct target of creas of type 2 diabetic Zucker diabetic fatty (ZDF) PPAR-γ ligand contributing to glucose transport rats. We also showed that liver type glucokinase (L- into liver in a condition when PAPR-γ expression GK) was directly upregulated by TZDs in its promoter is increased as in type 2 diabetes or in severe (Kim et al., 2004). Because L-GK functions very obesity. closely with GLUT2 in transporting glucose into liver, it is possible that TZDs can activate both GLUT2 and L-GK gene in a coordinate manner. Keywords: GLUT2; liver; promoter; PPAR-γ; rosiglita- To explore this possibility, we have dissected zone; type 2 diabetes mouse GLUT2 promoter and identified a PPRE in the -197/-184 region. We also demonstrated that PPAR-α 102 Exp. Mol. Med. Vol. 37(2), 101-110, 2005 is not a good inducer of GLUT2 expression whereas Primary hepatocytes preparation from mouse liver PPAR-γ stimulated the endogenous GLUT2 mRNA Hepatocytes were isolated from male ICR mouse (ap- expression in primary mouse hepatocytes when PPAR- proximately 30 g) by the collagenase perfusion meth- γ was ectopically expressed. Also, in the liver of ob/ od (Seglen, 1972). Dissociation into individual hepato- ob mice, where PPAR-γ expression is increased (Ed- cytes was performed in Dulbecco's modified Eagles' vardsson et al., 1999), the PPAR-γ ligand upre- medium (DMEM) containing 10% (v/v) heat inactiva- gulated the GLUT2 expression whereas PPAR-α li- ted fetal calf serum (FCS), 0.1 IU/ml insulin, 10 nM gand did not affect its expression, indicating that dexamethasone, 25 mM glucose, 100 U/ml penicillin PPAR- may contribute to lowering blood glucose le- γ G, 100 µg/ml streptomycin and 0.25 µg/ml amphoteri- vel by activating GLUT2 gene expression in the dia- cin B. For each hepatocyte preparation, cell viability betic liver. was checked by the exclusion of trypan blue. Materials and Methods Cell culture and transient transfection Alexander cells (Human epithelial hepatoma cell lines; Materials American Type Culture Collection number CRL-8024) were maintained as monolayer cultures and grown in Wy14,643 (20 mM in 19% BSA and 5% dimethyl sul- Dulbecco's modified Eagle's medium (DMEM) (Invitro- foxide), 9-cis retinoic acid (2 mM in 50% ethanol and gen) supplemented with 10% (v/v) fetal bovine serum, 50% dimethyl sulfoxide), and rosiglitazone (2 mM in 100 U/ml penicillin, and 100 µg/ml streptomycin. Plas- 5% BSA and 5% dimethly sulfoxide) were diluted to mid DNAs were purified using Qiagen Midiprep kit the final concentration of 20 µM, 1 µM, and 2 µM columns (Qiagen, Valencia, CA). Transient transfec- respectively. Wy14,643 and 9-cis retinoic acid were tion and luciferase assays were performed as descri- purchased from Sigma-Aldrich (St. Louis, MO). Rosi- bed previously (Cha et al., 2001). Briefly, cells were glitazone was kindly donated by GlaxoSmithKline, UK. plated in six-well tissue culture plates at a density of PPAR-α and PPAR-γ antibodies were purchased from 1×106 cells/well in 2 ml of medium. After a 20 h PPMX Perseus Proteomics Inc, Japan. attachment period, transfections with 0.5 µg of each construct of GLUT2 promoter and control vector were performed with LipofectAMINE PLUS reagent (Life Construction of plasmids Technologies, Carlsbad, CA), according to the ma- The promoter region (-1112/+1) of the mouse GLUT2 nufacturer's protocol. After 24 h transfection, the me- gene was cloned into the KpnI/XhoI site of the pGL3- dium was replaced by a medium containing Wy basic vector (Promega, Madison, WI) to generate 14,643 (20 µM, final concentration), rosiglitazone (2 pMGT2-1112. Deletion constructs of mouse GLUT2 pro- µM, final concentration) and 9-cis retinoic acid (1 µM, moter were prepared from pMGLUT2 (pMGT2-1112/ final concentration) respectively. Cells were cultured +1). pMGT2d-890 was prepared by excising out 222 further for 24 h and harvested in reporter lysis buffer bp fragment of SalI and EcoRI digestion. pMGT2d- (Promega). Luciferase data were expressed as luci- 389 was constructed by digesting with SalI and PstI ferase activity corrected by β-galactosidase activity in from the pMGT2-1112/+1. Other deletion constructs of the cell lysate. Each transfection was performed in tri- mouse GLUT2 promoter were prepared by digesting plicate and repeated three to five times. pMGT2-1112/+1 after EcoRI sites was generated by PCR-based site-directed mutagenesis kit (Stratagene, La Jolla, CA), and named pMGT2d-283, pMGT2d-166, Nuclear extracts preparation and pMGT2d-57, respectively. The PPRE truncated Nuclear extract from liver of male Sprague-Dawley construct, pMGT2d-283/-166, was also prepared from rats was prepared as described by Gorski et al. (Gor- pMGT2d-389 with the same method described above. ski et al., 1986). Protein concentration was deter- Mutant constructs pMGT2m-389mut1 (mut1), pMGT2m- mined by the method of Bradford (Bradford, 1976). 389mut2 (mut 2), and pMGT2m-389mut1+2 (mut1+2) were produced by introducing substitution mutations into pMGT2d-389 using site-directed mutagenesis. The Electrophoretic mobility shift assay (EMSA) sequences of constructs were confirmed by dideoxy- The oligonucleotide probe GLUT2-PPRE was labeled DNA sequencing method. Expression plasmids, pCMX- as described previously (Kim et al., 2002b). Ten pmo- mPPAR-α, pCMX-mPPAR-γ and pCMX-mRXR-α were les of single stranded sense oligonucleotide were la- kind gifts from Drs. R. M. Evans and D. J. Mangel- beled with [γ-32P] ATP using T4 polynucleotide kinase sdorf (Mangelsdorf et al., 1992; Kliewer et al., 1994). and annealed with 50 pmoles of unlabeled antisense Control vector, pCMX was prepared from pCMX- oligonucleotides. The resulting double stranded oli- mRXR-α by excising out the 1.5 kb fragment of gonucleotides were purified by Sephadex G50 spin mRXR-α cDNA. The dominant negative plasmid of column. The labeled probe (10,000 cpm) was in- NF-Y (△4NF-YA13m29; NF-Ym) was provided by Dr.