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Peroxisomal Proliferator–Activated Receptor- Upregulates Peroxisomal Proliferator–Activated Receptor-␥ Upregulates Glucokinase Gene Expression in ␤-Cells Ha-il Kim,1 Ji-Young Cha,1 So-Youn Kim,1 Jae-woo Kim,1 Kyung Jin Roh,2 Je-Kyung Seong,2 Nam Taek Lee,3 Kang-Yell Choi,1 Kyung-Sup Kim,1 and Yong-ho Ahn1 Thiazolidinediones, synthetic ligands of peroxisomal because of severe hyperglycemia (2,3). Adenovirus-medi- proliferator–activated receptor-␥ (PPAR-␥), improve ated expression of GLUT2 and GK in IL cells results in peripheral insulin sensitivity and glucose-stimulated gaining of glucose sensitivity (4). Thus, GLUT2 and GK are insulin secretion in pancreatic ␤-cells. To explore the important in glucose sensing of ␤-cells. However, GLUT2, role of PPAR-␥ in glucose sensing of ␤-cells, we have being a low-affinity, high-capacity glucose transporter, is ␤ ␤ dissected the -cell–specific glucokinase ( GK) pro- believed to play a more permissive role in glucose sensing, moter, which constitutes glucose-sensing apparatus in pancreatic ␤-cells, and identified a peroxisomal prolif- allowing rapid equilibration of glucose across the plasma erator response element (PPRE) in the promoter. The membrane. GK traps glucose in ␤-cells by phosphorylation GK-PPRE is located in the region between ؉47 and (5) and is the flux-controlling enzyme for glycolysis in␤ ؉68 bp. PPAR-␥/retinoid X receptor-␣ heterodimer ␤-cells (4). Thus, it serves as the gatekeeper for metabolic binds to the element and activates the ␤GK promoter. signaling, suggesting that GK rather than GLUT2 is directly The ␤GK promoter lacking or having mutations in PPRE ␥ ␥ responsible for the insulin secretion in response to in- cannot be activated by PPAR- . PPAR- activates the creasing blood glucose levels (5). ␤GK promoter in ␤-cells as well as non–␤-cells. Further- more, troglitazone increases endogenous GK expres- Thiazolidinediones (TZDs) are a new class of antidia- sion and its enzyme activity in ␤-cell lines. These results betic agents that act by improving insulin sensitivity in indicate that PPAR-␥ can regulate GK expression in various animal models of obesity and diabetes (6–9). The ␤-cells. Taking these results together with our previous biological effects of TZDs are exerted by binding to and work, we conclude that PPAR-␥ regulates gene expres- activating peroxisomal proliferator–activated receptor-␥ sion of glucose-sensing apparatus and thereby improves ␥ ␤ (PPAR- ). There is a strong correlation between TZD- glucose-sensing ability of -cells, contributing to the ␥ restoration of ␤-cell function in type 2 diabetic subjects PPAR- interaction and the antidiabetic action of TZDs; by troglitazone. Diabetes 51:676–685, 2002 the relative potency of TZDs for binding to PPAR-␥ and activation of PPAR-␥ in vitro correlates perfectly with their antidiabetic potency in vivo (10). Patients with a dominant-negative mutation in the PPAR-␥ gene show nsulin is the most important molecule among the severe hyperglycemia, which provides a genetic link be- regulators in glucose homeostasis. Glucose is the tween PPAR-␥ and type 2 diabetes (11). TZDs stimulate primary physiological stimulus for the regulation of adipocyte differentiation, preferentially generating smaller Iinsulin secretion in ␤-cells, the process that requires adipocytes that are more sensitive to insulin and produce glucose sensing. The glucose-sensing apparatus of ␤-cells lower levels of free fatty acids, tumor necrosis factor-␣, consists of glucose transporter isotype 2 (GLUT2) and and leptin (10,12). TZDs are also known to restore the glucokinase (GK), which play a critical role in glucose- functions of ␤-cells, reduce intracellular fat deposition, stimulated insulin secretion (GSIS) (1). ␤-Cell–specific and relieve ␤-cells from a lipotoxic environment (13,14). knockout of GLUT2 or GK results in infantile death There are reports that TZDs improve glucose-sensing ability in isolated islets of diabetic ZDF rats and increase ␤ From the 1Department of Biochemistry and Molecular Biology, the Institute of GLUT2 gene expression in -cells (8,13,15). These data Genetic Science, Yonsei University College of Medicine, Seoul, Korea; the suggest that the expression of genes involved in glucose 2Department of Laboratory Animal Medicine, Medical Research Center, Yon- ␤ sei University College of Medicine, Seoul, Korea; and the 3Department of sensing of pancreatic -cells may be modulated by Chemistry, Korea Military Academy, Seoul, Korea. PPAR-␥. However, the molecular targets of TZDs involved Address correspondence and reprint requests to Yong-ho Ahn, Department in their action on the physiological regulation of insulin of Biochemistry and Molecular Biology, Yonsei University College of Medi- cine, 134 Shinchon-dong, Seodaemoon-gu, Seoul 120-752, Korea. E-mail: secretion are yet to be identified (16). [email protected]. We previously reported the presence of peroxisomal Received for publication 6 September 2001 and accepted in revised form 26 November 2001. proliferator response element (PPRE) in the rat GLUT2 H.-I.K. and J.-Y.C. contributed equally to this work. promoter and suggested its possible significance to ex- ␤GK, ␤-cell–specific GK; DMEM, Dulbecco’s modified Eagle’s medium; plain the role of PPAR-␥ in restoring GSIS (15). However, EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; GK, glucokinase; GSIS, glucose-stimulated insulin secretion; LGK, rat liver–spe- the general belief that GK may be more important than cific GK; MODY, maturity-onset diabetes of the young; PDX-1, pancreatic GLUT2 in the glucose sensing of ␤-cells led us to explore duodenal homeobox gene-1; PPAR, peroxisomal proliferator–activated recep- ␤ ␤ tor; PPRE, peroxisomal proliferator response element; RPA, RNase protection the presence of PPRE in the -cell–specific GK ( GK) assay; RXR-␣, retinoid X receptor-␣; TZD, thiazolidinedione. gene. In this report, we identify a PPRE in the ␤GK gene 676 DIABETES, VOL. 51, MARCH 2002 H.-I. KIM AND ASSOCIATES and show that this element is responsible for the upregu- containing 10 mmol/l HEPES (pH 7.9), 60 mmol/l KCl, 10% glycerol (vol/vol), lation of GK expression by PPAR-␥ in ␤-cells. and 1 mmol/l dithiothreitol. Poly(dI-dC) (1 ␮g) was added to each reaction to suppress nonspecific binding. Anti–PPAR-␥ serum (2 ␮l) was added to the reaction for supershift assay. The protein-DNA complexes were resolved from the free probe by electrophoresis at 4°C on a 5% polyacrylamide gel in 0.5ϫ RESEARCH DESIGN AND METHODS TBE buffer (1ϫ TBE contains 9 mmol/l Tris, 90 mmol/l boric acid, and 20 Materials. Troglitazone was a gift from Sankyo (Tokyo, Japan). WY14643 was mmol/l EDTA, pH 8.0). The dried gels were exposed to X-ray film at Ϫ70°C purchased from Cayman Chemical (Ann Arbor, MI), and 9-cis retinoic acid with an intensifying screen. was purchased from Sigma-Aldrich (St. Louis, MO). Troglitazone concentra- The oligonucleotides used in EMSA were as follows: RGPϩ43/75, 5Ј- tion was adjusted to 10 mmol/l in 19% BSA (wt/vol), 5% DMSO (vol/vol). AGAGTTACCTGTTGCCTCATTACTCAAAAGCCA-3Ј; RGPϩ43/75m1, 5Ј-AGAGc WY14643 (20 mmol/l) and 9-cis retinoic acid (2 mmol/l) were prepared in 50% ccgggGTTGCCTCATTACTCAAAAGCCA-3Ј; RGPϩ43/75m2, 5Ј-AGAGTTACCT ethanol (vol/vol) and 50% DMSO (vol/vol), respectively. Expression plasmids GgatatcCATTACTCAAAAGCCA-3Ј; and RGPϩ43/75m3, 5Ј-AGAGTTACCTGTT pCMX-mPPAR-␣, pCMX-mPPAR-␥, pCMX-mRXR-␣, and pCMX-mRXR-␥ were GCCTCgaattcCAAAAGCCA-3Ј. The PPRE sequence is underlined, and mutated gifts from Drs. R.M. Evans and D.J. Mangelsdorf. Rat GK promoter region bases are shown in lowercase letters (21). spanning –1,003/ϩ196 bp of ␤-cell–specific gene (17) and –1,448/ϩ127 of RNA preparation, RNase protection assay, and RT-PCR. Total RNA was liver-specific gene (18) were cloned into pGL3 basic vector and named isolated from ␤-cell lines treated with 20 ␮mol/l troglitazone and 1 ␮mol/l pRGP-1003 and pRGL-1448, respectively. 5Ј serial deletion of ␤GK promoter 9-cis retinoic acid for 24 h using TRIzol reagent by the manufacturer’s reporter constructs pRGP-404, pRGP-128, pRGPϩ10, and pRGPϩ100 were protocol (Life Technologies). Production of probes and RNase protection constructed by amplifying rat GK promoter regions of Ϫ404/ϩ196, Ϫ128/ assay (RPA) were performed using Strip-EZ RNA probe synthesis kit and RPA ϩ196, ϩ10/ϩ196, and ϩ100/ϩ196 bp, respectively, and subcloning into pGL3 III kit (Ambion, Austin, TX) according to the manufacturer’s protocol. Briefly, basic vector. For construction of PPRE-truncated promoter reporter con- antisense RNA probes, which covered the ϩ370/ϩ696-bp region of rat ␤GK structs pRGPdϩ10/100, pRGPdϩ49/73, and pRGPdϩ73/100, KpnI sites were and the ϩ224/ϩ440-bp region of rat ␤-actin, were produced from introduced into the appropriate region by site-directed mutagenesis, and pCRGKP3SP6 and pCRactin using [32P]UTP and T7 RNA polymerase, then KpnI-digested fragments were excised. Mutant constructs pRGP-1003m1, template DNA and free nucleotides were removed by DNase I digestion and pRGP-1003m2, pRGP-1003m3, pRGP-1003m4, pRGP-1003m5, pRGP-1003m6, two successive ethanol precipitations. Total RNA (50 ␮g) and 300,000 cpm of and pRGP-1003mT were produced by introducing substitution mutations into probes were hybridized at 42°C for 24 h, then unhybridized RNA was digested pRGP-1003 using the QuickChange site-directed mutagenesis kit (Stratagene, by RNase A/RNase T1 mix. The remaining samples after RNase digestion were La Jolla, CA). pRGPϩ10m5 and pRGPϩ10mT were also produced by intro- precipitated and resuspended in 10 ␮l gel loading buffer. Samples were ducing substitution mutations into pRGPϩ10. pGPRRE3-tk-LUC was pro- incubated for 3 min at 94°C and subjected to electrophoresis on 5% denaturing duced by inserting three copies of the ϩ44/ϩ70-bp region of ␤GK gene into the polyacrylamide gel. The dried gels were exposed to X-ray film at –70°C with an SalI site of ptkLUC reporter. pCRGKP3SP6 and pCRactin were cloned by intensifying screen. inserting the PCR product amplifying the region ϩ370/ϩ696 bp of rat ␤GK For RT-PCR, first-strand cDNA was synthesized from 2 ␮g of total RNA in gene and ϩ224/ϩ440 bp of rat ␤-actin gene, respectively.
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