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• W2017487810 abstract "The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is involved in glucose homeostasis and synthetic PPARγ ligands, the thiazolidinediones, a new class of antidiabetic agents that reduce insulin resistance and, as a secondary effect, reduce hepatic glucose output. PPARγ is highly expressed in normal human pancreatic islet α-cells that produce glucagon. This peptide hormone is a functional antagonist of insulin stimulating hepatic glucose output. Therefore, the effect of PPARγ and thiazolidinediones on glucagon gene transcription was investigated. After transient transfection of a glucagon-reporter fusion gene into a glucagon-producing pancreatic islet cell line, thiazolidinediones inhibited glucagon gene transcription when PPARγ was coexpressed. They also reduced glucagon secretion and glucagon tissue levels in primary pancreatic islets. A 5′/3′-deletion and internal mutation analysis indicated that a pancreatic islet cell-specific enhancer sequence (PISCES) motif within the proximal glucagon promoter element G1 was required for PPARγ responsiveness. This sequence motif binds the paired domain transcription factor Pax6. When the PISCES motif within G1 was mutated into a GAL4 binding site, the expression of GAL4-Pax6 restored glucagon promoter activity and PPARγ responsiveness. GAL4-Pax6 transcriptional activity was inhibited by PPARγ in response to thiazolidinedione treatment also at a minimal viral promoter. These results suggest that PPARγ in a ligand-dependent but DNA binding-independent manner inhibits Pax6 transcriptional activity, resulting in inhibition of glucagon gene transcription. These data thereby define Pax6 as a novel functional target of PPARγ and suggest that inhibition of glucagon gene expression may be among the multiple mechanisms through which thiazolidinediones improve glycemic control in diabetic subjects. The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is involved in glucose homeostasis and synthetic PPARγ ligands, the thiazolidinediones, a new class of antidiabetic agents that reduce insulin resistance and, as a secondary effect, reduce hepatic glucose output. PPARγ is highly expressed in normal human pancreatic islet α-cells that produce glucagon. This peptide hormone is a functional antagonist of insulin stimulating hepatic glucose output. Therefore, the effect of PPARγ and thiazolidinediones on glucagon gene transcription was investigated. After transient transfection of a glucagon-reporter fusion gene into a glucagon-producing pancreatic islet cell line, thiazolidinediones inhibited glucagon gene transcription when PPARγ was coexpressed. They also reduced glucagon secretion and glucagon tissue levels in primary pancreatic islets. A 5′/3′-deletion and internal mutation analysis indicated that a pancreatic islet cell-specific enhancer sequence (PISCES) motif within the proximal glucagon promoter element G1 was required for PPARγ responsiveness. This sequence motif binds the paired domain transcription factor Pax6. When the PISCES motif within G1 was mutated into a GAL4 binding site, the expression of GAL4-Pax6 restored glucagon promoter activity and PPARγ responsiveness. GAL4-Pax6 transcriptional activity was inhibited by PPARγ in response to thiazolidinedione treatment also at a minimal viral promoter. These results suggest that PPARγ in a ligand-dependent but DNA binding-independent manner inhibits Pax6 transcriptional activity, resulting in inhibition of glucagon gene transcription. These data thereby define Pax6 as a novel functional target of PPARγ and suggest that inhibition of glucagon gene expression may be among the multiple mechanisms through which thiazolidinediones improve glycemic control in diabetic subjects. peroxisome proliferator-activated receptor peroxisome proliferator-activated receptor response element pancreatic islet cell-specific enhancer sequence reverse transcriptase cAMP-response element-binding protein-binding protein green fluorescent protein retinoid X receptor Peroxisome proliferator-activated receptor γ (PPARγ)1 is a member of the ligand-regulated nuclear hormone receptor superfamily (1Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2735) Google Scholar). Like other nuclear receptors, PPARγ comprises an amino-terminal ligand-independent transactivation domain (AF-1), a central DNA-binding domain, and a carboxyl-terminal ligand-binding domain that contains a second, ligand-dependent transactivation surface (AF-2) (1Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2735) Google Scholar). PPARγ binds as a heterodimer with the 9-cis-retinoic acid receptor, RXR, to response elements in target genes to activate transcription. A typical PPRE consists of a direct repeat of hexamer half-sites, TGACCT, spaced by one nucleotide (DR-1) (1Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2735) Google Scholar). PPAR and RXR occupy the 5′ and 3′ half-sites, respectively, and thus show a polarity in binding that is the opposite of that observed for other nuclear receptor-RXR heterodimers (1Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2735) Google Scholar). Like other nuclear receptors, there is evidence that PPARγ-RXR require the ligand-dependent recruitment of coactivator proteins like SRC-1, GRIP-1, pCIP, CBP, p300, DRIP205, and p120 (2Yang W. Rachez C. Freedman L.P. Mol. Cell. Biol. 2000; 20: 8008-8017Crossref PubMed Scopus (106) Google Scholar, 3Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1692) Google Scholar, 4Gampe R.T. Montana V.G. Lambert M.H. Miller A.B. Bledsoe R.K. Milburn M.V. Kliewer S.A. Willson T.M. Xu H.E. Mol. Cell. 2000; 5: 545-555Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, 5Monden T. Kishi M. Hosoya T. Satoh T. Wondisford F.E. Hollenberg A.N. Yamada M. Mori M. Mol. Endocrinol. 1999; 13: 1695-1703PubMed Google Scholar) to effectively stimulate gene transcription. This recruitment is dependent on allosteric alterations in the AF-2 helical domain. A “mouse trap” model of receptor activation has been proposed, in which the AF-2 helix closes on the ligand-binding site in response to ligand and establishes a transcriptionally active form of the receptor (3Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1692) Google Scholar). Cocrystal studies (3Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1692) Google Scholar, 4Gampe R.T. Montana V.G. Lambert M.H. Miller A.B. Bledsoe R.K. Milburn M.V. Kliewer S.A. Willson T.M. Xu H.E. Mol. Cell. 2000; 5: 545-555Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar) indicated that two highly conserved amino acids, Glu-469 in the AF-2 helix and Lys-301 in helix 3 of the ligand-binding domain, form a charge clamp that places a helical LXXLL motif of SRC-1 class of coactivators into a hydrophobic pocket in the receptor. In addition to stimulation of transcription, PPARγ has been shown to be capable of also negative regulation of gene transcription (6Ricote M. Li A.C. Willson T.M. Kelly C.J. Glass C.K. Nature. 1998; 391: 79-82Crossref PubMed Scopus (3260) Google Scholar, 7Li M. Pascual G. Glass C.K. Mol. Cell. Biol. 2000; 20: 4699-4707Crossref PubMed Scopus (353) Google Scholar, 8Jiang C. Ting A.T. Seed B. Nature. 1998; 391: 82-86Crossref PubMed Scopus (539) Google Scholar, 9Nawa T. Nawa M.T. Cai Y. Zhang C. Uchimura I. Narumi S. Numano F. Kitajima S. Biochem. Biophys. Res. Commun. 2000; 275: 406-411Crossref PubMed Scopus (57) Google Scholar, 10Uchimura K. Nakamuta M. Enjoji M. Irie T. Sugimoto R. Muta T. Iwamoto H. Nawata H. Hepatology. 2001; 33: 91-99Crossref PubMed Scopus (116) Google Scholar, 11Faveeuw C. Fougeray S. Angeli V. Fontaine J. Chinetti G. Gosset P. Delerive P. Maliszewski C. Capron M. Staels B. Moser M. Trottein F. FEBS Lett. 2000; 486: 261-266Crossref PubMed Scopus (153) Google Scholar, 12Han K.H. Chang M.K. Boullier A. Green S.R. Li A. Glass C.K. Quehenberger O. J. Clin. Invest. 2000; 106: 793-802Crossref PubMed Scopus (161) Google Scholar, 13Yang X.Y. Wang L.H. Chen T. Hodge D.R. Resau J.H. DaSilva L. Farrar W.L. J. Biol. Chem. 2000; 275: 4541-4544Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 14Ikeda Y. Sugawara A. Taniyama Y. Uruno A. Igarashi K. Arima S. Ito S. Takeuchi K. J. Biol. Chem. 2000; 275: 33142-33150Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). PPARγ has been suggested to be involved in a broad range of cellular functions, including adipocyte differentiation, inflammatory responses, and apoptosis, as well as in chronic diseases such as obesity, atherosclerosis, and cancer (15Kersten S. Desvergne B. Wahli W. Nature. 2000; 405: 421-424Crossref PubMed Scopus (1665) Google Scholar, 16Vamecq J. Latruffe N. Lancet. 1999; 354: 141-148Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). Of particular importance is its role in glucose homeostasis and type 2 diabetes mellitus (15Kersten S. Desvergne B. Wahli W. Nature. 2000; 405: 421-424Crossref PubMed Scopus (1665) Google Scholar, 16Vamecq J. Latruffe N. Lancet. 1999; 354: 141-148Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). Human genetic studies support an important role of PPARγ in mammalian metabolism (15Kersten S. Desvergne B. Wahli W. Nature. 2000; 405: 421-424Crossref PubMed Scopus (1665) Google Scholar, 17Altshuler D. Hirschhorn J.N. Klannemark M. Lindgren C.M. Vohl M.C. Nemesh J. Lane C.R. Schaffner S.F. Bolk S. Brewer C. Tuomi T. Gaudet D. Hudson T.J. Daly M. Groop L. Lander E.S. Nat. Genet. 2000; 26: 76-80Crossref PubMed Scopus (123) Google Scholar, 18Barroso I. Gurnell M. Crowley V.E.F. Agostini M. Schwabe J.M. Soos M.A. Masien G.U. Williams T.D.M. Lewis H. Schafer A.J. Chatterjee V.K.K. O'Rahilly S. Nature. 1999; 402: 880-883Crossref PubMed Scopus (1161) Google Scholar). Thus, dominant negative mutations in human PPARγ are associated with hypertension, severe insulin resistance, and diabetes mellitus (18Barroso I. Gurnell M. Crowley V.E.F. Agostini M. Schwabe J.M. Soos M.A. Masien G.U. Williams T.D.M. Lewis H. Schafer A.J. Chatterjee V.K.K. O'Rahilly S. Nature. 1999; 402: 880-883Crossref PubMed Scopus (1161) Google Scholar). These physiologic and pathophysiologic actions suggest that synthetic PPARγ ligands may be of use in the treatment of type 2 diabetes mellitus. Thiazolidinediones like rosiglitazone are PPARγ ligands and a new class of orally active antidiabetic drugs (19Saltiel A.R. Olefsky J.M. Diabetes. 1996; 45: 1661-1669Crossref PubMed Scopus (0) Google Scholar, 20Schoonjans K. Auwerx J. Lancet. 2000; 355: 1008-1010Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 21Olefsky J.M. J. Clin. Invest. 2000; 106: 467-472Crossref PubMed Scopus (508) Google Scholar). They decrease hepatic glucose output and reduce insulin resistance by increasing insulin-dependent peripheral glucose disposal (19Saltiel A.R. Olefsky J.M. Diabetes. 1996; 45: 1661-1669Crossref PubMed Scopus (0) Google Scholar). Thiazolidinediones thereby markedly decrease plasma glucose, insulin, and triglyceride levels in animal models of type II diabetes as well as in type II diabetic subjects (19Saltiel A.R. Olefsky J.M. Diabetes. 1996; 45: 1661-1669Crossref PubMed Scopus (0) Google Scholar). The antidiabetic effect of thiazolidinediones requires several days of treatment and does not produce overt hypoglycemia (19Saltiel A.R. Olefsky J.M. Diabetes. 1996; 45: 1661-1669Crossref PubMed Scopus (0) Google Scholar). Thiazolidinediones have been shown to decrease adipocyte tumor necrosis factor α/resistin secretion and circulating free fatty acid levels; to increase basal glucose uptake in 3T3-L1 adipocytes, L6 myocytes, and human muscle cultures derived from obese type II diabetic subjects; and to stimulate glucokinase gene transcription in HepG2 cells (19Saltiel A.R. Olefsky J.M. Diabetes. 1996; 45: 1661-1669Crossref PubMed Scopus (0) Google Scholar, 20Schoonjans K. Auwerx J. Lancet. 2000; 355: 1008-1010Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 21Olefsky J.M. J. Clin. Invest. 2000; 106: 467-472Crossref PubMed Scopus (508) Google Scholar, 22Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. 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The correlation betweenin vivo antihyperglycemic activity and in vitroPPARγ activity (29Willson T.M. Cobb J.E. Cowan D.J. Wiethe R.W. Correa I.D. Prakash S.R. Beck K.D. Moore L.B. Kliewer S.A. Lehmann J.M. J. Med. Chem. 1996; 39: 665-668Crossref PubMed Scopus (656) Google Scholar) suggests that thiazolidinediones act as antidiabetic agents by regulating the transcription of a subset of genes through PPARγ. However, the target genes involved are unclear. It has been shown recently that high levels of PPARγ are expressed in glucagon-producing α-cells of the endocrine pancreas (30Dubois M. Pattou F. Kerr-Conte J. Gmyr V. Vandewalle B. Desreumaux P. Auwerx J. Schoonjans K. Diabetologia. 2000; 43: 1165-1169Crossref PubMed Scopus (167) Google Scholar, 31Braissant O. Foufelle F. Scotto C. Dauca M. Wahli W. Endocriniology. 1996; 137: 354-366Crossref PubMed Scopus (0) Google Scholar, 32Zhou Y.T. Shimabukuro M. Wang M.Y. Lee Y. Higa M. Milburn J.L. Newgard C.B. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8898-8903Crossref PubMed Scopus (163) Google Scholar). The pancreatic islet hormone glucagon is a biologic antagonist of insulin. The effects of glucagon on blood glucose levels balance those of insulin; glucagon increases hepatic glucose production and opposes hepatic glucose storage, whereas insulin increases peripheral glucose uptake and opposes glucagon-mediated hepatic glucose production. The metabolic consequences of abnormal α-cell function in diabetes are well defined (33Unger R.H. Orci L. N. Engl. J. Med. 1981; 304: 1518-1524Crossref PubMed Scopus (211) Google Scholar, 34Unger R.H. Orci L. N. Engl. J. Med. 1981; 304: 1575-1580Crossref PubMed Scopus (129) Google Scholar, 35Lefèbvre P.J. Diabetes Care. 1995; 18: 715-730Crossref PubMed Scopus (97) Google Scholar). In addition to hyperglycemia, insulin resistance, and impaired β-cell function, relative hyperglucagonemia is a common feature of patients with type II diabetes (33Unger R.H. Orci L. N. Engl. J. Med. 1981; 304: 1518-1524Crossref PubMed Scopus (211) Google Scholar, 34Unger R.H. Orci L. N. Engl. J. Med. 1981; 304: 1575-1580Crossref PubMed Scopus (129) Google Scholar, 35Lefèbvre P.J. Diabetes Care. 1995; 18: 715-730Crossref PubMed Scopus (97) Google Scholar). The elevated glucagon levels in diabetes contribute to increased hepatic glucose output and hyperglycemia (33Unger R.H. Orci L. N. Engl. J. Med. 1981; 304: 1518-1524Crossref PubMed Scopus (211) Google Scholar, 34Unger R.H. Orci L. N. Engl. J. Med. 1981; 304: 1575-1580Crossref PubMed Scopus (129) Google Scholar, 35Lefèbvre P.J. Diabetes Care. 1995; 18: 715-730Crossref PubMed Scopus (97) Google Scholar). Consequently, inhibition of glucagon secretion has been shown to reduce fasting hyperglycemia in diabetic animals (36Freyse E.J. Becher T. El-Hag O. Knospe S. Göke B. Fischer U. Diabetes. 1997; 46: 824-828Crossref PubMed Scopus (24) Google Scholar) and patients (37Creutzfeldt W.O.C. Kleine N. Willms B. ℘rskov C. Holst J.J. Nauck M.A. Diabetes Care. 1996; 19: 580-586Crossref PubMed Scopus (308) Google Scholar, 38Drucker D.J. Diabetes. 1998; 47: 159-169Crossref PubMed Google Scholar). Effects on the expression of glucagon in pancreatic islets are therefore important aspects in the treatment of diabetes mellitus. Because PPARγ is expressed in glucagon-producing α-cells but its function has been unknown, in the present study the effect of PPARγ and thiazolidinediones on glucagon gene transcription was investigated. PPARγ and thiazolidinediones were found to inhibit glucagon gene transcription in pancreatic islet cells. They also reduced glucagon secretion and tissue levels in pancreatic islets. Mapping studies and the use of GAL4 fusion proteins indicate that PPARγ represses in a ligand-dependent but DNA binding-independent manner transactivation by the paired domain-containing transcription factor Pax6 leading to inhibition of glucagon gene transcription. This novel action of PPARγ assigns a function to PPARγ expressed in pancreatic islet α-cells and suggests that the mechanisms through which thiazolidinediones improve glycemic control in diabetic subjects may include the inhibition of glucagon gene expression. The plasmids pT81Luc (39Nordeen S.K. BioTechniques. 1988; 6: 454-457PubMed Google Scholar), −350GluLuc (40Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar), 5xGal4E1BLuc (41Krüger M. Schwaninger M. Blume R. Oetjen E. Knepel W. Naunyn Schmiedebergs Arch. Pharmacol. 1997; 356: 433-440Crossref PubMed Scopus (32) Google Scholar), −292GluLuc, −169GluLuc, −136GluLuc, −60GluLuc, −350/−48GluLuc, −350/−91GluLuc, −350/−150GluLuc, −350/−210GluLuc, (42Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar), −350(mutG1)GluLuc, pGAL4-Pax6 (43Grzeskowiak R. Amin J. Oetjen E. Knepel W. J. Biol. Chem. 2000; 275: 30037-30045Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), PPRELuc, pPPARγ, pRXRα (44Heinlein C.A. Ting H.J. Yeh S. Chang C. J. Biol. Chem. 1999; 274: 16147-16152Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), and pGAL4-PPARγ (45Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3461) Google Scholar) have been described previously. The plasmid pCMV-GFPtpz was purchased from Canberra-Packard (Dreieich, Germany). Total RNA was extracted from InR1-G9 cells using a commercial kit (RNeasy, Quiagen). For first strand cDNA synthesis, random hexamer primers (Amersham Biosciences, Inc.) were used. The RT enzyme was obtained from Invitrogen (Superscript II reverse transcriptase). For PCR amplification, the following primers were used: upstream primer, 5′-AGAGCTGACCCAATGGTTGC-3′; and downstream primer, 5′-ATCTCCGCCAACAGCTTCTC-3′ (EMBL/GenBank™/DDBJ accession no. Z30972) (size of the expected product: 421 bp). PCR without RT step served as control for DNA contamination. After agarose gel electrophoresis, the RT-PCR product was identified by extraction, subcloning (TA-cloning kit, Promega), and cycle sequencing (Thermo Sequenase fluorescent labeled primer cycle sequencing kit, Amersham Biosciences, Inc.; IRD-800 labeled primers, MWG Biotech, Ebersberg, Germany). The glucagon-producing pancreatic islet cell line InR1-G9 (46Takaki R. Ono J. Nakamura M. Yokogawa Y. Kumae S. Hiraoka T. Yamaguchi K. Hamaguchi K Uchida S. In Vitro Cell Dev. Biol. 1986; 22: 120-126Crossref PubMed Scopus (82) Google Scholar) was grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were trypsinized and transfected in suspension by the DEAE-dextran method (40Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar) with 2 μg of reporter gene plasmids and, when indicated, 1 μg of expression vector per 6-cm dish. Cotransfections were carried out with a constant amount of DNA, which was maintained by adding Bluescript (Stratagene, La Jolla). In all experiments 0.5 μg of cytomegalovirus-green fluorescent protein (GFP) (plasmid pCMV-GFPtpz) per 6-cm dish was cotransfected to check for transfection efficiency (the relative luciferase activities presented in the figures are derived from luciferase/GFP ratios). Twenty-four hours after transfection, cells were incubated in RPMI 1640 containing 0.5% bovine serum albumin and antibiotics as described above. Cell extracts (40Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar) were prepared 48 h after transfection. The luciferase assay was performed as described previously (40Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar). Green fluorescent protein was measured in the cell extracts using the FluoroCount™ microplate fluorometer (Packard). After the preparation of Langerhans pancreatic islets of NMRI mice (32Zhou Y.T. Shimabukuro M. Wang M.Y. Lee Y. Higa M. Milburn J.L. Newgard C.B. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8898-8903Crossref PubMed Scopus (163) Google Scholar), islets were cultured in RPMI medium supplemented with 5 mmglucose, 10% bovine serum albumin, 100 units/ml penicillin, and 100 μg/ml streptomycin. After 48 h, glucagon levels were measured in the supernatants and the islet extracts (40Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar) by radioimmunoassay using a commercial kit (IBL, Hamburg, Germany). Rosiglitazone was kindly provided by SmithKline Beecham (Worthing, United Kingdom); darglitazone and englitazone (CP-72,467–02, sodium salt) was provided by Pfizer Inc. (Groton, CT). A stock solution (100 mm) was prepared in Me2SO. Controls received the solvent only. PPARγ was found by RT-PCR to be expressed in the glucagon-producing pancreatic islet cell line InR1-G9 (data not shown). In normal pancreatic islets, the expression of PPARγ is very high, approximately two thirds of the expression level in white adipose tissue (30Dubois M. Pattou F. Kerr-Conte J. Gmyr V. Vandewalle B. Desreumaux P. Auwerx J. Schoonjans K. Diabetologia. 2000; 43: 1165-1169Crossref PubMed Scopus (167) Google Scholar). In contrast, InR1-G9 cells express low levels of PPARγ such that activation of a PPAR-dependent promoter (PPRELuc) required transfection of a PPARγ expression plasmid (Fig.1). This cell line therefore allowed a direct assessment of the role of PPARγ in glucagon gene transcription. Similarly, low level expression of PPARγ in cell lines derived from tissues with high level expression has been reported previously (6Ricote M. Li A.C. Willson T.M. Kelly C.J. Glass C.K. Nature. 1998; 391: 79-82Crossref PubMed Scopus (3260) Google Scholar, 13Yang X.Y. Wang L.H. Chen T. Hodge D.R. Resau J.H. DaSilva L. Farrar W.L. J. Biol. Chem. 2000; 275: 4541-4544Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). To study the effect of PPARγ and thiazolidinediones on glucagon gene transcription, 350 base pairs of the 5′-flanking region of the rat glucagon gene were fused to the luciferase reporter gene (construct −350GluLuc) (40Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar). This glucagon promoter fragment is sufficient to confer tissue-specific gene expression (47Philippe J. Drucker D.J. Knepel W. Jepeal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 8: 4877-4888Crossref PubMed Scopus (116) Google Scholar) and regulation of gene transcription by cAMP-, calcium-, protein kinase C-, and insulin-induced signaling pathways (40Schwaninger M. Lux G. Blume R. Oetjen E. Hidaka H. Knepel W. J. Biol. Chem. 1993; 268: 5168-5177Abstract Full Text PDF PubMed Google Scholar, 42Fürstenau U. Schwaninger M. Blume R. Kennerknecht I. Knepel W. Mol. Cell. Biol. 1997; 17: 1805-1816Crossref PubMed Scopus (22) Google Scholar, 43Grzeskowiak R. Amin J. Oetjen E. Knepel W. J. Biol. Chem. 2000; 275: 30037-30045Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 48Schwaninger M. Blume R. Oetjen E. Lux G. Knepel W. J. Biol. Chem. 1993; 268: 23111-23115Abstract Full Text PDF PubMed Google Scholar, 49Schwaninger M. Blume R. Krüger M. Lux G. Oetjen E. Knepel W. J. Biol. Chem. 1995; 270: 8860-8866Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 50Knepel W. Chafitz J. Habener J.F. Mol. Cell. Biol. 1990; 10: 6799-6804Crossref PubMed Scopus (67) Google Scholar, 51Oetjen E. Diedrich T. Eggers A. Eckert B. Knepel W. J. Biol. Chem. 1994; 269: 27036-27044Abstract Full Text PDF PubMed Google Scholar, 52Fürstenau U. Schwaninger M. Blume R. Jendrusch E.M. Knepel W. J. Biol. Chem. 1999; 274: 5851-5860Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In the absence of a cotransfected PPARγ expression plasmid, treatment of InR1-G9 cells with the thiazolidinedione rosiglitazone at concentrations up to 100 μm had no effect on glucagon promoter activity (data not shown). Additionally, the cotransfection of an expression plasmid encoding PPARγ alone had no effect on −350GluLuc activity (94 ± 3% of controls, n = 6). However, when a PPARγ expression plasmid was transfected into these cells, rosiglitazone inhibited glucagon gene transcription (Fig.2). Thus rosiglitazone inhibits glucagon gene transcription by a PPARγ-dependent mechanism. Inhibition of glucagon gene transcription by rosiglitazone was concentration-dependent with an IC50 value of ∼300 nm (Fig. 2). Cotransfection of an expression vector encoding RXRα together with PPARγ did not alter the concentration-response curve for inhibition by rosiglitazone of −350GluLuc activity (data not shown). These concentrations of rosiglitazone are similar to those that activated a PPARγ-dependent promoter (Fig. 1). The maximum inhibition of glucagon gene transcription by rosiglitazone was ∼40% (Fig. 2). Like rosiglitazone, two other thiazolidinediones, darglitazone and englitazone, also inhibited glucagon gene transcription (Fig.3). To assess the effect of thiazolidinediones in a natural context, the effect of thiazolidinediones on glucagon secretion and glucagon tissue levels was investigated in primary pancreatic islets. After 48 h of treatment with rosiglitazone, glucagon secretion from isolated pancreatic islets was inhibited by 44% (Fig. 4). Englitazone and darglitazone showed a similar inhibition (Fig. 4). Furthermore, glucagon tissue levels were significantly reduced by treatment with rosiglitazone, englitazone, or darglitazone (Fig. 4). These data indicate that PPARγ inhibits glucagon gene transcription in response to binding of thiazolidinediones. Thiazolidinediones also reduce glucagon tissue levels and secretion in pancreatic islets.Figure 2Inhibition of glucagon gene transcription by rosiglitazone and PPARγ. Plasmid −350GluLuc was transfected into InR1-G9 cells together with pPPARγ. Rosiglitazone was added 24 h before harvest. Luciferase activity is expressed as percentage of the mean value of the activity measured in the untreated controls. Values are means ± S.E. of three independent experiments, each done in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Inhibition of glucagon gene transcription by the thiazolidinediones darglitazone and englitazone. InR1-G9 cells were transfected with −350GluLuc and pPPARγ. They were treated with rosiglitazone (Rosi, 10 μm), darglitazone (Dar, 30 μm), or englitazone (Engl, 100 μm) for 24 h before harvest. Luciferase activity is expressed as percentage of the mean value, in each experiment, of the activity measured in the untreated controls. Values are means ± S.E. of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Inhibition by thiazolidinediones of glucagon secretion and glucagon tissue levels in primary pancreatic islets.Isolated mouse pancreatic islets were treated with rosiglitazone (Rosiglit, 30 μm), englitazone (Englit, 100 μm) darglitazone (Darglit, 30 μm), or the solvent (control) for 48 h. Glucagon secretion and tissue levels are expressed as percentage of the mean value, in each experiment, of the levels measured in the respective control. Values are means ± S.E. of five independent experiments, each done in duplicate. *,p < 0.005 (Student's t test).View Large Image Figure ViewerDownload Hi-res image Download (PPT) PPARγ is known" @default.
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• W2017487810 title "Repression of Glucagon Gene Transcription by Peroxisome Proliferator-activated Receptor γ through Inhibition of Pax6 Transcriptional Activity" @default.
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