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• W2071978660 abstract "Insulin biosynthesis and secretion are critical for pancreatic β-cell function, but both are impaired under diabetic conditions. We have found that hyperglycemia induces the expression of the basic helix-loop-helix transcription factor c-Myc in islets in several different diabetic models. To examine the possible implication of c-Myc in β-cell dysfunction, c-Myc was overexpressed in isolated rat islets using adenovirus. Adenovirus-mediated c-Myc overexpression suppressed both insulin gene transcription and glucose-stimulated insulin secretion. Insulin protein content, determined by immunostaining, was markedly decreased in c-Myc-overexpressing cells. In gel-shift assays c-Myc bound to the E-box in the insulin gene promoter region. Furthermore, in βTC1, MIN6, and HIT-T15 cells and primary rat islets, wild type insulin gene promoter activity was dramatically decreased by c-Myc overexpression, whereas the activity of an E-box mutated insulin promoter was not affected. In HeLa and HepG2 cells c-Myc exerted a suppressive effect on the insulin promoter activity only in the presence of NeuroD/BETA2 but not PDX-1. Both c-Myc and NeuroD can bind the E-box element in the insulin promoter, but unlike NeuroD, the c-Myc transactivation domain lacked the ability to activate insulin gene expression. Additionally p300, a co-activator of NeuroD, did not function as a co-activator of c-Myc. In conclusion, increased expression of c-Myc in β-cells suppresses the insulin gene transcription by inhibiting NeuroD-mediated transcriptional activation. This mechanism may explain some of the β-cell dysfunction found in diabetes. Insulin biosynthesis and secretion are critical for pancreatic β-cell function, but both are impaired under diabetic conditions. We have found that hyperglycemia induces the expression of the basic helix-loop-helix transcription factor c-Myc in islets in several different diabetic models. To examine the possible implication of c-Myc in β-cell dysfunction, c-Myc was overexpressed in isolated rat islets using adenovirus. Adenovirus-mediated c-Myc overexpression suppressed both insulin gene transcription and glucose-stimulated insulin secretion. Insulin protein content, determined by immunostaining, was markedly decreased in c-Myc-overexpressing cells. In gel-shift assays c-Myc bound to the E-box in the insulin gene promoter region. Furthermore, in βTC1, MIN6, and HIT-T15 cells and primary rat islets, wild type insulin gene promoter activity was dramatically decreased by c-Myc overexpression, whereas the activity of an E-box mutated insulin promoter was not affected. In HeLa and HepG2 cells c-Myc exerted a suppressive effect on the insulin promoter activity only in the presence of NeuroD/BETA2 but not PDX-1. Both c-Myc and NeuroD can bind the E-box element in the insulin promoter, but unlike NeuroD, the c-Myc transactivation domain lacked the ability to activate insulin gene expression. Additionally p300, a co-activator of NeuroD, did not function as a co-activator of c-Myc. In conclusion, increased expression of c-Myc in β-cells suppresses the insulin gene transcription by inhibiting NeuroD-mediated transcriptional activation. This mechanism may explain some of the β-cell dysfunction found in diabetes. basic helix-loop-helix pancreatic and duodenal homeobox factor-1 green fluorescent protein plaque-forming unit transactivation domain cytomegalovirus reverse transcription chloramphenicol acetyltransferase The development of type 2 diabetes is usually associated with pancreatic β-cell dysfunction occurring together with insulin resistance. Usually β-cells can compensate for insulin resistance by increasing insulin secretion, but insufficient compensation leads to glucose intolerance. Once hyperglycemia becomes apparent, β-cell function deteriorates (1.Weir G.C. Laybutt D.R. Kaneto H. Bonner-Weir S. Sharma A. Diabetes. 2001; 50 (suppl.): 154-159Crossref Google Scholar). The adverse effects of chronic hyperglycemia on β-cells, called glucose toxicity, have been demonstrated by various in vivo (2.Bonner-Weir S. Trent D.F. Weir G.C. J. Clin. Invest. 1983; 71: 1544-1553Crossref PubMed Scopus (414) Google Scholar, 3.Tokuyama Y. Sturis J. DePaoli A.M. Takeda J. Stoffel M. Tang J. Sun X. Polonsky K.S. Bell G.I. Diabetes. 1995; 44: 1447-1457Crossref PubMed Google Scholar, 4.Zangen D.H. Bonner-Weir S. Lee C.H. Latimer J.B. Miller C.P. Habener J.F. Weir G.C. Diabetes. 1997; 46: 258-264Crossref PubMed Scopus (127) Google Scholar, 5.Jonas J.-C. Sharma A. Hasenkamp W. Iikova H. Patane G. Laybutt R. Bonner-Weir S. Weir G.C. J. Biol. Chem. 1999; 274: 14112-14121Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar) and in vitro studies (6.Sharma A. Olson L.K. Robertson R.P. Stein R. Mol. Endocrinol. 1995; 9: 1127-1134Crossref PubMed Google Scholar,7.Poitout V. Olson K. Robertson R.P. J. Clin. Invest. 1996; 97: 1041-1046Crossref PubMed Scopus (128) Google Scholar). After chronic exposure to hyperglycemia, insulin gene transcription and glucose-stimulated insulin secretion are suppressed. c-Myc is a basic helix-loop-helix (bHLH)1 transcription factor and has an important influence on cell cycle progression, cell differentiation, and the process of apoptosis (8.Evan G.I. Littlewood T.D. Curr. Opin. Genet. Dev. 1993; 3: 44-49Crossref PubMed Scopus (359) Google Scholar, 9.Evan G.I. Littlewood T.D. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1362) Google Scholar, 10.Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1391) Google Scholar, 11.Laybutt, D. R., Weir, G. C., Kaneto, H., Lebet, J., Palmiter, R. D., Sharma, A., and Bonner-Weir, S., Diabetes, in pressGoogle Scholar, 12.Pelengaris S. Rudolph B. Littlewood T. Curr. Opin. Genet. Dev. 2000; 10: 100-105Crossref PubMed Scopus (176) Google Scholar). Under chronic hyperglycemic conditions in vivo, the expression of many β-cell-associated genes is decreased (3.Tokuyama Y. Sturis J. DePaoli A.M. Takeda J. Stoffel M. Tang J. Sun X. Polonsky K.S. Bell G.I. Diabetes. 1995; 44: 1447-1457Crossref PubMed Google Scholar, 4.Zangen D.H. Bonner-Weir S. Lee C.H. Latimer J.B. Miller C.P. Habener J.F. Weir G.C. Diabetes. 1997; 46: 258-264Crossref PubMed Scopus (127) Google Scholar, 5.Jonas J.-C. Sharma A. Hasenkamp W. Iikova H. Patane G. Laybutt R. Bonner-Weir S. Weir G.C. J. Biol. Chem. 1999; 274: 14112-14121Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar), but, in contrast, the expression of some suppressed genes is markedly up-regulated. In normal adult islets, the c-Myc expression level is very low, but c-Myc is induced in diabetic rats following partial pancreatectomy and in rats made hyperglycemic with glucose clamps (5.Jonas J.-C. Sharma A. Hasenkamp W. Iikova H. Patane G. Laybutt R. Bonner-Weir S. Weir G.C. J. Biol. Chem. 1999; 274: 14112-14121Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 13.Jonas J.-C. Laybutt D.R. Steil G.M. Trivedi N. Pertusa J.G. Casteele M.V.D. Weir G.C. Henquin J.-C. J. Biol. Chem. 2001; 276: 35375-35381Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The changes in c-myc gene expression are correlated with graded levels of hyperglycemia. Thus, we hypothesize that induction of c-Myc expression is involved in the β-cell dysfunction of diabetes. Insulin enhancer elements, E-box and A-box, play an important role in regulating cell-specific expression of the insulin gene (14.Karlsson O. Edlund T. Moss J.B. Rutter W.J. Walker M.D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8819-8823Crossref PubMed Scopus (184) Google Scholar, 15.Crowe D.T. Tsai M.-J. Mol. Cell. Biol. 1989; 9: 1784-1789Crossref PubMed Scopus (103) Google Scholar, 16.Whelan J. Cordle S.R. Henderson E. Weil P.A. Stein R. Mol. Cell. Biol. 1990; 10: 1564-1572Crossref PubMed Google Scholar, 17.German M.S. Moss L.G. Wang J. Rutter W.J. Mol. Cell. Biol. 1992; 12: 1777-1788Crossref PubMed Google Scholar, 18.German M.S. Wang J. Mol. Cell. Biol. 1994; 14: 4067-4075Crossref PubMed Scopus (150) Google Scholar, 19.Robinson G.L.W.G. Peshavaria M. Henderson E. Shieh S.-Y. Tsai M.-J. Teitelman G. Stein R. J. Biol. Chem. 1994; 269: 2452-2460Abstract Full Text PDF PubMed Google Scholar). NeuroD, also known as BETA2, binds to the E-box (20.Naya F.J. Stellrecht C.M.M. Tsai M.-J. Genes Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (525) Google Scholar), and the pancreatic and duodenal homeobox factor-1 (PDX-1), also known as IDX-1/STF-1/IPF1, binds to the A-box (21.Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (775) Google Scholar, 22.Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Crossref PubMed Google Scholar, 23.Miller C.P. McGehee R.E. Habener J.F. EMBO J. 1994; 13: 1145-1156Crossref PubMed Scopus (378) Google Scholar). These two transcription factors are very important for insulin gene transcription. NeuroD, a member of the bHLH transcription factor family, is expressed in pancreatic and intestinal endocrine cells and neural tissue. PDX-1, a member of the homeodomain-containing transcription factor family, is expressed in the pancreas and duodenum and plays a major role in pancreatic development (24.Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 37: 606-609Crossref Scopus (1573) Google Scholar, 25.Ahlgren U. Jonsson J. Edlund H. Development. 1996; 122: 1409-1416Crossref PubMed Google Scholar, 26.Offield M.F. Jetton T.L. Labosky P. Ray M. Stein S. Magnuson M. Hogan B.L.M. Wright C.V.E. Development. 1996; 122: 983-985Crossref PubMed Google Scholar, 27.Kaneto H. Miyagawa J. Kajimoto Y. Yamamoto K. Watada H. Umayahara Y. Hanafusa T. Matsuzawa Y. Yamasaki Y. Higashiyama S. Taniguchi N. J. Biol. Chem. 1997; 272: 29137-29143Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), in β-cell differentiation (28.Guz Y. Montminy M.R. Stein R. Leonard J. Gamer L.W. Wright C.V.E. Teitelman G. Development. 1995; 121: 11-18Crossref PubMed Google Scholar, 29.Bonner-Weir S. Taneja M. Weir G.C. Tatarkiwicz K. Song K.-H. Sharma A. O'Neil J.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7999-8004Crossref PubMed Scopus (913) Google Scholar), and in maintaining normal β-cell function probably by regulating multiple important β-cell genes (30.Watada H. Kajimoto Y. Umayahara Y. Matsuoka T. Kaneto H. Fujitani Y. Kamada Y. Kawamori R. Yamasaki Y. Diabetes. 1996; 45: 1478-1488Crossref PubMed Google Scholar, 31.Waeber G. Thompson N. Nicod P. Bonny C. Mol. Endocrinol. 1996; 10: 1327-1334Crossref PubMed Scopus (326) Google Scholar, 32.Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (786) Google Scholar, 33.Wang H. Maechler P. Ritz-Laser B. Hagenfeldt K.A. Ishihara H. Philippe J. Wollheim C.B. J. Biol. Chem. 2001; 276: 25279-25286Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Clinically, mutations in NeuroD (34.Malecki M.T. Jhala U.S. Antonellis A. Fields L. Doris A. Orban T. Saad M. Warram J.H. Montiminy M. Krolewski A.S. Nat. Genet. 1999; 23: 323-328Crossref PubMed Scopus (515) Google Scholar) and in PDX-1 cause maturity-onset diabetes of the young (35.Stoffers D.A. Ferrer J. Clarke W.L. Habener J.F. Nat. Genet. 1997; 17: 138-139Crossref PubMed Scopus (8) Google Scholar, 36.Stoffers D.A. Zinkin N.T. Stanojevic V. Clarke W.L. Habener J.F. Nat. Genet. 1997; 15: 106-110Crossref PubMed Scopus (936) Google Scholar). Mice homozygous for the null mutation in NeuroD have a striking reduction in the number of β-cells, develop severe diabetes, and die perinatally (37.Naya F.J. Huang H. Qiu Y. Mutoh H. DeMayo F. Leiter A.B. Tsai M.-J. Genes Dev. 1997; 11: 2323-2334Crossref PubMed Scopus (852) Google Scholar). The PDX-1-knockout mice are apancreatic and also develop fatal perinatal hyperglycemia (24.Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 37: 606-609Crossref Scopus (1573) Google Scholar), while heterozygous PDX-1-knockout mice have impaired glucose tolerance (38.Dutta S. Bonner-Weir S. Montminy M. Wright C. Nature. 1998; 392: 560Crossref PubMed Scopus (118) Google Scholar). In this study, we examined the effects of c-Myc on insulin gene transcription and found that increased expression of c-Myc in β-cells suppresses the insulin gene transcription by inhibiting NeuroD-mediated transcriptional activation. Islets were isolated from pancreata of 200–250-g male Sprague-Dawley rats (Taconic Farms, Germantown, NY) with collagenase digestion. Briefly, the common bile duct was cannulated and injected with 6 ml of cold M199 medium containing 1.5 mg/ml collagenase (Roche Molecular Biochemicals). The islets were separated on Histopaque 1077 (Sigma) density gradient. The washed islets were picked individually under a dissecting microscope and cultured in RPMI medium (11 mm glucose, supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin sulfate) in a humidified atmosphere of 5% CO2at 37 °C. All animal procedures were approved by the Animal Care Committee of the Joslin Diabetes Center. A recombinant adenovirus expressing c-Myc was prepared using the AdEasy system (kindly provided by Dr. Bert Vogelstein, Johns Hopkins Oncology Center) (39.He T.-C. Zhou S. DaCosta L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3255) Google Scholar). In brief, the c-Myc encoding region (kindly provided by Dr. Richard D. Palmiter, University of Washington) was cloned into the XbaI-EcoRV site of a shuttle vector pAdTrack-CMV. To produce homologous recombination, 1.0 μg of linearized c-Myc-containing plasmid and 0.1 μg of the adenoviral backbone plasmid, pAdEasy-1, were introduced into electrocompetent Escherichia coli BJ5183 cells with electroporation (2,500 V, 200 ohms). Then the resultant plasmid was re-transformed into E. coli XL-Gold Ultracompetent Cells (Stratagene, La Jolla, CA). The plasmid was linearized with PacI and then transfected into the adenovirus packaging cell line 293 using LipofectAMINE (Invitrogen) which was maintained in Dulbecco's modified Eagle's medium. Ten days after transfection, cell lysate was obtained from the 293 cells. The cell lysate was added to 293 cells again, and when most of the cells were killed by the adenovirus infection and detached, cell lysate was obtained again (This process was repeated three times.). Control adenovirus expressing green fluorescent protein (Ad-GFP) was prepared in the same manner. To determine viral titers, confluent 293 cells were infected with a 1:10,000 dilution of the final lysate containing Ad-Myc (with GFP). After an 18-h incubation, the effective titer was determined by the following formula: 107 × the average number of GFP-positive cells per field (×100 magnification), which was considered equivalent to plaque-forming units (PFU)/ml. This number was considered to be proportional to the number of infective particles in the original lysates. Isolated rat islets (∼500 islets) were infected with Ad-Myc (with GFP) or Ad-GFP (without c-Myc), using a 1-h exposure to 50 μl of the adenovirus (1 × 108 PFU/ml). One hour after infection the islets were cultured for 3 days in 3 ml of RPMI medium in 6-cm bacteriologic Petri dishes. Total RNA was extracted from islets using Trizol (Invitrogen). After quantification by spectrophotometry, 500 ng of RNA was heated at 85 °C for 3 min and then reverse-transcribed (RT) into cDNA with 160 μm dNTP, 50 ng of random hexamers, 10 mmdithiothreitol, and 200 units of Superscript II RNase H−reverse transcriptase (Invitrogen). The reactions took place for 10 min at 25 °C, 60 min at 42 °C, and 10 min at 95 °C. Polymerization reactions were performed in a Perkin-Elmer 9700 Thermocycler in a 50-μl reaction volume containing 3 μl of cDNA (20-ng RNA equivalents), 160 μm cold dNTPs, 2.5 μCi of [α-32P]dCTP (3000 Ci/mmol), 10 pmol of appropriate oligonucleotide primers, 1.5 mm MgCl2, and 5 units of AmpliTaq Gold DNA polymerase (PerkinElmer Life Sciences). The oligonucleotide primers and cycle number used for multiplex PCR were as follows. Insulin: (forward) TCT TCT ACA CAC CCA TGT CCC, (reverse) GGT GCA GCA CTG ATC CAC, 15 cycles; GLUT2: (forward) TGG GTT CCT TCC AGT TCG, (reverse) AGG CGT CTG GTG TCG TAT G, 20 cycles; glucokinase: (forward) TGA CAG AGC CAG GAT GGA G, (reverse) TCT TCA CGC TCC ACT GCC, 25 cycles; Kir6.2: (forward) CAT GGA GAA CGG TGT GGG, (reverse) CAG ATA GGA GGT GCG GGC, 25 cycles; SUR: (forward) ATC ACG GAA GGA GGG GAG, (reverse) TTC CGG CTT GTC GAA CTC, 28 cycles; LDH: (forward) ACA GTT GTT GGG GTT GGT G, (reverse) CCG GCT CTC TCC CTC TTG, 25 cycles; c-Myc: (forward) AGT TGG ACA GTG GCA GGG, (reverse) ACA GGA TGT AGG CGG TGG, 28 cycles; PDX-1: (forward) CGG ACA TCT CCC CAT ACG, (reverse) AAA GGG AGA TGA ACG CGG, 20 cycles; NeuroD: (forward) GCA AAG GTT TGT CCC AGC, (reverse) ACG TGG AAG ACG TGG GAG, 20 cycles. The thermal cycle profile employed a 10-min denaturing step at 94 °C followed by the number of cycles and an extension step of 10 min at 72 °C. In each step, the gene products of interest were amplified with an internal control gene (rRNA, cyclophilin, α-tubulin, or TATA-binding protein (TBP)) to correct for experimental variations between samples. The oligonucleotide primers were as follows. Cyclophilin: (forward) AAC CCC ACC GTG TTC TTC, (reverse) TGC CTT CTT TCA CCT TCC C; α-tubulin: (forward) CTC GCA TCC ACT TCC CTC, (reverse) ATG CCC TCA CCC ACG TAC; TBP: (forward) ACC CTT CAC CAA TGA CTC CTA TG, (reverse) ATG ATG ACT GCA GCA AAT CGC. The primers for rRNA was purchased from AMBION (Austin, TX). The amplimers were separated on a 6% polyacrylamide gel in 1 × TBE buffer (45 mm Tris-HCl, 45 mmboric acid, 1 mm EDTA). The gel was dried, and the amount of [α-32P]dCTP incorporated into each amplimer was measured with a PhosphorImager and quantified with the ImageQuant software (Molecular Dynamics, Sunnyvale, CA). To ensure the validity of the measurement of mRNA levels by semiquantitative-radioactive multiplex PCR, control experiments were performed using normal rat islet cDNA to show that the amount of each amplimer obtained in a multiplex PCR was independent of the presence of other primers, excluding the possibility of strong interferences between primers as we reported previously (5.Jonas J.-C. Sharma A. Hasenkamp W. Iikova H. Patane G. Laybutt R. Bonner-Weir S. Weir G.C. J. Biol. Chem. 1999; 274: 14112-14121Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). In brief, the number of cycles was adjusted to be in the exponential phase of the amplification of each product, and we verified that the amount of each PCR product in a multiplex reaction increases linearly with the amount of starting cDNA (5–40-ng RNA equivalents), ensuring that changes in the ratio of PCR product to control gene product truly reflect a change in mRNA abundance of that gene relative to the control gene. Whole cell extracts were obtained from islets infected with Ad-Myc or Ad-GFP or uninfected islets. After treatment with lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40), supernatants were collected. Ten micrograms of cell extracts were fractionated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membranes (Immun-BlotTM PVDF Membrane, Bio-Rad) using transfer buffer containing 20% methanol, 25 mm Tris base, and 192 mm glycine (300 mA, 2 h). After blocking the membranes at room temperature for 1 h in 50 mm Tris-HCl, 150 mm NaCl, 0.1% Tween 20 with 5% nonfat dry milk, the membranes were incubated at 4 °C overnight in TBS buffer (50 mm Tris-HCl, 150 mm NaCl) containing a 1:1000 dilution of rabbit anti-c-Myc antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and washed three times in TBS buffer with 0.1% Tween 20 (TBS-T). The membranes were then incubated for 1 h at room temperature in TBS containing a 1:1000 dilution of anti-rabbit IgG antibody (Bio-Rad) coupled to horseradish peroxidase, followed by three 10-min washings with TBS-T. Immunoreactive bands were made visible by incubation with LumiGLO (Cell Signaling, Beverly, MA) and exposed to x-ray film (Eastman Kodak Co.). Glucose-stimulated insulin secretion was determined by static incubation. Isolated rat islets (50 islets) were preincubated for 30 min in 2 ml of HEPES-balanced Krebs-Ringer bicarbonate buffer and then incubated for 60 min in the same buffer supplemented with 0.5% bovine serum albumin and either 2.8 mm or 16.7 mm glucose. The insulin secreted into the medium was determined with a radioimmunoassay kit (Linco Research, St. Charles, MO) using rat insulin as the standard. Islets were fixed for 60 min in 10% buffered formalin, enrobed in 2% agar to keep the pellet together through processing and embedding, immersed in the same fixative for another 90 min, washed, and stored in 0.1 m phosphate buffer until routine embedding in paraffin. Sections of these pellets were incubated at 4 °C overnight with guinea pig anti-insulin antibody (Linco Research) diluted 1:100 in PBS containing 1% bovine serum albumin and then incubated for 1 h at room temperature with Texas Red-conjugated donkey anti-guinea pig IgG (1:100) (Jackson Immunochemicals). Nuclear extracts were obtained from uninfected islets and islets (∼500 islets) infected with Ad-Myc and Ad-GFP. Cells were treated with 1 ml of hypotonic buffer (20 mm HEPES, pH 7.9, 20 mm NaF, 1 mmEDTA, 1 mm EGTA, 1 mmNa4P2O7, 1 mmNa3VO4, 1 mm dithiothreitol); then 50 μl of high salt buffer (420 mm NaCl and 20% glycerol in hypotonic buffer) was added to the pellet, followed by 1 h of incubation at 4 °C. The supernatants were used as nuclear extracts. Two micrograms of nuclear extract were incubated with 2 μg of poly(dI-dC) at room temperature in 20 μl of reaction buffer. The reaction buffers for binding to A-box and E-box were Buffer 1 (10 mm HEPES, pH 7.8, 0.1 mm EDTA, 75 mm KCl, 2.5 mm MgCl2, 1 mm dithiothreitol, 3% Ficoll) and Buffer 2 (25 mm HEPES, pH 7.8, 0.2 mm EDTA, 150 mm KCl, 5 mm dithiothreitol, 9% glycerol), respectively. The binding reaction was initiated by adding32P-labeled double-stranded oligonucleotide probes. Double-stranded oligonucleotides reproducing the rat insulin gene PDX-1 and NeuroD binding region and surrounding sequences (ACG TCC TCT TAA GAC TCT AAT TAC CCT ACG T and ACG TTC TGG CCA TCT GCT GAT CCT ACG T) were used as binding probes. In some of the binding assays, anti-PDX-1 antiserum (30.Watada H. Kajimoto Y. Umayahara Y. Matsuoka T. Kaneto H. Fujitani Y. Kamada Y. Kawamori R. Yamasaki Y. Diabetes. 1996; 45: 1478-1488Crossref PubMed Google Scholar) or anti-NeuroD antibody (Santa Cruz) was added to the reaction mixture 1 h before addition of the DNA probes. After the binding reactions, samples were analyzed by separation on 6% polyacrylamide gel in 1 × TBE buffer (45 mm Tris-HCl, 45 mm boric acid, 1 mm EDTA) followed by autoradiography. βTC1, HIT-T15, HepG2, and HeLa cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin sulfate in a humidified atmosphere of 5% CO2 at 37 °C. MIN6 cells were grown in the same conditions except for using Dulbecco's modified Eagle's medium (25 mm glucose) and 20% fetal calf serum. The cells were replated 24 h before transfection; then 1.0 μg of c-Myc expression plasmid (or the empty vectors), 1.0 μg of rat insulin II promoter-reporter (luciferase) plasmid (40.Sharma A. Stein R. Mol. Cell. Biol. 1994; 14: 871-879Crossref PubMed Google Scholar) containing 238-bp 5′-flanking sequences of the rat insulin II promoter region, 1.0 μg of the PDX-1 (41.Peshavaria M. Henderson E. Sharma A. Wright C.V. Stein R. Mol. Cell. Biol. 1997; 17: 3987-3996Crossref PubMed Scopus (85) Google Scholar) and NeuroD (40.Sharma A. Stein R. Mol. Cell. Biol. 1994; 14: 871-879Crossref PubMed Google Scholar) expression plasmids (or the empty vectors), and 0.5 μg of pSV-β-galactosidase control vector (Promega, Madison, WI) were co-transfected into the cells with the LipofectAMINE reagent (Invitrogen) using the conditions recommended by the manufacturer. NeuroD:c-Myc-TAD (amino acids 1–143) and c-Myc:NeuroD-TAD (amino acids 189–355) fusion plasmids were made by PCR using NeuroD and c-Myc expression plasmids as templates; the resulting clones were verified by sequencing. These plasmids were transfected as above. For transfection into primary islets, 1.0 μg of c-Myc expression plasmid (or the empty vectors), 1.0 μg of rat insulin I promoter-reporter (CAT) plasmid (kindly provided by Dr. Michael German, University of California-San Francisco) (18.German M.S. Wang J. Mol. Cell. Biol. 1994; 14: 4067-4075Crossref PubMed Scopus (150) Google Scholar) containing the 345-bp 5′-flanking sequences of the rat insulin I promoter region, and 0.5 μg of pSV-β-galactosidase control vector were co-transfected into the islets (∼100 islets) as described previously (42.Sander M. Griffen S.C. Huang J. German M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11572-11577Crossref PubMed Scopus (46) Google Scholar) with some modification. In brief, the plasmid DNA mixture was incubated with 10 μl of LipofectAMINE reagent, 100 μl of control adenovirus (Ad-GFP) (1 × 108 PFU/ml), and 100 μl of OPTI-MEM medium for 30 min, and then the DNA/LipofectAMINE/adenovirus mixture was then added to the cells. Forty eight hours after the transfections, cells were harvested for luciferase and β-galactosidase assays. Preparations of cellular extracts were assayed using a luciferase assay system (Promega) and a CAT assay system (Promega). For the luciferase assay, light emission was measured with Monolight 3010 Luminometer (BD PharMingen, San Diego, CA). β-Galactosidase assays were performed with the β-galactosidase enzyme assay system (Promega). The luciferase and CAT results were normalized with respect to transfection efficiency assessed from the results of the β-galactosidase assays. All results are presented as mean ± S.E. in three or four independent experiments. Statistical analysis was performed using the unpaired Student's t test. To evaluate the possible implications of c-Myc induction in β-cell dysfunction, isolated rat pancreatic islets from Sprague-Dawley rats were infected with c-Myc-expressing adenovirus (Ad-Myc) or control adenovirus (Ad-GFP). Fig. 1A shows representative islets 3 days after infection with Ad-Myc. Total RNA was isolated 3 days after infection and used for RT-PCR followed by densitometric analyses. As shown in Fig. 1B, infection with Ad-Myc led to an increase in c-myc mRNA expression in islets as compared with uninfected cells or cells infected with Ad-GFP. Also, as shown in Fig. 1C, induction of c-Myc protein level was confirmed by Western blotting. To examine the effects of c-Myc overexpression on glucose-stimulated insulin secretion, static incubations were performed using the islets exposed to Ad-Myc (or Ad-GFP). Three days after infection with Ad-Myc, glucose-stimulated insulin secretion was decreased, although adenovirus infection itself (Ad-GFP) did not affect insulin secretion (Fig. 1D). To examine whether the insulin protein level is decreased by c-Myc overexpression, immunostaining for insulin was performed after infection with Ad-Myc (or Ad-GFP). Fig. 2shows representative immunostaining 3 days after infection with the adenovirus. As shown in Fig. 2A, many islet cells were c-Myc (GFP)-positive. The percentage of non-β-cells was quite low in islets after infection with either Ad-Myc or Ad-GFP (Fig. 2A,lower panel). It should be pointed out that the mantle pattern for non-β-cells is somewhat different from the more even distribution found in sections of pancreas. It appears that some mantle cells are lost and even redistributed during the process of islet isolation with collagenase and subsequent culture. Therefore, most of the infected cells are likely to be β-cells. As shown in Fig. 2A, upper panel, insulin expression was clearly detected in islets after Ad-GFP infection (right panel) but was markedly reduced after Ad-Myc infection (middle panel). These results clearly suggest that c-Myc overexpression, but not adenovirus infection itself, suppresses the insulin expression. There was no obvious difference in non-β-cell hormone staining between Ad-Myc (Fig. 2A, lower, middle panel) and Ad-GFP group (right panel), although we cannot deny the possibility that c-Myc has some less obvious effects on non-β-cells. Furthermore, as shown in Fig. 2B, upper panel, insulin immunostaining was markedly reduced in the cells expressing c-Myc, whereas non-β-cell hormones (glucagon, somatostatin, pancreatic polypeptide) were detected even in c-Myc-overexpressing cells (Fig. 2B, lower panel). Therefore, we assume that c-Myc overexpression suppresses insulin biosynthesis, leading to lower insulin content and the secretion shown in Fig. 1D. To evaluate the possible implications of c-Myc induction for β-cell dysfunction, we examined the effects of c-Myc on β-cell-associated gene expression. As shown in Fig. 3A, 3 days after the infection, total RNA was isolated and used for RT-PCR followed by densitometric analyses. The amount of insulin mRNA was remarkably decreased in the c-Myc-overexpressing islets whereas no decrease was observed in the Ad-GFP-infected cells. GLUT2 and glucokinase mRNA levels were also moderately decreased by c-Myc overexpression, whereas the expression levels of other β-cell-specific genes,Kir6.2 and SUR, did not change. Additionally, lactate dehydrogenase, which is known to be transactivated by c-Myc, was induced in islets exposed to Ad-Myc. To explore the mechanism for c-Myc-mediated reduction of insulin gene expression, we examined expression levels of NeuroD and PDX-1, both of which are very important transcription factors for insulin gene expression. As shown in Fig. 3B, the expression level of NeuroD was not affected by c-Myc overexpression. The PDX-1 expression level showed a tendency to be r" @default.
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• W2071978660 title "Induction of c-Myc Expression Suppresses Insulin Gene Transcription by Inhibiting NeuroD/BETA2-mediated Transcriptional Activation" @default.
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