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• W2020804923 abstract "The role of insulin signaling in pancreatic β cells has become increasingly apparent. Stably transformed insulinoma cell lines (MIN6) were created with small interfering RNA resulting in the reduction of insulin receptor (IR) expression up to 80% (insulin receptor knockdown, IRKDΔ80). Functionally perturbed IR signaling was confirmed with the absence of insulin-stimulated insulin receptor substrate 1 tyrosine phosphorylation. Additionally, Akt phosphorylation was reduced and responded poorly to glucose stimulation. Gene expression profiling revealed that reduced IR expression was associated with alterations in expression of >1,500 genes with diverse functions. IRKD cells exhibited low rate of proliferation due to delay in transition from G0/G1 to S phase, whereas susceptibility to apoptosis did not differ from that of control cells. Insulin content was reduced in proportion to the reduction of IR. IRKD cells maintained glucose responsiveness as measured by NAD(P)H generation, whereas Ca2+ responses and insulin secretion were enhanced. IRKDΔ80 and control cells were treated with glucose (25 mm) or insulin (100 nm) for 45 min, and gene expression profiles were assessed. Transcriptional activation of several hundred early response genes common to both glucose and insulin stimulation was observed in control cells. In IRKDΔ80 cells, insulin failed to activate any genes as anticipated. Importantly, glucose stimulation of gene expression in IRKDΔ80 cells showed that most genes previously activated by glucose were no longer activated, suggesting a major autocrine/paracrine effect of insulin on glucose-regulated gene expression. On the other hand, there were a number of glucose-regulated genes in the IRKDΔ80 cells that were not previously observed in control cells, suggesting a feedback regulation of insulin signaling on glucose-regulated gene expression. These results demonstrate important roles of the insulin receptor in islet β cell gene expression and function and may serve to elucidate molecular defects in animal models with diminished β cell insulin signaling. The role of insulin signaling in pancreatic β cells has become increasingly apparent. Stably transformed insulinoma cell lines (MIN6) were created with small interfering RNA resulting in the reduction of insulin receptor (IR) expression up to 80% (insulin receptor knockdown, IRKDΔ80). Functionally perturbed IR signaling was confirmed with the absence of insulin-stimulated insulin receptor substrate 1 tyrosine phosphorylation. Additionally, Akt phosphorylation was reduced and responded poorly to glucose stimulation. Gene expression profiling revealed that reduced IR expression was associated with alterations in expression of >1,500 genes with diverse functions. IRKD cells exhibited low rate of proliferation due to delay in transition from G0/G1 to S phase, whereas susceptibility to apoptosis did not differ from that of control cells. Insulin content was reduced in proportion to the reduction of IR. IRKD cells maintained glucose responsiveness as measured by NAD(P)H generation, whereas Ca2+ responses and insulin secretion were enhanced. IRKDΔ80 and control cells were treated with glucose (25 mm) or insulin (100 nm) for 45 min, and gene expression profiles were assessed. Transcriptional activation of several hundred early response genes common to both glucose and insulin stimulation was observed in control cells. In IRKDΔ80 cells, insulin failed to activate any genes as anticipated. Importantly, glucose stimulation of gene expression in IRKDΔ80 cells showed that most genes previously activated by glucose were no longer activated, suggesting a major autocrine/paracrine effect of insulin on glucose-regulated gene expression. On the other hand, there were a number of glucose-regulated genes in the IRKDΔ80 cells that were not previously observed in control cells, suggesting a feedback regulation of insulin signaling on glucose-regulated gene expression. These results demonstrate important roles of the insulin receptor in islet β cell gene expression and function and may serve to elucidate molecular defects in animal models with diminished β cell insulin signaling. Pancreatic β cells dynamically adapt to their environment (1Bell G.I. Polonsky K.S. Nature. 2001; 414: 788-791Crossref PubMed Scopus (412) Google Scholar, 2Kahn S.E. J. Clin. Endocrinol. Metab. 2001; 86: 4047-4058Crossref PubMed Scopus (583) Google Scholar). Changes in plasma glucose concentration along with various hormones and growth factors have been shown to be major determinants of insulin secretion, biosynthesis, and islet mass (3Hugl S.R. White M.F. Rhodes C.J. J. Biol. Chem. 1998; 273: 17771-17779Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 4Cousin S.P. Hugl S.R. Myers Jr., M.G. White M.F. Reifel-Miller A. Rhodes C.J. Biochem. J. 1999; 344: 649-658Crossref PubMed Scopus (81) Google Scholar). The islet β cell responds to these changes in its environment by altering its transcriptional responses, and these changes in gene expression ultimately result in altered mass and function. However, the signaling pathways activated by these environmental changes and the ensuing transcriptional events that mediate these biological responses are only beginning to be elucidated. Specifically, the relationships between the signaling pathways activated by glucose and those activated by growth factors such as insulin remain unclear. The roles of insulin as a growth factor in the modulation of β cell mass and function have been implied by the results of recent experiments. Glucose stimulation of β cells in culture has been shown to result in the activation of the IR as does the application of exogenous insulin, suggesting that insulin secreted from β cells binds to its receptor eliciting a physiological response (5Rothenberg P.L. Willison L.D. Simon J. Wolf B.A. Diabetes. 1995; 44: 802-809Crossref PubMed Google Scholar, 6Xu G.G. Rothenberg P.L. Diabetes. 1998; 47: 1243-1252PubMed Google Scholar). There have been many in vivo as well as in vitro studies attempting to clarify the roles of insulin signaling in β cell function; however, no consensus has yet been achieved (reviewed in Ref. 7Leibiger I.B. Leibiger B. Berggren P.O. FEBS Lett. 2002; 532: 1-6Crossref PubMed Scopus (89) Google Scholar and references therein). Some have suggested that insulin inhibits glucose-stimulated insulin secretion (8Elahi D. Nagulesparan M. Hershcopf R.J. Muller D.C. Tobin J.D. Blix P.M. Rubenstein A.H. Unger R.H. Andres R. N. Engl. J. 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Chem. 1999; 71: 5551-5556Crossref PubMed Scopus (38) Google Scholar, 14Xu G.G. Gao Z.Y. Borge Jr., P.D. Jegier P.A. Young R.A. Wolf B.A. Biochemistry. 2000; 39: 14912-14919Crossref PubMed Scopus (54) Google Scholar, 15Srivastava S. Goren H.J. Diabetes. 2003; 52: 2049-2056Crossref PubMed Scopus (26) Google Scholar, 16Kulkarni R.N. Bruning J.C. Winnay J.N. Postic C. Magnuson M.A. Kahn C.R. Cell. 1999; 96: 329-339Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 17Kulkarni R.N. Winnay J.N. Daniels M. Bruning J.C. Flier S.N. Hanahan D. Kahn C.R. J. Clin. Investig. 1999; 104: R69-R75Crossref PubMed Scopus (239) Google Scholar, 18Otani K. Kulkarni R.N. Baldwin A.C. Krutzfeldt J. Ueki K. Stoffel M. Kahn C.R. Polonsky K.S. Am. J. Physiol. 2004; 286: E41-E49Crossref PubMed Scopus (146) Google Scholar). Most recently, Da Silva Xavier et al. (19Da Silva Xavier G. Qian Q. Cullen P.J. Rutter G.A. Biochem. J. 2004; 377: 149-158Crossref PubMed Scopus (69) Google Scholar) reported no glucose-stimulated insulin secretion in the absence of insulin signaling. Furthermore, the potential importance of autocrine/paracrine insulin signaling in pancreatic β cells, especially on β cell mass, was highlighted by the β cell-specific insulin receptor (IR) 1The abbreviations used are: IR, insulin receptor; βIRKO, β cell-specific insulin receptor knock-out; siRNA, small interfering RNA; FBS, fetal calf serum; IRKD, insulin receptor knockdown; RT, reverse transcription; qRT, quantitative RT; CI, confidence intervals; BrdUrd, bromodeoxyuridine; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; PI3-kinase, phosphatidylinositol 3-kinase; ERK, extracellular signal-regulated kinase; IRS, insulin receptor substrate. knock-out (βIRKO) mouse model (16Kulkarni R.N. Bruning J.C. Winnay J.N. Postic C. Magnuson M.A. Kahn C.R. Cell. 1999; 96: 329-339Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar) as well as in IR substrate 2 (IRS-2) null mice (11Kubota N. Tobe K. Terauchi Y. Eto K. Yamauchi T. Suzuki R. Tsubamoto Y. Komeda K. Nakano R. Miki H. Satoh S. Sekihara H. Sciacchitano S. Lesniak M. Aizawa S. Nagai R. Kimura S. Akanuma Y. Taylor S.I. Kadowaki T. Diabetes. 2000; 49: 1880-1889Crossref PubMed Scopus (430) Google Scholar, 20Withers D.J. Gutierrez J.S. Towery H. Burks D.J. Ren J.M. Previs S. Zhang Y. Bernal D. Pons S. Shulman G.I. Bonner-Weir S. White M.F. Nature. 1998; 391: 900-904Crossref PubMed Scopus (1339) Google Scholar), both of which developed impairment of β cell growth and overt glucose intolerance. However, in the case of the βIRKO mouse model, the conclusion that insulin signaling in the β cell is important for controlling β cell mass is confounded by hypothalamic reduction of the insulin receptor with concomitant tendency to obesity and insulin resistance that may have secondary harmful effects on the β cell. Additionally, Wicksteed et al. (21Wicksteed B. Alarcon C. Briaud I. Lingohr M.K. Rhodes C.J. J. Biol. Chem. 2003; 278: 42080-42090Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) recently demonstrated that neither endogenous or exogenous insulin affected insulin secretion, proinsulin translation, preproinsulin mRNA levels, or total protein synthesis in primary cultures of rodent islets for either short (1 h) or long (24 h) incubation. Thus together these studies leave open the question of whether insulin, acting through autocrine/paracrine effects, is a modulator of β cell function and mass. Because it is difficult to dissect molecular defects in animal models that result in reduction of the small amount of islet tissue, we approached this problem by studying glucose-responsive insulinoma cells in culture. In these studies, we sought to define the roles of insulin signaling through its receptor in β cell and the effects of glucose on these processes. Creation of stable insulinoma cell lines expressing small interfering RNA (siRNA) to reduce the synthesis of the IR allowed us to examine the effects of reduced IR signaling on β cell function. Stable reduction of IR led to alterations in expression of an unexpected number of genes. Additionally, we observed a role of the IR in proliferation, insulin content, glucose-stimulated insulin secretion, and glucose-regulated early gene expression. Construction of siRNA Expressing Plasmids—A polIII-mediated small interfering RNA expressing plasmid vector system (pSUPER vector) was constructed according to the methods described by Brummelkamp et al. (22Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3968) Google Scholar). 64-base DNA oligonucleotides corresponding to sense target sequence, hairpin loop, and antisense target sequence were synthesized (Integrated DNA Technologies, Coraville, IA), annealed together, and then ligated into BglII and HindIII digested pSUPER vector. Plasmids were purified with Wizard Plus Maxipreps kit (Promega, Madison, WI). A target sequence against mouse IR was found, 5′-ACTGCATGGTTGCCCATGA-3′, which yielded a satisfactory loss of function. A control oligonucleotide with the same GC content and no corresponding mammalian gene, 5′-GCTACAGTAGACGGAATCG-3′, was utilized as a scrambled control. Cell Culture, Transfection of Insulinoma Cells, and Selection of Stably Transfected Clones—The MIN6 insulinoma cell line was a kind gift of Dr. Jun-ichi Miyazaki (Osaka University, Osaka, Japan). MIN6 cells were maintained in Dulbecco's modified Eagle's medium containing 25 mm glucose supplemented with 15% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml l-glutamine, and 5 μl/liter β-mercaptoethanol in humidified 5% CO2, 95% air at 37 °C (23Miyazaki J. Araki K. Yamato E. Ikegami H. Asano T. Shibasaki Y. Oka Y. Yamamura K. Endocrinology. 1990; 127: 126-132Crossref PubMed Scopus (1054) Google Scholar). Parental MIN6 cells used for plasmid transfection were between passages 24 and 26. Polyamine transfection reagent TransIT™-LT1 (Mirus Corporation, Madison, WI) was used to transfect the insulinoma cells according to the company's instructions. A total of 10 μg of pSUPER vector and 40 μlof TransIT-LT1 were used for each 10-cm plates. 1 μg of pCDNA 3.1 plasmid, which contains a neomycin cassette, was also transfected to each plate at the same time to facilitate selecting stably transfected cells with G418 (a neomycin derivative). The transfected cells were first selected with culture medium containing 500 μg/ml G418 (Mediatech, Herndon, VA) for 4 weeks, and then isolated colonies of the surviving cells (defined as passage 4) were transferred to a 6-well tissue culture plate and maintained in culture medium with 200 μg/ml G418. Protein levels and mRNA expression were tested at passage 7–8, and clones were further maintained. MIN6 cells transfected with empty pSUPER vector were designated as Con(E), those with scrambled target sequence were referred to as Con(S), and those with reduced IR expression were designated as insulin receptor knockdown (IRKD). Con(E), Con(S), and IRKD cells were propagated by weekly passage and were used for experiments herein between passages 9 and 18, which corresponded to passages 33 and 42 of parental MIN6 cells. Quantitative RT-PCR (qRT-PCR)—Total RNA was purified, and 1 μg was used to prepare cDNA, primed with random hexamers, and reverse-transcribed with Superscript II (Invitrogen) according to the manufacturer's protocol. qRT-PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye (ABI) as described previously (24Morrison T.B. Weis J.J. Wittwer C.T. BioTechniques. 1998; 24: 954-962PubMed Google Scholar, 25Wittwer C.T. Herrmann M.G. Moss A.A. Rasmussen R.P. BioTechniques. 1997; 22: 130-138Crossref PubMed Scopus (1137) Google Scholar) using the ABI 7000 sequence detection system (Applied Biosystems). For comparison of transcript levels between samples, a standard curve of cycle thresholds for serial dilutions of a cDNA sample was established and then used to calculate the relative abundance of each gene. Values were then normalized to the relative amounts of 18 S ribosomal RNA, which were obtained from a similar standard curve. This control was chosen after observing that 18 S rRNA levels correlated well with cyclophilin and tubulin mRNA levels when MIN6 cells were subject to different glucose concentrations for various durations including ones used in these studies. All of the PCR reactions were performed as at least replicates of four. Standard error of the quantity of transcript normalized to the amount of 18 S ribosomal RNA was calculated from a formula with consideration of error propagation. When gene expression levels of two conditions were compared, the ratio was expressed with standard error calculated from the same formula. Specificity of each primer pair was confirmed by melting curve analysis and agarose-gel electrophoresis of PCR products. Sequences of primers used in this study are as follows: insulin receptor, forward 5′-TTTGTCATGGATGGAGGCTA-3′ and reverse 5′-CCTCATCTTGGGGTTGAACT-3′; immediate early response 2 (Ier2), forward 5′-CAGCGATTTGAGCGACAGTA-3′ and reverse 5′-GGGTCCACAGTTCAGGAGAC-3′; early growth response 1 (Egr1), forward 5′-CGAATCTGCATGCGTAACTT-3′ and reverse 5′-GCAAACTTCCTCCCACAAAT-3′; inhibitor of DNA binding 2 (Id2), forward 5′-GGACGACCCGATGAGTCT-3′ and reverse 5′-TGCTGGGCACCAGTTCCTT-3′; transducer of ErbB-2.1 (Tob1), forward 5′-GAAGAATAGTGGCCGTAGCA-3′ and reverse 5′-TTCAGGAGGTGGTTCACATT-3′; DNA segment, Chr4, Wayne State University 53, forward 5′-TTCGCCTGAGTGAGAAAGAT-3′ and reverse 5′-CAAGTCGAAGTTGGCTGTTC-3′; cyclin E1, forward 5′-TCGTTACATGGCATCACAAC-3′ and reverse 5′-AAACTGGTGCAACTTTGGAG-3′; Foxo3, forward 5′-ACTCCAAGACCTGCTTGCTT-3′ and reverse 5′-GGTGCTAGCCTGAGACATCA-3′; p21CIP1, forward 5′-CCTGACAGATTTCTATCACTCCA-3′ and reverse 5′-CAGGCAGCGTATATCAGGAG-3′; and cyclin D2, forward 5′-TTTCCTCTGGCCATGAATTA-3′ and reverse 5′-CAGCTTGGAAGCTAGGAACA-3′. Immunoprecipitation and Western Blotting—Cells were incubated in medium containing 5 mm glucose and 2% FBS for 18 h (defined as “unstimulated” state) followed by treatment of 25 mm glucose or 100 nm insulin for indicated duration of time. Cells were lysed with buffer (PBS, 1% Triton X-100, 1 mm EDTA, 1 mm EGTA, 0.01 m dithiothreitol, 1 mm Na3VO4, and a tablet of Complete protease inhibitor mixture (Roche Applied Science)). For IRS-1 immunoprecipitation, 100 μg of cell lysates were subjected to immunoprecipitation with anti-IRS-1 rabbit polyclonal antibody (BD Transduction Laboratories, San Diego, CA) and immobilized on protein G-Sepharose beads. For Western blotting, total proteins or immunoprecipitates were separated by electrophoresis though 10% polyacrylamide, 0.1% SDS gels and transferred to polyvinylidene difluoride membranes followed by immunoblotting. Immunodetection was performed with Western Lightning (PerkinElmer Life Sciences) following the manufacturer's protocol. Antibodies used in this study were as follows: anti-insulin receptor β subunit (BD Transduction Laboratories); anti-β actin (Sigma); anti-phosphospecific (Ser-473) Akt; anti-Akt; and anti-phosphotyrosine (Cell Signaling, Boston, MA). Labeling of RNA Transcripts for Microarray, Hybridization, and Data Acquisition—First strand cDNA was generated by oligo(dT)-primed reverse transcription (Superscript II; Invitrogen) utilizing the 3DNA Array 50 kit (Genisphere) (26Manduchi E. Scearce L.M. Brestelli J.E. Grant G.R. Kaestner K.H. Stoeckert Jr., C.J. Physiol. Genomics. 2002; 10: 169-179Crossref PubMed Scopus (56) Google Scholar). Modified oligo(dT) primers were utilized in which a fluorophore/dendrimer-specific oligonucleotide sequence was attached to the 5′ end of the dT primer. Two hybridizations were carried out in a sequential manner. The primary hybridization was performed by adding 38 μl of sample to the microarray under a supported glass coverslip (Erie Scientific) at 50 °C for 16–20 h at high humidity in the dark. Secondary hybridizations were carried out using the complimentary capture reagents provided in the 3DNA Array 50 kit. Slides were scanned on a PerkinElmer ScanArray Express HT scanner to detect Cy3 and Cy5 fluorescence. Laser power was kept constant for Cy3/Cy5 scans, and photomultiplier tube was varied for each experiment based on background fluorescence. Gridding and analysis of images were performed using QuantArray (PerkinElmer Life Sciences). Each spot was defined on a pixel-by-pixel basis using a modified Mann-Whitney statistical test. Microarray Sample Pairing—To assess gene expression alteration by comparing two conditions, transcripts from each sample were directly compared using EPCon-pancreatic cDNA microarray with ∼11,000 probes (27Kaestner K.H. Lee C.S. Scearce L.M. Brestelli J.E. Arsenlis A. Le P.P. Lantz K.A. Crabtree J. Pizarro A. Mazzarelli J. Pinney D. Fischer S. Manduchi E. Stoeckert Jr., C.J. Gradwohl G. Clifton S.W. Brown J.R. Inoue H. Cras-Meneur C. Permutt M.A. Diabetes. 2003; 52: 1604-1610Crossref PubMed Scopus (50) Google Scholar). For each sample pair, transcripts from one condition were labeled with either Cy3 or Cy5 and hybridized with the Cy5- or Cy3-labeled transcripts from the partner condition. Another pair of RNAs was also labeled-inverting dyes. Those pairs of hybridization were repeated twice. When unstimulated MIN6-Con and IRKDΔ80 cells were compared, four microarrays were used. For assessing transcriptional responses either to glucose or insulin treatment, a total of eight microarrays per cell line were used. Microarray Normalization and Statistical Analysis—In the microarray studies performed herein, normalization and statistical analyses were performed as follows. Only probes with a signal intensity of 2.0-fold or greater above the corresponding background intensity in both channels were chosen for calculation of fold changes. The intensity of each spot was adjusted by subtracting background intensities. The log ratio of Cy5 and Cy3 channel intensity of each spot was then calculated, and the median of the log ratio from all of the probes was subtracted from each log ratio (28Quackenbush J. Nat. Genet. 2002; 32: 496-501Crossref PubMed Scopus (1501) Google Scholar). The log ratios from four pairs of “dye flip” hybridizations were utilized to estimate probe-specific measurement variation (29Yang I.V. Chen E. Hasseman J.P. Liang W. Frank B.C. Wang S. Sharov V. Saeed A.I. White J. Li J. Lee N.H. Yeatman T.J. Quackenbush J. Genome Biol. 2002; 3: 062.1-062.12Google Scholar). Probe-specific measurement variation was subtracted from log fold change to calculate the final normalized log fold change (probe-specific normalization). From the pairing scheme, four direct ratios were obtained for each comparison. These ratios were used to calculate the average fold change as well as the mean ± S.E. 95% confidence intervals (CI, two standard errors away from the average fold change) were calculated with attention to the change of distribution when converting log fold change to fold change (30Chen Y. Kamat V. Dougherty E.R. Bittner M.L. Meltzer P.S. Trent J.M. Bioinformatics. 2002; 18: 1207-1215Crossref PubMed Scopus (158) Google Scholar). Criteria were set to assess changes in gene expression that included a fold change of 1.35 or more compared with “unstimulated.” This criterion was selected after observing that the mean variance of all of the probes was 0.15, and the false positive rate was estimated to be 0.0032%. With similar multiple hybridizations and cDNA microarrays, the fold change of 1.3–1.4 was utilized to identify significant gene expression change in other studies (31Woo A.L. Gildea L.A. Tack L.M. Miller M.L. Spicer Z. Millhorn D.E. Finkelman F.D. Hassett D.J. Shull G.E. J. Biol. Chem. 2002; 277: 49036-49046Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 32Dunckley T. Lukas R.J. J. Biol. Chem. 2003; 278: 15633-15640Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 33Ferrante Jr., A.W. Thearle M. Liao T. Leibel R.L. Diabetes. 2001; 50: 2268-2278Crossref PubMed Scopus (36) Google Scholar). Additionally, significant fold changes required that the 95% CI of the fold change, two standard errors away from the fold change, excluded 1. Thus any fold changes meeting these criteria could be considered significant with a p value of <0.05. Microarray Annotation and Gene Ontology Functions—Annotation for the clones on the arrays were performed as reported previously using BLASTn analysis against the public databases (34Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70762) Google Scholar, 35Wheeler D.L. Church D.M. Lash A.E. Leipe D.D. Madden T.L. Pontius J.U. Schuler G.D. Schriml L.M. Tatusova T.A. Wagner L. Rapp B.A. Nucleic Acids Res. 2002; 30: 13-16Crossref PubMed Scopus (196) Google Scholar). Gene ontology functions were gathered through the Source Repository (source.stanford.edu/) (36Diehn M. Sherlock G. Binkley G. Jin H. Matese J.C. Hernandez-Boussard T. Rees C.A. Cherry J.M. Botstein D. Brown P.O. Alizadeh A.A. Nucleic Acids Res. 2003; 31: 219-223Crossref PubMed Scopus (354) Google Scholar) and classified according to the categories provided by the Gene Ontology Consortium (www.geneontology.org/) (37Ashburner M. Ball C.A. Blake J.A. Botstein D. Butler H. Cherry J.M. Davis A.P. Dolinski K. Dwight S.S. Eppig J.T. Harris M.A. Hill D.P. Issel-Tarver L. Kasarskis A. Lewis S. Matese J.C. Richardson J.E. Ringwald M. Rubin G.M. Sherlock G. Nat. Genet. 2000; 25: 25-29Crossref PubMed Scopus (27278) Google Scholar). Hierarchical Clustering—Hierarchical clusters were performed using the Genesis software version 1.3 (Institute for Biomedical Engineering, Graz University of Technology, Graz, Austria) (38Sturn A. Quackenbush J. Trajanoski Z. Bioinformatics. 2002; 18: 207-208Crossref PubMed Scopus (1475) Google Scholar). Heat maps generated by hierarchical clustering were created using the average linkage clustering (Euclidean distances) directly from the logarithmic ratios (base e), and the intensity of those colors indicates the degree of fold changes in natural log scale (above 1.5 or below -1.5 fold changes are saturated). BrdUrd Incorporation—Two days before the procedure, cells were seeded onto 25-mm glass coverslips at a density of 2 × 106 cells/ml. Medium was changed once 24 h before the procedure, and then during the last hour of culturing, BrdUrd (10 μm) was added to the medium. Detection of BrdUrd was carried out with immunohistochemistry utilizing anti-BrdUrd monoclonal antibody according to the manufacturer's recommendations (Roche Diagnostics, Indianapolis, IN). Cells were stained with 1 μg/ml 4′,6-diamidino-2-phenylindole for identifying nucleus. At least four different fields from each coverslip were selected to count at least 2,000 cells to calculate the rate of BrdUrd-positive cells. Shown are the results from six independent samples from each of the cell lines. Cell Cycle Analysis with Flow Cytometry—Cells were lifted off the plates with trypsin-EDTA and were resuspended in PBS containing 1% FBS and then fixed with 70% ethanol. Cells were resuspended again in PBS containing 30 μg/ml propidium iodine and 250 mg/ml RNase A and further incubated at 4 °C for 1 h before analysis with a FACSCalibur laser-based flow cytometer (BD Biosciences). The cell cycle phase distribution was analyzed with FLOWJO software (Tree Star, Ashland, OR). Apoptosis Assays—The terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) technique was used to detect DNA strand breaks formed during apoptosis (39Gavrieli Y. Sherman Y. Ben-Sasson S.A. J. Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (9156) Google Scholar). Cells on coverslips were fixed with 4% paraformaldehyde for 45min at room temperature and then permeabilized with 1% Triton X-100. After a rinse with PBS, cells were incubated with fluorescein isothiocyanate (FITC)-labeled dUTP in the presence of enzyme terminal deoxynucleotidyltransferase for 1 h at 37 °C. Coverslips were mounted on glass slides in mounting medium containing the counterstain propidium iodide (2.5 μg/ml) and visualized using a fluorescent microscope. At least 500 cells/field were scored in a blinded fashion to determine the percentage of TUNEL-positive cells. Four fields per slide were used, and six independent slides were used for the final result. For annexin V assay (40Span L.F. Pennings A.H. Vierwinden G. Boezeman J.B. Raymakers R.A. de Witte T. Cytometry. 2002; 47: 24-31Crossref PubMed Scopus (79) Google Scholar), the cells were lifted off the plate using trypsin-EDTA. After washing, the cells were incubated with a label containing annexin V linked to FITC. FITC-positive cells/propidium iodide cells were analyzed by flow cytometry. Insulin Secretion—Cells (5 × 105 cells/well, 24-well plate) were seeded and cultured regularly for 2 days. The cells were preincubated for 30 min in HEPES-balanced Krebs-Ringer bicarbonate buffer (119 mm NaCl, 4.74 mm KCl, 2.54 mm CaCl2, 1.19 mm MgCl2, 1.19 mm KH2PO4, 25 mm NaHCO3, and 10 mm HEPES, pH 7.4) containing 0.5% BSA with 5 mm glucose and then incubated for 2 h with various concentrations of glucose or KCl. Released insulin was measured by radioimmunoassay using rat insulin as standard. The cellular protein content or DNA content was also used to normalize the amount of insulin secretion. Measurement of Insulin Content—Cells (5 × 105 cells/well, 24-well plate) were seeded and grown overnight. The medium was removed, and the cells were washed twice with ice-cold PBS and extracted with acid ethanol (15 mm/liter HCl, 75% ethanol) for 18 h at 4 °C. After clarification of the extracts by centrifugation at 15,000 × g at 4 °C, the insulin concentration was measured by radioimmunoassay with rat insulin as a standard. Measurement of NAD(P)H Production—NAD(P)H autofluorescence was measured essentially as described previously (41Johnson J.D. Ahmed N.T. Luciani D.S. Han Z. Tran H. Fujita J. Misler S. Edlund H. Polonsky K.S. J. Clin. Investig. 2003; 111: 1147-1160Crossref PubMed Scopus (303) Google Scholar, 42Pontoglio M. Sreenan S. Roe M. Pugh W. Ostrega D. Doyen A. Pick A.J. Baldwin A. Velho G. Froguel P. Levisetti M. Bonner-Weir S. Bell G.I. Yaniv M. Polonsky K.S. J. Clin. Investig. 1998; 101: 2" @default.
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• W2020804923 date "2005-02-01" @default.
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• W2020804923 title "Reduced Expression of the Insulin Receptor in Mouse Insulinoma (MIN6) Cells Reveals Multiple Roles of Insulin Signaling in Gene Expression, Proliferation, Insulin Content, and Secretion" @default.
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