Glucose and Insulin Treatment of Insulinoma Cells Results in Transcriptional Regulation of a Common Set of Genes Mitsuru Ohsugi,1 Corentin Cras-Me´neur,1 Yiyong Zhou,1 Wesley Warren,2 Ernesto Bernal-Mizrachi,1 and M. Alan Permutt1 Glucose and insulin are important regulators of islet ␤-cell growth and function by activating signaling path- ways resulting in transcriptional changes that lead to ancreatic islet ␤-cells can be regulated by multi- adaptive responses. Several immediate early genes have ple stimuli, including nutrients and growth fac- been shown to be rapidly induced by glucose-activated tors. ␤-Cell proliferation and function are depolarization in islet ␤-cells. The current studies ad- controlled by plasma glucose concentration and dress aspects of glucose-regulated transcription: 1) the P by growth factors acting via multiple intracellular signal- number and characteristics of these genes, 2) if depo- larization is the major mechanism, and 3) if glucose- ing pathways (1). Changes in gene expression that result stimulated insulin secretion is responsible, because from the activation of these signaling pathways are likely insulin per se can activate transcription. Here, the responsible for the adaptation of ␤-cells to physiological expression profiles of glucose-responsive insulinoma and pathological states. However, large gaps in our knowl- cells 45 min after the addition of glucose, KCl to induce edge currently exist regarding the changes in gene expres- depolarization, or insulin were assessed by endocrine sion and the molecular mechanisms mediating these ␤-cell pancreas cDNA microarrays. Glucose activated more responses to nutrients and growth factors. than 90 genes, representing diverse gene ontology func- Some genes likely to be involved in chronic glucose tions, and most were not previously known to be glucose ␤ responsive. KCl activated 80% of these same glucose- regulation of islet -cell mass or function have been regulated genes and, along with the effects of pretreat- identified (2–6). We have focused on early signaling events ment with diazoxide, suggested that glucose signaling is initiated by glucose treatment of insulinoma cells that mediated primarily via depolarization. There were >150 result in rapid transient activation of a number of imme- genes activated by insulin, and remarkably 71% were diate early genes (IEGs). These include Egr1, Egr2, c-fos, also regulated by glucose. Preincubation with a phos- and c-jun, known to respond to growth factor stimulation phatidylinositol (PI) 3-kinase inhibitor resulted in al- in a number of other tissues (7). The signaling pathways most total inhibition of depolarization and insulin- for induction of IEGs exhibit considerable stimulus and activated transcriptional responses. Thus, through gene expression profiling, these data demonstrate that glu- tissue specificity and in general involve activation of cose and insulin rapidly activate a PI 3-kinase pathway, kinase/phosphatase cascades (8). Initial glucose-mediated resulting in transcription of a common set of genes. This signaling can represent the first step in elucidating long- is consistent with glucose activation of gene transcrip- term changes in gene expression and islet physiology. tion either directly or indirectly through a paracrine/ These signaling pathways, limited to the initial kinase/ autocrine effect via insulin release. These results phosphatase cascades, are critical for understanding how illustrate that expression gene profiling can contribute the ␤-cell responds to its environment. The events occur- ␤ to the elucidation of important -cell biological func- ring from the time the stimulus reaches the ␤-cell until the tions. Diabetes 53:1496–1508, 2004 signal is transmitted to the nucleus to activate or repress transcription of a particular set of genes may be crucial in understanding the defects in islet growth in diabetic From the 1Division of Endocrinology, Metabolism, and Lipid Research, subjects or the adverse consequences of glucose toxicity Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri; and the 2Genome Sequencing Center, Washington Univer- on ␤-cell function. sity School of Medicine, St. Louis, Missouri. IEGs are often transcription factors that in turn activate Address correspondence and reprint requests to M. Alan Permutt, MD, Division of Endocrinology, Metabolism, and Lipid Research, Washington expression of downstream target genes, thus generating University School of Medicine, 660 S. Euclid Ave., Campus Box 8127, St. Louis, distinct biological responses by inducing specific long- MO 63110. E-mail: [email protected]. term programs of gene expression. In pancreatic ␤-cells, Received for publication 5 March 2004 and accepted 19 March 2004. M.O. and C.C.-M. contributed equally to this study. activation of expression of these IEGs was shown to ϩ Additional information for this article can be found in an online appendix at depend on depolarization activation of voltage-gated Ca2 http://diabetes.diabetesjournals.org. channels and subsequent influx of extracellular Ca2ϩ. This DMEM, Dulbecco’s modified Eagle’s medium; EPCon, Endocrine Pancreas 2ϩ Consortium; EST, expressed sequence tag; FBS, fetal bovine serum; GO, gene resulted in activation of Ca -regulated kinases, including ontology; IEG, immediate early gene; KATP channel, ATP-sensitive potassium calmodulin-dependent kinase IV and protein kinase A, channel; PI, phosphatidylinositol; qRT-PCR, quantitative RT-PCR; SSC, so- dium chloride–sodium citrate. leading to phosphorylation and activation of several tran- © 2004 by the American Diabetes Association. scription factors (cAMP-responsive element binding pro- 1496 DIABETES, VOL. 53, JUNE 2004 M. OHSUGI AND ASSOCIATES FIG. 1. A: Experimental scheme. MIN6 insulinoma cells were incubated in 5 mmol/l glucose, 5% FBS DMEM, for 18 h before stimulation. The “unstimulated” sample was harvested with- out any addition. Stimulated samples were harvested, and total RNA was extracted 45 min after addition of 25 mmol/l glucose, 50 mmol/l KCl, or 100 nmol/l insulin. B: Pairing scheme. RNA from four samples was labeled with either Cy3 or Cy5 fluorescent dye, and then a pair of samples was hybridized to a cDNA microarray. A graphic representation of the pairing scheme is shown here. An arrow indicates hybrid- ization to a cDNA microarray and RNA labeling with Cy3 or Cy5 fluorescent dye as indicated. Two double-sided arrows with different colors of arrowheads indicate dye-flip hybrid- ization. C: Distribution of the standard deviations for a “self-vs.-self” experiment derived from 9,700 cDNA probe as described in RESEARCH DESIGN AND (12 ؍ microarrays (n METHODS tein, serum response factor, and Elk-1) (9,10). The results the present experiments extend our knowledge of IEGs of these experiments defined the rapid glucose-signaling regulated by glucose, by KCl-induced depolarization, and pathways for a small number of IEGs whose transcription by insulin through use of high-resolution custom cDNA is rapidly activated by glucose, but the results now pose microarrays that contain clones from the Endocrine Pan- additional questions addressed by the current study. creas Consortium (EPCon: http://www.cbil.upenn.edu/ Animal models perfused with glucose for 4–5 days, or EPConDB). The arrays used for these experiments contain transgenic animals overexpressing a particular gene (11), up to 9,700 cDNAs with Ͼ3,000 novel clones not currently result in more readily measured physiological changes, yet available on commercial arrays (12–14). The results of this the sequence of molecular events leading to these physi- work suggest that glucose activation of IEGs is mediated ological changes are difficult to discern. This result high- primarily via depolarization and that glucose and insulin lights the desirability of beginning to dissect these activate an overlapping set of genes. Further, both of these mechanisms using other models. Thus, we designed ex- growth stimuli appear to activate transcription through a periments using insulinoma cells to elucidate early tran- phosphatidylinositol (PI) 3-kinase–dependent pathway. scriptional responses to islet growth factors. The results of These results further illustrate how monitoring expression DIABETES, VOL. 53, JUNE 2004 1497 1498 TABLE 1 GENE REGULATION IN PANCREATIC Gene regulation by glucose, ranked according to fold change, is compared with that by KCl-induced depolarization and with insulin treatment GenBank accession LocusLink G Ͼ U G Ͼ U K Ͼ U K Ͼ U I Ͼ U I Ͼ U number ID Name Symbol ratio 95% CI ratio 95% CI ratio 95% CI Top 50 glucose upregulated genes (54 clones including duplicates) AA537033 13653 Early growth response 1 Egr1 3.91 (2.55–5.99) 7.42 (4.86–11.33) 0.93 (0.77–1.12) AA958974 15936 Immediate early response 2 Ier2 2.92 (2.57–3.33) 7.89 (6.82–9.13) 0.99 (0.86–1.14) BI790969 15901 Inhibitor of DNA binding 1 Idb1 2.55 (2.36–2.76) 2.35 (2.07–2.68) 0.98 (0.88–1.09) AA869400 15936 Immediate early response 2 Ier2 2.54 (2.33–2.77) 6.68 (5.83–7.66) 0.94 (0.84–1.05) AI646026 15937 Immediate early response 3 Ier3 2.25 (2.13–2.39) 1.55 (1.41–1.7) 0.90 (0.84–0.96) AA119154 233895 cDNA sequence BC006909 BC006909 2.07 (1.83–2.35) 5.81 (4.5–7.5) 0.95 (0.88–1.02) W10821 1.84 (1.52–2.23) 3.77 (3.07–4.62) 0.93 (0.85–1.01) ␤ -CELLS BI319352 74155 RIKEN cDNA 1300002F13 gene 1300002F13Rik 1.69 (1.5–1.9) 1.42 (1.27–1.59) 1.09 (0.96–1.24) AA123373 15902 Inhibitor of DNA binding 2 Idb2 1.61 (1.52–1.71) 1.47 (1.4–1.55) 1.28 (1.23–1.32)