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First published online 1 November 2005
doi: 10.1242/jcs.02600

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Glucose represses connexin36 in insulin-secreting cells

¹ Department of Internal Medicine, Laboratory of Molecular Biology 19-135S, University Hospital, CHUV-1011 Lausanne, Switzerland
² Department of Chemical Sciences, University of Catania, 95125 Catania, Italy

View larger version (37K): [in a new window]   Fig. 1. Levels of Cx36 mRNA and protein are decreased by glucose in insulin-secreting cells. (A) Quantitative RT-PCR analysis showed a decrease of Cx36 mRNA in insulin-secreting cell lines and pancreatic rat islets after a 24 hour incubation at high (20 mM) versus low (2 mM) glucose concentration. Rat islets, ßTC3 cells and MIN6-B1 cells display a 40% decrease in Cx36 transcript whereas the INS-1 and INS-1E cell lines present a 60% decrease in Cx36 mRNA level. Data are means ± s.e.m. of four independent experiments. Relative Cx36 transcripts levels were normalized to the ribosomal protein L27. (B) Northern blots showed a dose-dependent decrease of Cx36 mRNA in INS-1 cells after 24 hours. By contrast, the level of the macrophage inhibitory factor (MIF) encoding mRNA, used as a positive control, is increased by glucose. Quantitative assessment of Cx36 mRNA confirmed that glucose induces a 60% reduction of Cx36 transcript expression. Data are means ± s.e.m. of five independent experiments. (C) Western-blot analysis normalized with tubulin and ß-actin showed a dose-dependent decrease of Cx36 in INS-1 cells after 24 hours. Densitometric analysis indicated that Cx36 protein levels were decreased by 70% in presence of a high glucose concentration. Data are means ± s.e.m. of six independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001.

View larger version (32K): [in a new window]   Fig. 2. Cx36 downregulation by glucose is reversible and occurs at the transcriptional level. (A) INS-1 cells preincubated for 24 hours at 2 mM, 10 mM and 20 mM glucose were further incubated at 10 mM glucose for 24 hours. Densitometric analysis of three independent northern blots revealed that Cx36 expression levels returned to control values after 8 hours. Mean (± s.e.m.) of three independent experiments. ***, P<0.001; **, P<0.01. (B) After a 24 hour preincubation at 2 mM, 10 mM or 20 mM glucose, INS-1 cells were exposed to 5 µg ml-1 actinomycin D for 1 hour, 2 hours, 3 hours or 5 hours. Quantitative assessment of Cx36 mRNA levels (northern blot) showed that glucose had no effect on the stability of the Cx36 transcript.

View larger version (37K): [in a new window]   Fig. 3. Effects of various nutrients on Cx36 levels. Cells were incubated for 24 hours in the presence of glucose (2 mM or 20 mM), L-glucose (20 mM), 3-O-methyl-D-glucose (20 mM), 2-deoxy-D-glucose (20 mM), glucosamine (20 mM) or leucine (10 mM). Quantitative assessment of western blot demonstrated that 2-deoxy-D-glucose induced a 50% decrease in Cx36 levels. Data are means (± s.e.m.) from six independent experiments made in duplicate. **, P<0.01; ***, P<0.001.

View larger version (14K): [in a new window]   Fig. 4. ß-Cell depolarization and MAPK pathway are not involved in Cx36 regulation. INS-1 cells were incubated for 24 hours at 2 mM or 20 mM glucose with or without 20 mM KCl, 10 µM nifedipine, 100 nM PMA and 50 µM PD98059, as indicated. Quantitative assessment (± s.e.m.) of six independent experiments revealed that none of these compounds influence Cx36 regulation by glucose. ***, P<0.001.

View larger version (58K): [in a new window]   Fig. 5. The cAMP-PKA pathway mediates the effect of glucose on Cx36 levels. INS-1 cells were incubated for 24 hours at 2 mM glucose with or without 20 µM forskolin or at 20 mM glucose with or without 10 µM H89. (A) Western-blot analysis showed that forskolin had a similar effect to glucose on Cx36 expression, whereas H89 fully blunted the effects of glucose and forskolin. Data are means ± s.e.m. of densitometric analyses of six independent experiments made in duplicate. **, P<0.01, ***, P<0.001. (left) Immunolabeling of Cx36 (Cx36 recognized by fluorescein-conjugated secondary antibodies) resulted in a punctate membrane labeling in INS-1 cells (white tab represents 50 µm). (a) 2 mM glucose; (b) 20 mM glucose; (c) 2 mM glucose, 20 µM forskolin; (d) 20 mM glucose, 10 µM H89. Pictures are representative of four independent experiments. (right) Quantitations of the fluorescent dots were performed under the microscope on many images by counting the immunofluorescent dots in a square containing 100 cells. Data are means ± s.e.m. of four independent experiments. ***, P<0.001.

View larger version (19K): [in a new window]   Fig. 6. Glucose increases cAMP levels in INS-1 cells. INS-1 cells were incubated for 24 hours at either 2 mM or 20 mM glucose with or without 10 µM nifedipine or 20 µM forskolin, as indicated. Measurements of the intracellular cAMP levels showed that glucose increased cAMP levels independently of Ca2+ entry. Data are means ± s.e.m. of three independent experiments made in duplicate. **, P<0.01; ***, P<0.001.

View larger version (34K): [in a new window]   Fig. 7. Glucose decreases the transcriptional activity of the human Cx36 promoter through a CRE. (A) Progressive deletions of the human Cx36 promoter were transfected into INS-1 cells. After transfection, cells were incubated at 2 mM or 20 mM glucose. The –1079 to –316 region of the Cx36 promoter drives glucose responsiveness. The mutation of a CRE (pGL3-1079m) located in this region abolished the inhibitory effect of glucose on the luciferase activity. Results are means ± s.e.m. of four experiments performed in triplicate. Luciferase activities were normalized using the pRLSV40renilla construct. *, P<0.05. NRSE, neuron-restrictive silencing element. (B) Alignments of CREs: the TGACG core region (bold) of the consensus CRE is 100% conserved between the three species. HIP CRE1, human insulin promoter CRE 1; HIP CRE 2, human insulin promoter CRE 2; RIP1 CRE, rat insulin promoter 1 CRE.

View larger version (90K): [in a new window]   Fig. 8. Glucose and PKA activation enhance the binding activity of CREM1 to the human Cx36 CRE motif in INS-1 cells. Electrophoretic mobility shift assays (EMSAs) were performed using the Cx36 CRE and nuclear extracts (NE) from INS-1 cells cultured at either 2 mM or 20 mM glucose with or without forskolin or H89. For the competition assay, a 100-fold excess of the unlabelled oligonucleotides were used (cold competitor x100). mut, mutated Cx36 CRE; wt, wild-type Cx36 CRE. Compared with the free probe, two slow-migrating complexes (C1 and C2) were detected. The levels of binding are increased in presence of high levels of glucose or forskolin and reduced by H89 treatment. For the supershift analysis, nuclear extract from INS-1 cells cultured at either 2 mM or 20 mM glucose were exposed to specific antibodies directed against CRE modulator protein 1 (CREM-1), activating transcription factor 3 (ATF3), CCAAT/enhancer-binding protein ß (C/EBPß), phosphorylated CRE-binding protein (P-CREB), RE-1-silencing transcription factor (REST, also known as NRSF). CREM-1 and P-CREB supershifted the C1 complexes. Data are representative of four independent experiments.

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