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ICER-1γ Overexpression Drives Palmitate-mediated Connexin36 Down-regulation in Insulin-secreting Cells

2007; Elsevier BV; Volume: 283; Issue: 9 Linguagem: Inglês

10.1074/jbc.m708181200

ISSN

1083-351X

Autores

Florent Allagnat, Florian Alonso, David Martín, Amar Abderrahmani, Gérard Waeber, Jacques‐Antoine Haefliger,

Tópico(s)

Heme Oxygenase-1 and Carbon Monoxide

Resumo

Channels formed by the gap junction protein connexin36 (Cx36) contribute to the proper control of insulin secretion. We investigated the impact of chronic hyperlipidemia on Cx36 expression in pancreatic β-cells. Prolonged exposure to the saturated free fatty acid palmitate reduced the expression of Cx36 in several insulin-secreting cell lines and isolated mouse islets. The effect of palmitate was fully blocked upon protein kinase A (PKA) inhibition by H89 and (Rp)-cAMP, indicating that the cAMP/PKA pathway is involved in the control of Cx36 expression. Palmitate treatment led to overexpression of the inducible cAMP early repressor (ICER-1γ), which bound to a functional cAMP-response element located in the promoter of the CX36 gene. Inhibition of ICER-1γ overexpression prevented the Cx36 decrease, as well as the palmitate-induced β-cell secretory dysfunction. Finally, freshly isolated islets from mice undergoing a long term high fat diet expressed reduced Cx36 levels and increased ICER-1γ levels. Taken together, these data demonstrate that chronic exposure to palmitate inhibits the Cx36 expression through PKA-mediated ICER-1γ overexpression. This Cx36 down-regulation may contribute to the reduced glucose sensitivity and altered insulin secretion observed during the pre-diabetic stage and in the metabolic syndrome. Channels formed by the gap junction protein connexin36 (Cx36) contribute to the proper control of insulin secretion. We investigated the impact of chronic hyperlipidemia on Cx36 expression in pancreatic β-cells. Prolonged exposure to the saturated free fatty acid palmitate reduced the expression of Cx36 in several insulin-secreting cell lines and isolated mouse islets. The effect of palmitate was fully blocked upon protein kinase A (PKA) inhibition by H89 and (Rp)-cAMP, indicating that the cAMP/PKA pathway is involved in the control of Cx36 expression. Palmitate treatment led to overexpression of the inducible cAMP early repressor (ICER-1γ), which bound to a functional cAMP-response element located in the promoter of the CX36 gene. Inhibition of ICER-1γ overexpression prevented the Cx36 decrease, as well as the palmitate-induced β-cell secretory dysfunction. Finally, freshly isolated islets from mice undergoing a long term high fat diet expressed reduced Cx36 levels and increased ICER-1γ levels. Taken together, these data demonstrate that chronic exposure to palmitate inhibits the Cx36 expression through PKA-mediated ICER-1γ overexpression. This Cx36 down-regulation may contribute to the reduced glucose sensitivity and altered insulin secretion observed during the pre-diabetic stage and in the metabolic syndrome. The fine-tuning of insulin secretion in response to nutrient stimulation relies on a closely coordinated functioning of pancreatic β-cells. The cell-to-cell communication mediated by gap junction channels contributes to synchronization of β-cell clusters and has been demonstrated to be essential for the proper regulation of storage and release of insulin, both in vitro and in vivo (for review see Ref. 1Michon L. Nlend Nlend R. Bavamian S. Bischoff L. Boucard N. Caille D. Cancela J. Charollais A. Charpantier E. Klee P. Peyrou M. Populaire C. Zulianello L. Meda P. Biochim. Biophys. Acta. 2005; 1719: 82-101Crossref PubMed Scopus (69) Google Scholar). Gap junctions are specific membrane structures consisting of aggregates of intercellular channels interconnecting the cytoplasms of neighboring cells and providing them with a direct pathway for sharing ions, nutrients, and intracellular messengers. We and others (2Le Gurun S. Martin D. Formenton A. Maechler P. Caille D. Waeber G. Meda P. Haefliger J.A. J. Biol. Chem. 2003; 278: 37690-37697Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 3Moreno A.P. Berthoud V.M. Perez-Palacios G. Perez-Armendariz E.M. Am. J. Physiol. 2004; 288: E948-E956Google Scholar, 4Serre-Beinier V. Le Gurun S. Belluardo N. Trovato-Salinaro A. Charollais A. Haefliger J.A. Condorelli D.F. Meda P. Diabetes. 2000; 49: 727-734Crossref PubMed Scopus (141) Google Scholar, 5Theis M. Mas C. Doring B. Degen J. Brink C. Caille D. Charollais A. Kruger O. Plum A. Nepote V. Herrera P. Meda P. Willecke K. Exp. Cell Res. 2004; 294: 18-29Crossref PubMed Scopus (60) Google Scholar) have demonstrated that only one connexin, the constitutive unit of these channels, is expressed in insulin-secreting cells, connexin36 (Cx36 2The abbreviations used are:Cx36connexin36FFAfree fatty acidPKAcAMP-dependent protein kinase AChIPchromatin immunoprecipitationhGHhuman growth hormoneRTreverse transcriptionCREMcAMP-response element modulatorCREcAMP-response elementNCnormal chowHFhigh fatICER-1γinducible cAMP early repressor 1ASantisense. for 36 kDa), and that this connexin plays a critical role in β-cell function. In addition, modulation of the Cx36 levels results in impaired glucose-induced insulin secretion, indicating that Cx36 must be expressed at a very precise level to maintain a normal insulin secretion (2Le Gurun S. Martin D. Formenton A. Maechler P. Caille D. Waeber G. Meda P. Haefliger J.A. J. Biol. Chem. 2003; 278: 37690-37697Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 6Caton D. Calabrese A. Mas C. Serre-Beinier V. Charollais A. Caille D. Zufferey R. Trono D. Meda P. J. Cell Sci. 2003; 116: 2285-2294Crossref PubMed Scopus (39) Google Scholar). Thus, changes in the Cx36 expression levels might impair β-cell function and hence be involved in the pathophysiology of diabetes mellitus. connexin36 free fatty acid cAMP-dependent protein kinase A chromatin immunoprecipitation human growth hormone reverse transcription cAMP-response element modulator cAMP-response element normal chow high fat inducible cAMP early repressor 1 antisense. Type 2 diabetes partly ensues from β-cell failure to compensate for peripheral insulin resistance (for review see Ref. 7Prentki M. Nolan C.J. J. Clin. Investig. 2006; 116: 1802-1812Crossref PubMed Scopus (1329) Google Scholar). Abnormalities in both glucose and lipid metabolism contribute to the pathogenesis of this condition and in particular to the inexorable decline of β-cell function (8Poitout V. Robertson R.P. Endocrinology. 2002; 143: 339-342Crossref PubMed Scopus (550) Google Scholar, 9Robertson R.P. Harmon J. Tran P.O. Poitout V. Diabetes. 2004; 53: 119-124Crossref PubMed Google Scholar). Recently, we have demonstrated that long term exposure to a high concentration of glucose resulted in a reduced expression of Cx36 in insulin-secreting cells (10Allagnat F. Martin D. Condorelli D.F. Waeber G. Haefliger J.A. J. Cell Sci. 2005; 118: 5335-5344Crossref PubMed Scopus (48) Google Scholar). Although hyperglycemia is undoubtedly a major contributor to the onset of β-cell failure, chronic elevation in circulating free fatty acids (FFAs) also accompany the progression to type 2 diabetes (11Bergman R.N. Ader M. Trends Endocrinol. Metab. 2000; 11: 351-356Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 12Boden G. Curr. Diab. Rep. 2005; 5: 167-170Crossref PubMed Scopus (50) Google Scholar, 13Wyne K.L. Am. J. Med. 2003; 115: 29-36Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 14Zhou Y.P. Grill V.E. J. Clin. Investig. 1994; 93: 870-876Crossref PubMed Scopus (631) Google Scholar). Indeed, prolonged exposure of β-cells to elevated concentrations of FFAs, both in vivo and in vitro, leads to elevated basal secretion and blunted response to glucose, both defects being reminiscent of the diabetic state (14Zhou Y.P. Grill V.E. J. Clin. Investig. 1994; 93: 870-876Crossref PubMed Scopus (631) Google Scholar, 15Bollheimer L.C. Skelly R.H. Chester M.W. McGarry J.D. Rhodes C.J. J. Clin. Investig. 1998; 101: 1094-1101Crossref PubMed Scopus (227) Google Scholar, 16Haber E.P. Procopio J. Carvalho C.R. Carpinelli A.R. Newsholme P. Curi R. Int. Rev. Cytol. 2006; 248: 1-41Crossref PubMed Scopus (86) Google Scholar, 17Haber E.P. Ximenes H.M. Procopio J. Carvalho C.R. Curi R. Carpinelli A.R. J. Cell Physiol. 2003; 194: 1-12Crossref PubMed Scopus (127) Google Scholar, 18McGarry J.D. Dobbins R.L. Diabetologia. 1999; 42: 128-138Crossref PubMed Scopus (496) Google Scholar, 19Zhou Y.P. Grill V. J. Clin. Endocrinol. Metab. 1995; 80: 1584-1590Crossref PubMed Google Scholar, 20Zhou Y.P. Grill V.E. Diabetes. 1995; 44: 394-399Crossref PubMed Scopus (93) Google Scholar). Here we report the effects of a prolonged exposure of insulin-secreting cells to various FFAs on Cx36 expression, and we document the generation of a selective decrease of Cx36 expression by saturated FFAs. This decrease was concomitant with increased ICER-1γ (inducible cAMP early repressor) expression, and CX36 promoter studies revealed that ICER-1γ mediates the inhibitory effect of palmitate on Cx36 expression. We demonstrated that ICER-1γ blockade prevented the deleterious effect of palmitate on β-cell secretion, providing a mechanistic explanation for the altered β-cell function observed after a prolonged exposure to FFA. Finally, we further observed in vivo that mice fed a high fat diet for 15 weeks displayed a marked decrease in Cx36 levels that correlates with increased levels of circulating FFAs. Materials—Glucose, palmitate, methyl ester palmitate, stearate, oleate, linoleate, and (Rp)-cAMP were purchased from Sigma, and compound C and H89 were from Calbiochem. All experiments using FFAs were performed in the absence of serum at 5 mm glucose for INS-1E cells and islets, and 11 mm glucose for MIN6-B1 at a final concentration of 1% bovine serum albumin. The pSV-ICER expression plasmid (21Molina C.A. Foulkes N.S. Lalli E. Sassone-Corsi P. Cell. 1993; 75: 875-886Abstract Full Text PDF PubMed Scopus (526) Google Scholar) was provided by Dr. Regazzi, University of Lausanne, Switzerland. The plasmid encoding ICER antisense (ICER AS) was constructed by inserting PCR-amplified fragment of ICER from pSVICER. Primers used were as follows: 5′-AGAAAGTCTAGACATGGCTGTAACTGGAGATGAA-3′ (sense) and 5′-ACTGTGCAGGATCCCTGGTGAGGCAGC-3′ (antisense). The PCR fragment was inserted between the BamHI and XbaI cloning sites of the pcDNA3 vector (22Abderrahmani A. Cheviet S. Ferdaoussi M. Coppola T. Waeber G. Regazzi R. EMBO J. 2006; 25: 977-986Crossref PubMed Scopus (54) Google Scholar). Preparation of FFAs—FFAs were prepared as described previously (23Beeharry N. Lowe J.E. Hernandez A.R. Chambers J.A. Fucassi F. Cragg P.J. Green M.H. Green I.C. Mutat. Res. 2003; 530: 27-33Crossref PubMed Scopus (69) Google Scholar). Briefly, stock palmitic acid or methyl ester palmitic acid solution in methanol (80 mmol/liter) was conjugated to fatty acid-free bovine serum albumin in a 3:1 molar ratio at 37 °C for at least 1 h prior to treatment. The oleic and linoleic acid used were purchased pre-conjugated to albumin (Fluka Chemie). The FFAs concentrations were measured using the NEFA C kit (Wako). Cell Lines—The rat insulinoma cell line INS-1E (kindly provided by Dr. Pierre Maechler, CMU, University of Geneva) was maintained in the complete RPMI 1640 medium as described previously (2Le Gurun S. Martin D. Formenton A. Maechler P. Caille D. Waeber G. Meda P. Haefliger J.A. J. Biol. Chem. 2003; 278: 37690-37697Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 24Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Crossref PubMed Scopus (748) Google Scholar, 25Merglen A. Theander S. Rubi B. Chaffard G. Wollheim C.B. Maechler P. Endocrinology. 2004; 145: 667-678Crossref PubMed Scopus (476) Google Scholar). MIN6-B1 (kindly provided by Dr. Philippe Halban, CMU, University of Geneva) were cultured in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum, 25 mm glucose, 71 μm 2-mercaptoethanol, 2 mm glutamine, 100 units/ml penicillin, and 100 mg/liter streptomycin (26Lilla V. Webb G. Rickenbach K. Maturana A. Steiner D.F. Halban P.A. Irminger J.C. Endocrinology. 2003; 144: 1368-1379Crossref PubMed Scopus (115) Google Scholar). Cells were kept at 37 °C in a humidified incubator under 5% CO2. Mouse Models and Mouse Islets Isolation—Our institutional review committee for animal experiments approved all the procedures for mouse care, surgery and euthanasia. Adult male C57BL6 mice were submitted to a 15-week high fat diet (72% fat, 28% protein, and <1% carbohydrate, low nitrates, 30% lard, 20% corn oil from Unité d'Alimentation Rationnelle, Epinaysur-Orge, France) (27Burcelin R. Dolci W. Thorens B. Metabolism. 1999; 48: 252-258Abstract Full Text PDF PubMed Scopus (100) Google Scholar, 28Cook S. Hugli O. Egli M. Menard B. Thalmann S. Sartori C. Perrin C. Nicod P. Thorens B. Vollenweider P. Scherrer U. Burcelin R. Diabetes. 2004; 53: 2067-2072Crossref PubMed Scopus (106) Google Scholar). Mice fed normal chow or a high fat diet were anesthetized by inhalation of 5% halothane (Arovet AG), killed by cervical dislocation, and immediately used for pancreas sampling. Mouse islets of Langerhans were isolated from the pancreas by collagenase digestion, filtered on a 100 μm cell strainer (BD Biosciences), and cultured in INS-1E medium, as described previously (10Allagnat F. Martin D. Condorelli D.F. Waeber G. Haefliger J.A. J. Cell Sci. 2005; 118: 5335-5344Crossref PubMed Scopus (48) Google Scholar). Blood Chemistry—Plasma concentrations of glucose were measured in the fed state in conscious mice using the Ascencia DEX2® blood glucose meter (Bayer). The NEFA (nonesterified fatty acids) and cholesterol concentrations were measured using the NEFA C kit (Wako) and the cholesterol CHOD-PAP method (Axonlab), according to manufacturer's instructions. Plasma insulin levels were determined using the ultrasensitive mouse insulin enzyme-linked immunosorbent assay (Mercodia). RNA Isolation and Quantitative RT-PCR (Lightcycler©)—Cells were homogenized in the Tripure isolation reagent (Roche Diagnostics), and total RNA was extracted using the kit procedure. Mouse islets mRNA were isolated using nucleospin RNA II columns (Macherey-Nagel). Transcripts (1 μg) were reverse-transcribed using ImProm-2 Reverse transcription System (Promega). Quantitative PCR was performed using the SYBR® Premix ExTaq™ (Takara) in a Lightcycler Instrument (Roche Diagnostics). cDNAs were amplified using the following primers: rat Cx36, 5′-ATACAGGTGTGAATGAGGGAGGATG-3′(sense) and 5′-TGGAGGGTGTTACAGATGAAAGAGG-3′(antisense); rat ribosomal protein L-27, 5′-GATCCAAGATCAAGTCCTTTGTG-3′(sense) and 5′-CTGGGTCTCTGAACACATCCT-3′(antisense); insulin 5′-TGGCTTCTTCTACACACC-3′(sense) and 5′-TCTAGTTGCAGTAGTTCT-3′(antisense); ICER-1γ, 5′-CTGGGTCTCTGAACACATCCT-3′(sense) 5′-CACCTTGTGGCAAAGCAGTA-3′(antisense); and acetyl-CoA carboxylase, 5′-CACGTTCAGAGCGAGAGATG-3′(sense) and 5′-ATGATGGCTCGGATGAAGAA-3′(antisense). Western Blotting—INS-1E cells were solubilized by sonication in SDS-buffer (62.5 mm Tris-EDTA, pH 6.8, SDS 5%). Protein content was measured using a detergent-compatible BCA protein assay kit (Pierce). Western blots were carried out as described previously (10Allagnat F. Martin D. Condorelli D.F. Waeber G. Haefliger J.A. J. Cell Sci. 2005; 118: 5335-5344Crossref PubMed Scopus (48) Google Scholar). Membranes were saturated for 1 h in TBS containing 5% milk and 0.05% Tween 20 prior to overnight hybridization at 4 °C with rabbit polyclonal antibodies against Cx36 (2Le Gurun S. Martin D. Formenton A. Maechler P. Caille D. Waeber G. Meda P. Haefliger J.A. J. Biol. Chem. 2003; 278: 37690-37697Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 29Martin D. Tawadros T. Meylan L. Abderrahmani A. Condorelli D.F. Waeber G. Haefliger J.A. J. Biol. Chem. 2003; 278: 53082-53089Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), diluted 1:200, monoclonal anti α-tubulin antibodies (Fluka Chemie; diluted 1:2,000), and rabbit polyclonal anti CREM-1 sc440 (Santa Cruz Biotechnology; diluted 1:500). After incubation at room temperature (1 h) with the convenient secondary antibody conjugated to horseradish peroxidase (Fluka Chemie; diluted 1:20,000), membranes were revealed by enhanced chemiluminescence (ECL, Amersham Biosciences). Densitometric analyses of immunolabeled proteins (Western blots films) were performed using the ImageQuant software (Amersham Biosciences). Transient Transfection and Luciferase Assays—INS-1E cells plated in 24-well dishes were transiently transfected using Lipofectamine 2000 reagent (Invitrogen) at a DNA/lipid ratio of 1:1 according to manufacturer's instructions. Transfection assay consisted of a mixture of the luciferase reporter plasmid containing the mouse luciferase gene under control of different fragments of the human CX36 promoter, together with an empty vector (pCDNA3), or a plasmid allowing constitutive expression of ICER-1 (21Molina C.A. Foulkes N.S. Lalli E. Sassone-Corsi P. Cell. 1993; 75: 875-886Abstract Full Text PDF PubMed Scopus (526) Google Scholar) or an ICER antisense plasmid (22Abderrahmani A. Cheviet S. Ferdaoussi M. Coppola T. Waeber G. Regazzi R. EMBO J. 2006; 25: 977-986Crossref PubMed Scopus (54) Google Scholar), and the internal control plasmid pRL-SV40 (Promega). 24 h after transfection, the cells were incubated in presence or absence of 0.2 mm palmitate. 24 h later, PLB (passive lysis buffer, Promega) cell extracts were prepared for dual luciferase reporter assays (Promega) in a Turner TD-20/20 luminometer. Promoter activity was normalized by the Renilla activity of the pRLSV40, as described previously (10Allagnat F. Martin D. Condorelli D.F. Waeber G. Haefliger J.A. J. Cell Sci. 2005; 118: 5335-5344Crossref PubMed Scopus (48) Google Scholar). Chromatin Immunoprecipitation (ChIP) Assay—INS-1E cells (106 cells) were cross-linked with 1% formaldehyde at room temperature for 10 min. The cross-linking reaction was stopped by adding glycine to a final concentration of 125 nmol/liter. Cells were collected and resuspended in SDS lysis buffer containing 1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.1. Cell pellets were lysed and sonicated to obtain the desired chromatin length (∼500 bp). Protein concentration of the cell supernatants was determined by BCA protein assay (Pierce), and the samples were diluted to 1 mg/ml in ChIP dilution buffer (1% Triton X-100, 2 mm EDTA, 150 mm NaCl, 20 mm Tris-HCl, pH 8.1, and protease inhibitors). Samples were precleared by incubation with blocked protein A-Sepharose (Amersham Biosciences) for at least 1 h at 4 °C. The precleared chromatin lysates were immunoprecipitated overnight at 4 °C with either polyclonal rabbit antibodies specific to CREM-1, c-Myc (9E10, Santa Cruz Biotechnology), or human REST (29Martin D. Tawadros T. Meylan L. Abderrahmani A. Condorelli D.F. Waeber G. Haefliger J.A. J. Biol. Chem. 2003; 278: 53082-53089Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The DNA-protein-antibody complexes were collected by addition of protein A-Sepharose for 2 h at 4 °C. Then the DNA-protein complexes were washed twice in low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, 150 mm NaCl), and once in high salt final wash buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, 500 mm NaCl). The complexes were eluted from the Sepharose beads by incubation at room temperature for 15 min in 1% SDS and 0.1 m NaHCO3. Eluates were heated for at least 4 h at 65 °C to reverse the formaldehyde cross-linking and precipitated overnight at –20 °C. The pellets were diluted in TE, RNase A- and proteinase K-treated, and purified using the PCR product cleanup kit (Roche Diagnostics) according to manufacturer's instruction. DNA was submitted to classic PCR amplification or LightCycler (1 cycle at 95 °C for 3 min, followed by 32 cycles at 94 °C for 25 s, 58 °C for 25 s, 72 °C for 25 s, and finally 1 cycle at 72 °C for 3 min), using specific primers 5′-CACCAGCGTGTCTGTTCCT-3′ (sense) and 5′-ATCTTGCGGTCTGAGGGAG-3′ (antisense). Human Growth Hormone (hGH) Secretion—INS-1E cells plated in 24-wells dishes were transiently cotransfected using Lipofectamine 2000 (Invitrogen) with a construct encoding the hGH (Nicholls), together with an empty vector (pCDNA3), a plasmid permitting constitutive expression of ICER-1 (21Molina C.A. Foulkes N.S. Lalli E. Sassone-Corsi P. Cell. 1993; 75: 875-886Abstract Full Text PDF PubMed Scopus (526) Google Scholar), an antisense ICER construct (22Abderrahmani A. Cheviet S. Ferdaoussi M. Coppola T. Waeber G. Regazzi R. EMBO J. 2006; 25: 977-986Crossref PubMed Scopus (54) Google Scholar), or a Cx36 coding plasmid (2Le Gurun S. Martin D. Formenton A. Maechler P. Caille D. Waeber G. Meda P. Haefliger J.A. J. Biol. Chem. 2003; 278: 37690-37697Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). 24 h later, the cells were cultured in presence or absence of 0.2 mm palmitate for 48 h. Then the cells were washed and preincubated for 1 h in KRBH (Krebs-Ringer/bicarbonate/HEPES) buffer (140 mm NaCl, 3.6 mm KCl, 0.5 mm NaH2PO4, 0.5 mm MgSO4, 1.5 mm CaCl2, 2 mm NaHCO3, 10 mm HEPES, and 0.1% bovine serum albumin). The medium was then removed, and the cells were incubated for 30 min in KRBH added with 2 mm glucose (basal condition) or in KRBH containing 20 mm glucose, 10 μm forskolin, and 100 μm isobutylmethylxanthine (stimulatory condition). The total amount of hGH produced by transfected cells and the fraction released into the medium during the incubation period were determined by enzyme-linked immunosorbent assay (Roche Diagnostics). Statistical Analyses—Data were expressed as mean ± S.E. Difference between means were assessed by Student's t test. In experiments involving the comparison of more than two experimental conditions, analyses of variance were performed. All computations were carried out with the JMP software, version 3.2.2 (SAS Institute, Cary NC). The α level of all tests was set at 0.05. Statistical significance was defined at a value of p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Palmitate, but Not Oleate or Linoleate, Represses Cx36 Expression in Insulin-secreting Cells—Pancreatic β-cells were cultured at 6 mm glucose without serum in the absence or presence of saturated palmitate (C 16:0), monounsaturated oleate (C 18:1), or polyunsaturated linoleate (C 18:2) for 48 h. Palmitate (0.2 mm), but not linoleate (0.2 mm) or oleate (0.2 mm), induced a 40% reduction in Cx36 mRNA expression levels in the rat β-cell line INS-1E (Fig. 1A). This effect was more pronounced in the mouse β-cell line MIN6-B1 (50%) and in isolated mouse islets (70%). As a positive control, acetyl-CoA carboxylase mRNA levels, which are known to be decreased after chronic exposure to FFAs (30Brun T. Assimacopoulos-Jeannet F. Corkey B.E. Prentki M. Diabetes. 1997; 46: 393-400Crossref PubMed Google Scholar), were reduced by about 50% in response to each FFA after 48 h (Fig. 1B). To ascertain the absence of a significant effect of linoleate or oleate on Cx36 expression, INS-1E cells were incubated in presence of each FFA in the 50–400 μm range. Elevation of the oleate and linoleate concentrations up to 0.4 mm did not alter Cx36 protein expression in INS-1E cells (Fig. 2B). Conversely, Western blot analyses performed on INS-1E cells cultured for 48 h in the presence of increasing levels of palmitate revealed that palmitate dose-dependently decreased Cx36 expression (Fig. 2A, left panel). Quantitative assessment of Western blot analyses (Fig. 2B) revealed that 0.2 mm was the lowest palmitate concentration inducing the maximal effect on Cx36 expression and was therefore the condition used in subsequent experiments.FIGURE 2Cx36 down-regulation by palmitate is dose-dependent and reversible. A, INS-1E cells were cultured for 48 h in the presence of increasing concentrations of palmitate (left panel) or stearate (right panel), as indicated. The blots are representative of three independent experiments. B, quantitative assessment of Western blot analyses on INS-1E cells cultured in the presence of increasing concentrations of palmitate (black square), stearate (empty square), oleate (black circle), or linoleate (empty circle). Results are means ± S.E. of four independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus INS-1E control. C, INS-1E cells were cultured for 48 h in the presence of palmitate (0.2 mm) or methyl ester palmitate (0.2 mm), and Cx36 levels were assessed by Western blot. Upper panel, representative Western blot; lower panel, quantitative assessment of Western blot analyses. Results are means ± S.E. of four independent experiments. *, p < 0.05; **, p < 0.01 versus INS-1E control. D, INS-1E cells preincubated for 24 h in presence of 0.2 mm palmitate were further incubated for 24 h in absence of palmitate (palmitate reversibility). Upper panel, representative Western blot; lower panel, quantitative assessment of Western blot analyses. Results are means ± S.E. of three independent experiments. **, p < 0.01 versus INS-1E control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effect of palmitate was reversible, as INS-1E cells preincubated for 24 h in presence of 0.2 mm palmitate and incubated for another 24 h in absence of palmitate showed control levels of Cx36 (Fig. 2D). To determine whether this regulation process was specific to saturated FFAs, we tested the effect of stearate and observed that this saturated FFA decreased the Cx36 levels to the same extent as did palmitate (Fig. 2, A and B). Metabolism of palmitate has been shown to be required to elicit acetyl-CoA carboxylase down-regulation (30Brun T. Assimacopoulos-Jeannet F. Corkey B.E. Prentki M. Diabetes. 1997; 46: 393-400Crossref PubMed Google Scholar) and β-cell apoptosis (23Beeharry N. Lowe J.E. Hernandez A.R. Chambers J.A. Fucassi F. Cragg P.J. Green M.H. Green I.C. Mutat. Res. 2003; 530: 27-33Crossref PubMed Scopus (69) Google Scholar). To test whether palmitate metabolism is mandatory to the regulation of Cx36 expression, INS-1E cells were treated with palmitic acid methyl ester, a nonmetabolizable analogue of palmitate that is not activated into a fatty acyl-CoA in the cytosol (31Parker S.M. Moore P.C. Johnson L.M. Poitout V. Metabolism. 2003; 52: 1367-1371Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 32Briaud I. Harmon J.S. Kelpe C.L. Segu V.B. Poitout V. Diabetes. 2001; 50: 315-321Crossref PubMed Scopus (254) Google Scholar, 33Diakogiannaki E. Dhayal S. Childs C.E. Calder P.C. Welters H.J. Morgan N.G. J. Endocrinol. 2007; 194: 283-291Crossref PubMed Scopus (59) Google Scholar). As shown in Fig. 2C, 0.2 mm methyl palmitate induced a significant 25% decrease in Cx36 levels, suggesting that palmitate oxidation is not required for the Cx36 decrease. Mechanism(s) of Palmitate-mediated Cx36 Down-regulation—We have recently demonstrated that glucose inhibits Cx36 expression through activation of the cAMP/PKA pathway (10Allagnat F. Martin D. Condorelli D.F. Waeber G. Haefliger J.A. J. Cell Sci. 2005; 118: 5335-5344Crossref PubMed Scopus (48) Google Scholar). INS-1E cells were exposed to palmitate, in the presence of the PKA inhibitor H89 or the specific membrane-permeable inhibitor of PKA activation (Rp)-cAMP. As shown in Fig. 3A, both compounds prevented the Cx36 decrease elicited by palmitate, suggesting that the cAMP/PKA pathway mediates the palmitate effect on Cx36 expression. Recent findings established that ICER-1γ is overexpressed after prolonged exposure to FFAs (34Zhou Y.P. Marlen K. Palma J.F. Schweitzer A. Reilly L. Gregoire F.M. Xu G.G. Blume J.E. Johnson J.D. J. Biol. Chem. 2003; 278: 51316-51323Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Here palmitate, but neither oleate nor linoleate, induced a 3-fold increase in ICER-1γ mRNA levels in INS-1E cells and isolated mouse islets (Fig. 3B). Western blot analyses using an antibody directed against CREM-1 detected the two major repressive isoforms of CREM expressed in β-cells, ICER-1 and ICER-1γ (35Inada A. Yamada Y. Someya Y. Kubota A. Yasuda K. Ihara Y. Kagimoto S. Kuroe A. Tsuda K. Seino Y. Biochem. Biophys. Res. Commun. 1998; 253: 712-718Crossref PubMed Scopus (32) Google Scholar), immunolocalized approximately at 17 and 14 kDa, respectively, and confirmed that ICER-1γ is the major isoform expressed in INS-1E cells. Palmitate-treated INS-1E cells displayed twice as much ICER-1 and ICER-1γ protein after 48 h, in comparison with untreated cells (Fig. 3C). As a positive control, 20 mm glucose treatment resulted in a 4-fold induction of ICER-1 and ICER-1γ content. The palmitate-mediated overexpression of ICER-1 and ICER-1γ was fully blocked in the presence of H89 (Fig. 3C). Of note, glucose or FFAs had no effect on CREM-1 expression (around 45 kDa). We also studied the potential additive effects of glucose and palmitate on Cx36 and ICER-1γ expression. Glucose and palmitate did not synergize in reducing Cx36 expression or inducing ICER-1γ expression at high glucose concentration (20 mm) (Fig. 4A). However, at 10 mm glucose, palmitate further decreased Cx36 mRNA levels in INS-1E cells. These data were clearly inversely correlated with the levels of ICER-1γ mRNA, although the palmitate-mediated increase in ICER-1γ levels in the presence of 10 or 20 mm glucose did not reach statistical significance, as compared with untreated cells (Fig. 4B). Oleate treatment, which had no effect on Cx36 and ICER-1γ expression at low glucose, remained ineffective in presence of higher glucose concentrations. We have previously demonstrated that the human and rodent cx36 promoters contain a highly conserved cAMP-response element (CRE) located between bases –566 and –556 upstream of the transcription start site of the cx36 gene (10Allagnat F. Martin D. Condorelli D.F. Waeber G. Haefliger J.A. J. Cell Sci. 2005; 118:

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