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Stimulation of Acetyl-Coa Carboxylase Gene Expression by Glucose Requires Insulin Release and Sterol Regulatory Element Binding

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Stimulation of Acetyl-Coa Carboxylase Gene Expression by Glucose Requires Insulin Release and Sterol Regulatory Element Binding Stimulation of Acetyl-CoA Carboxylase Gene Expression by Glucose Requires Insulin Release and Sterol Regulatory Element Binding Protein 1c in Pancreatic MIN6 ␤-Cells Chrysovalantis Andreolas,1 Gabriela da Silva Xavier,1 Frederique Diraison,1 Chao Zhao,1 Aniko Varadi,1 Fernando Lopez-Casillas,2 Pascal Ferre´,3 Fabienne Foufelle,3 and Guy A. Rutter1 Acetyl-CoA carboxylase I (ACCI) is a key lipogenic enzyme whose induction in islet ␤-cells may contribute to glucolipotoxicity. Here, we provide evidence that levated glucose concentrations stimulate the enhanced insulin release plays an important role in the transcription of several genes involved in glu- activation of this gene by glucose. Glucose (30 vs. 3 cose metabolism in islet ␤-cells (1–6). These mmol/l) increased ACCI mRNA levels ϳ4-fold and stim- Einclude the liver isoform of pyruvate kinase ulated ACCI (pII) promoter activity >30-fold in MIN6 (L-PK) (1,7) and acetyl-CoA carboxylase I (ACCI) (3). ACCI cells. The latter effect was completely suppressed by is transcribed from two promoters (PI and PII) (8), both of blockade of insulin release or of insulin receptor signal- which are subject to control by nutrients (9). Although ing. However, added insulin substantially, but not com- ACCI catalyzes an essential step in fatty acid synthesis in pletely, mimicked the effects of glucose, suggesting that lipogenic tissues, this is usually a minor pathway of intracellular metabolites of glucose may also contribute glucose usage in ␤-cells (3). However, malonyl-CoA, the to transcriptional stimulation. Mutational analysis of immediate product of ACCI, may play a signaling role in the ACCI promoter, and antibody microinjection, re- vealed that the effect of glucose required sterol re- glucose-stimulated insulin secretion (10,11). sponse element binding protein (SREBP)-1c. Moreover, We have recently reported that preproinsulin and L-PK adenoviral transduction with dominant-negative–acting gene expression is regulated by glucose in MIN6 ␤-cells SREBP1c blocked ACCI gene induction, whereas consti- largely through the activated release of endogenous insu- tutively active SREBP1c increased ACCI mRNA levels. lin (12), consistent with findings in primary ␤-cells (13). Finally, glucose also stimulated SREBP1c transcription, Moreover, homozygous inactivation of the insulin receptor although this effect was independent of insulin release. specifically in islet ␤-cells leads to diabetes in mice (14), These data suggest that glucose regulates ACCI gene emphasizing the probable importance of insulin for the expression in the ␤-cell by complex mechanisms that regulation of genes in the ␤-cell (15). However, the molec- may involve the covalent modification of SREBP1c. ular mechanisms through which gene expression is regu- However, overexpression of SREBP1c also decreased lated by insulin in this cell type are poorly understood (5). glucose-stimulated insulin release, implicating SREBP1c In particular, the identity of the transcription factors ␤ induction in -cell lipotoxicity in some forms of type 2 involved remains a matter of controversy (2,9). diabetes. Diabetes 51:2536–2545, 2002 Sterol regulatory element binding proteins (SREBPs) (1) are members of the basic/helix-loop-helix/leucine zipper family of transcription factors and play a central role in the regulation of lipid synthesis in other mammalian tissues (16,17). Alternative transcriptional start sites and splicing give rise to two isoforms of SREBP1: SREBP1a and SREBP1c (16). SREBP1c, which is preferentially ex- pressed in liver and adipose tissue, has recently been From the 1Department of Biochemistry, University of Bristol, Bristol, U.K., the implicated in the regulation by insulin of lipogenic genes in 2Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico; and 3Unit 465 INSERM, Centre Biomedical des Cord- these tissues (18,19). Thus, in isolated hepatocytes, insulin eliers, Paris, France. increases the level of SREBP1c mRNA (18), the endo- Address correspondence and reprint requests to Professor G.A. Rutter, ϳ plasmic-reticulum–bound precursor (Mr 125), and the Department of Biochemistry, University of Bristol, Bristol, BS8 1TD, U.K. ϳ E-mail: [email protected]. NH2-terminal fragment of SREBP1 (Mr 50) present in the Received for publication 1 August 2001 and accepted in revised form 3 May nucleus. Moreover, SREBP1c is indispensable for the 2002. L C.A., G.d.S.X., and F.D. contributed equally to this work. induction by insulin of the fatty acid synthase and -PK and ACCI, acetyl-CoA carboxylase I; FAS, fatty acid synthase; L-PK, liver isoform ACCI genes in the liver (18). Finally, expression of the of pyruvate kinase; SRE, sterol response element; SREBP, sterol regulatory constitutively active NH2-terminal fragment of SREBP1c element binding protein; SREBP1c CA, constitutively active NH2-terminal fragment of SREBP1c; SREBP1c DN, dominant-negative–acting form of (corresponding to amino acids 1–403) was sufficient to SREBP1c. activate glucokinase gene expression in the absence of 2536 DIABETES, VOL. 51, AUGUST 2002 C. ANDREOLAS AND ASSOCIATES insulin and, at low glucose concentrations, in liver cells corresponding to the rat sequence (20) were used: forward, 5Ј-GGAGCCATG (19). GATTGCACATT; reverse, 5Ј-AGGAAGGCTTCCAGAGAGGA, corresponding Recent studies have also shown that SREBP1c mRNA is to amino acids 40–59 and 211–230 of mature SREBP1c. Oligonucleotide primers for ␤-actin mRNA were used as described (29). Primers for the present at low levels in islets from normal rats and at amplification of ACCI were as follows: forward, 5Ј-AACTATGAGGTGGATCG elevated levels in diabetic fa/fa rats (20). Islets from these GAG; reverse, 5Ј-TCCATACCGCATTACCATGC, corresponding to nucleotides animals also show enhanced deposition of triglyceride 4021–4040 and 4481–4500, respectively, of human ACCI cDNA (accession no. (21). However, up to now, neither the presence of SREBP1 U19822) (30). PCR products were separated on 2% agarose gels containing protein nor its regulation or functional importance has ethidium bromide (0.5 ␮g/ml) and quantitated by digital imaging (ImageQuant; ␤ Molecular Dynamics). been explored in pancreatic -cells. 6 ␤ Immunoblot (Western) analysis. MIN6 cells (1.2 ϫ 10 ) were extracted into We show here that, in clonal MIN6 -cells, the effects of radio-immunoprecipitation assay buffer comprising PBS supplemented with glucose on the ACCI promoter are likely to be mediated by 1.0% (vol/vol) Nonidet P40, 0.5% Naϩ deoxycholate, and 0.1% (wt/vol) sodium the release of insulin. Moreover, suppression of SREBP1c dodecylsulfate. Protein concentration was determined with a commercially function with a dominant-negative–acting form of the available kit using BSA as standard (BCA, category no. 23225; Pierce, Rockford, IL). To obtain nuclear extracts, cells were lysed by swelling in protein completely inhibits glucose-induced transcription ϩ of the ACCI gene, whereas overexpression of the nuclear hypotonic buffer (10 nmol/l HEPES, pH 7.9, 10 mmol/l KCl, and 0.1 mmol/l Na EDTA) as described (31). Proteins were separated by SDS-PAGE (7.5% wt/vol) fragment of SREBP1c to high levels activated ACCI gene and transferred to immobilon membranes using a discontinuous buffer system expression and promoter activity. These results suggest as described (32). Blots were probed with monoclonal antibody to SREBP1 that insulin-stimulated changes in the activity of nuclear (1:1,000 dilution) and revealed with horseradish peroxidase–conjugated anti- SREBP1c are important for the regulation of the ACCI mouse IgG (1:10,000) using an enhanced chemiluminescence detection system gene by glucose in islets, but leave open the possibility of (Roche Diagnostics). Plasmids. To generate the SREBP1c promoter.luciferase vector (pSREBP.Luc ), involvement from other factors. FF genomic DNA was isolated from MIN6 cells by lysis in TRI reagent. The immediate 5Ј flanking region of the SREBP1c gene, lying between exons 1 and RESEARCH DESIGN AND METHODS 2 (33), was amplified by PCR using the primer pair: forward, 5Ј-TTTTAGA Ϫ574 Ј ϩ25 Materials. Culture media and antibiotics were obtained from Life Technolo- TCT GGATCCAGAACTGGATCATCAG; reverse, 5 -TTTTAAGCTT CTAG gies (Paisley, U.K.). Other reagents were from Sigma (Poole, Dorset, U.K.) or GGCGTGCAGACGCTAC (BglII and HindIII sites are underlined; superscript BDH (Poole). indicates nucleotide positions with respect to transcriptional start site). PCR Methods was performed in a total volume of 50 ␮l, comprising 0.3 ␮g DNA, 0.2 mmol/l Cell culture. MIN6 cells (22) were used between passages 20 and 30 and nucleotide-triphosphate, 20 pmol of each primer, and 0.875 units Taq poly- grown in Dulbecco’s modified Eagle’s medium containing 15% (vol/vol) merase (Expand High Fidelity). After purification on a 2% (wt/vol) agarose gel, heat-inactivated FCS, 25 mmol/l glucose, 2 mmol/l glutamine, 50 ␮mol/l the 636-bp product was subcloned upstream of humanized firefly luciferase ␤-mercaptoethanol, 100 IU/ml penicillin, and 100 mg/ml streptomycin in a cDNA in plasmid pGL3 basic (Promega). Correct amplification and fragment humidified atmosphere at 37°C with 5% CO2, unless specified otherwise. MIN6 insertion were confirmed by automated sequencing. Plasmid pACCIpII- Ϫ cells were maintained and infected with viruses at 25 mmol/l glucose. Cells 1082.LucFF contained the immediate flanking region ( 1082 to 62) of the were transferred to medium containing 3 mmol/l glucose 16 h before exploring ACCI(pII) promoter in plasmid pGL3, as reported by Luo and Kim (34). the subsequent effects of glucose. Ϫ Plasmid pACCIpII-285.LucFF contained the fragment 285 to 62 upstream of Amplification of recombinant adenoviruses and cell infection. Adenovi- firefly luciferase and corresponded to deletion mutant pPII-CAT4 (35). pACCIpII- ruses expressing constitutively active SREBP1c (amino acids 1–403, wild-type 217.Luc was generated by subcloning the 291 bp AvaI-SalI fragment from 320 FF sequence) or dominant-negative (1–403, Y A) SREBP1c were constructed pII-1082-Luc into the SmaI site of pGL3-basic. pCMV.Luc was as described and amplified as described (18,23). Virus particles were purified on CsCl FF FF earlier (36).
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