Stimulation of Acetyl-CoA Carboxylase Expression by Glucose Requires Release and Sterol Regulatory Element Binding 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 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 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 (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 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). The expression plasmid for Renilla luciferase (pRL.CMV) was gradients before infection (24) at a multiplicity of infection of 30 viral particles purchased from Promega. cDNAs encoding the NH -terminal fragment of per cell. Infection efficiency was judged using enhanced green fluorescent 2 SREBP1c (1–403), or the mutated (Y320A) form, in plasmid pcDNA3 (Invitro- protein, also present in the adenoviral genome; Ͼ95% individual MIN6 cells were infected after 24–48 h. In all experiments, adenovirus expressing gen) were obtained from Dr. B. Spiegelman (Harvard Medical School, Boston, enhanced green fluorescent protein cDNA only (null) was used as control. MA). Assay of insulin secretion. MIN6 cells (1.5 ϫ 105) were perifused (0.5 PCR mutagenesis of plasmid pACCIpII-285.LucFF was carried out using the ml/min) at 37°C in Krebs-Ringer bicarbonate HEPES buffer (1 ml) comprising QuikChange Site-Directed Mutagenesis Kit (Stratagene), according to the manufacturer’s protocol. The following oligonucleotide primers (with mu- 125 mmol/l NaCl, 3.5 mmol/l KCl, 1.5 mmol/l CaCl2, 0.5 mmol/l MgSO4, 0.5 ϩ mmol/l KH2 PO4, 2.5 mmol/l NaHCO3, and 10 mmol/l HEPES-Na , pH 7.4, and tated nucleotides in bold) were used: distal sterol response element (SRE) Ј Ј containing initially 3 mmol/l glucose and equilibrated with 95:5 O2:CO2. mutant: forward, 5 -GGTACCGAGCTCGGAAACTCGGCATCGCCTCACG-3 ; Fractions (0.5 ml) were collected and stored at Ϫ20°C. Total cellular insulin reverse, 5Ј-CGTGAGGCGATGCCGAGTTTCCGAGCTCGGTACC-3Ј; proximal was extracted in acidified ethanol (25). Radioimmunoassay was performed by SRE mutant: forward, 5Ј-CGGCATCGCCGACCGTCGCCCCGCCCCG-3Ј; re- competition with 125I-labeled rat insulin (Linco Research, St. Charles, MO) verse, 5Ј-CGGGGCGGGGCGACGGTCGGCGATGCCG-3Ј. (24). Micro-injection and single-cell imaging of gene expression. Intranuclear Assay of lipid deposition with oil red O. Staining of neutral lipid in MIN6 micro-injection of plasmids was performed using an Eppendorf 5121/5246 cells was performed as described (26). Lipid-specific staining was visualized micromanipulator/microinjector (7,37) at plasmid concentrations of 0.1 mg/ml with a Leica TCS NT confocal microscope (excitation at 564 nm). (LucFF-based vectors) or 0.05 mg/ml (pCMV.RL, pcDNA3.SREBP1c [1–403]/ Semiquantitative RT-PCR. Total RNA was isolated by cell lysis in TRI Y320A) in injection buffer (2 mmol/l Tris-HCl, 0.2 mmol/l Naϩ EDTA, pH 8.0) Reagent (Sigma). First-strand cDNA synthesis was performed as described (38). SREBP1 antibody was dialyzed into injection buffer and co-injected into (27). RT reactions (1 h) were performed at 42°C using 3.5 ␮g RNA (determined the nuclear compartment at 0.1 mg/ml as described (39). Individual experi- from A assuming 0.025 A ⅐ ␮gϪ1 ⅐ mlϪ1), oligo (15)-dT primers (1.25 ␮g), and 260 ments involved the injection of 100–200 separate cells per condition, with an RT (500 units, Moloney-murine leukemia virus; Promega) in a total volume of 50 ␮l. PCRs were also performed in a total volume of 50 ␮l, comprising 5 ␮l efficiency of 5–20% productive injection. Cells were imaged 6 h after micro- cDNA product, 0.2 mmol/l nucleotide triphosphate, 20 pmol of each primer, injection and cultured under the conditions given in the figure legends. and 0.875 units Taq polymerase (Expand High Fidelity; Roche Diagnostics, Photon-counting imaging was performed in single living cells using an Lewes, U.K.). Endogenous SREBP1c cDNA was amplified using primers Olympus IX-70 inverted microscope (ϫ10 air objective, 0.4 numerical aper- corresponding to the murine exon 1c sequence (28): forward, 5ЈATCGGCGCG ture) and an intensified charge-coupled device camera (Photek; Lewes, East GAAGCT GTCGGGGTAGCGTC; reverse, 5Ј ACTGTCTTGGTTGTTGATGAGC Sussex, U.K.) (7,40). TGGAGCAT (predicted product size, 116 bp). For SREBP 1a, the following Electrophoretic mobility shift assay. Assays were performed on nuclear primers were used: forward, 5Ј-TAGTCCGAAGCCGGGTGGGCGCCGGCGC extracts (see above) according to the study by Daniel and Kim (35). CAT; reverse, 5Ј- GATGTCGTTCAAAACCGCTGTGTGTCCAGTTC (predicted Statistics. Data are expressed as means Ϯ SE. Comparison of groups was product size, 106 bp). To amplify exogenously expressed SREBP1c, primers performed with a one-tailed Student’s t test.

DIABETES, VOL. 51, AUGUST 2002 2537 ROLE OF SREBP1c IN PANCREATIC ISLET ␤-CELLS

FIG. 1. Regulation of ACCI gene expression by insulin and glucose. A: IN6 cells were incubated for 24 h at 3 mmol/l glucose and subsequently for 6 h at 3 or 30 mmol/l glucose, plus the indicated concentrations of insulin. Extraction of total RNA and semiquantitative PCR was performed as given in RESEARCH DESIGN AND METHODS. The results of a typical experiment (upper panel) and combined data from three separate experiments

(lower panel) are shown. B: MIN6 cells were co-microinjected with plasmids ACCIpII-1082.LucFF and pCMV.RL (see RESEARCH DESIGN AND METHODS) before culture for6hateither 3 or 30 mmol/l glucose. Where indicated, insulin was added to 20 nmol/l, and KCl was added to 13.0 mmol/l (vs. 5.5 mmol/l), 100 ␮mol/l verapamil (verap), and 200 ␮mol/l diazoxide (diazo). ACCI (pII) promoter activity, measured as the ratio of firefly:Renilla ␮m) and is presented as the means ؎ SE for 50 ؍ reniformis luciferase activity, was then determined by single-cell imaging (images; scale bar 20–60 productively injected cells in each case (three to five separate experiments). Neither a control promoter (cytomegalovirus) nor empty promoter vector (pGL3, Promega) was affected by [glucose] in this assay (not shown). C: Cells were microinjected with plasmids and incubated as in B, with the added presence of clonidine (5 ␮mol/l) or insulin (20 nmol/l) as indicated. D: Cells were microinjected with luciferase reporter plasmids plus either empty pcDNA3 or phosphatase and tensin homologue (PTEN)-expressing vector, as shown, before incubation and luciferase assays. *P < 0.05 and ***P < 0.001 for the effect of the indicated stimulus vs. 3 mmol/l glucose; ##P < 0.01 and ###P < 0.001 for the effect of the indicated agents vs. control.

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FIG. 2. Deletion analysis of the ACCI pro- moter. Single MIN6 cells were microinjected with the indicated promoter constructs plus pCMV.RL plus either IgG or anti-SREBP1 antibody (SREBP1-ab) (1 mg/ml) as shown. Normalized promoter activity was measured after a 6-h culture at the indicated glucose concentrations.

RESULTS of the phosphoinositide 3Јphosphatase phosphatase and Regulation of ACCI gene expression by glucose and tensin homologue (43) (Fig. 1D). insulin. ACCI mRNA was strongly (approximately four- Role of SREBP1c in the activation of the ACCI fold) induced by incubation of MIN6 cells for6hat30vs. promoter by glucose. To determine which cis-acting 3 mmol/l glucose (Fig. 1A) or at 3 mmol/l glucose in the element(s) was involved in its regulation, we next per- presence of insulin (Ͼ1 nmol/l). Glucose- and insulin- formed 5Ј deletion analysis of the ACCI (pII) promoter. induced changes in ACCI mRNA level largely reflected a Truncation of the promoter as far as nucleotide Ϫ285 and stimulation of mRNA synthesis, and 30 mmol/l glucose consequent removal of proximal SREs (an E-box and an caused a Ͼ30-fold activation of an ϳ1-kb fragment of the Sp1 binding site) had no effect on ACCI promoter activity ACCI (pII) promoter assayed in single cells after microin- at 30 mmol/l glucose but led to a small increase in basal jection and imaging of a firefly luciferase reporter con- promoter activity (Fig. 2). By contrast, further removal of Ϫ Ϫ struct (plasmid ACCI pII-1082.LucFF; Fig. 1B). nucleotides 285 to 217 and thus two SRE half-sites (44) We previously reported that incubation of MIN6 cells led to a marked (Ͼ85%) reduction in glucose-stimulated under the conditions used here increases insulin levels, promoter activity (Fig. 2). measured in the medium at the end of the 6-h incubation, To explore the potential role of SREBP1, acting at the from ϳ7 to 14 nmol/l (12). To determine whether the SRE half-sites, we next co-microinjected a monoclonal effects of high [glucose] on ACCI mRNA levels and pro- antibody specific for SREBP1 (Fig. 2, right panel). This led moter activity could largely be explained by activated to a complete inhibition of the stimulation by 30 mmol/l insulin release, we suppressed the latter with the L-type glucose of the full-length (Ϫ1,082 kb) and Ϫ285-tuncated Ca2ϩ channel blocker, verapamil (41), or with an opener of ACCI promoters (Fig. 2, top). By contrast, anti-SREBP1 ATP-sensitive Kϩ channels, diazoxide. Either agent com- antibodies had no further inhibitory effect on the construct pletely inhibited ACCI (pII) promoter activation by 30 in which the two SRE half-sites had been removed (trun- mmol/l glucose (Fig. 1B). To exclude the possibility that cation at Ϫ217 bp; Fig. 2). these drugs acted via a decrease in intracellular free To further dissect the role of the two SRE half-sites, we [Ca2ϩ] rather than a blockade of insulin release per se, we mutated each of these individually (44). Mutation of either ␣ determined the effect of the 2-adrenoreceptor agonist, site led to a strong glucose-independent induction of clonidine, which inhibits glucose-induced insulin secretion promoter activity (Fig. 2). Suggesting that SREBP1c may but not increases in [Ca2ϩ] (12,42). Clonidine completely bind to either half-site as a monomer (44,45), anti-SREBP1 reversed the stimulation of ACCI promoter activity by 30 antibodies caused substantial inhibition of the promoter mmol/l glucose, and this effect was rescued by 20 nmol/l mutated at either SRE half-site (Fig. 2). insulin (Fig. 1C). Our next experiments explored the impact of SREBP1 We next explored the possibility that a factor other than inhibition on the activity of the endogenous ACCI gene. insulin, present within secretory vesicles and cosecreted Suggesting that SREBP1c is probably the predominant with the hormone (e.g., ATP), may be responsible for SREBP1 isoform in MIN6 ␤-cells, SREBP1c mRNA was triggering ACCI promoter activation. Arguing against this clearly quantifiable after 20 PCR cycles, whereas SREBP1a possibility, the effects of 30 mmol/l glucose were com- mRNA was undetectable after 20 PCR cycles and only pletely suppressed by blockade of signaling by insulin, but weakly detectable after 25 cycles (data not shown). Cor- not ATP receptors, with the phosphatidylinositol 3Ј kinase respondingly, infection with an adenovirus expressing a inhibitor, LY294002 (not shown), or through coexpression dominant-negative–acting form of SREBP1c (SREBP1c

DIABETES, VOL. 51, AUGUST 2002 2539 ROLE OF SREBP1c IN PANCREATIC ISLET ␤-CELLS

FIG. 3. Regulation of ACCI gene expression by SREBP1c DN. A: MIN6 cells were infected with the indicated adenoviruses before incubation at the given glucose concentrations. ACCI and ␤-actin mRNA levels were assessed by semiquantitative RT-PCR. Means (؎SE) of three separate experiments are shown. B: ACCI (pII) promoter activity was measured in single cells (see Fig. 1) co-injected with either empty pcDNA3 or ؎ plasmid pcDNA3.SREBP Y320A (SREBP1c DN) as indicated. Luminescence ratio of typical single cells (left panel) and combined data (means ␮m. *P < 0.05 and ***P < 0.001 for 30 ؍ SE) from five experiments involving 20–60 productively injected cells is shown (right panel). Scale bar the effect of 30 mmol/l glucose; #P < 0.05 and ##P < 0.01 for the effect of SREBP1c DN vs. control plasmid.

DN) (residues 1–403, Y320A), which is unable to bind DNA (pII) promoter activity (Fig. 4B) at either glucose concen- (46), completely suppressed the glucose-induced increase tration. in ACCI mRNA (Fig. 3A). This inhibition could be ascribed We next determined whether the effects of SREBP1c CA to the blockade of ACCI transcription because microinjec- on ACCI gene expression might be due to enhanced insulin tion of a plasmid encoding SREBP1c DN totally abrogated secretion. As evidence against this possibility, SREBP1 CA the glucose-induced increase in ACCI (pII) promoter ac- expression caused a near-complete abolition of glucose- tivity (Fig. 3B). stimulated insulin release (Fig. 4C, right panel) and was Regulation of ACCI expression by constitutively ac- associated with a marked accumulation of intracellular tive SREBP1c. To test whether an increase in SREBP1c lipid (Fig. 4C, left panel). amount or activity in response to glucose may be respon- Regulation of SREBP1c expression by glucose and sible for the stimulation of the ACCI gene, MIN6 cells were insulin. Elevated glucose concentrations (30 vs. 3 mmol/l) infected with an adenovirus bearing the constitutively increased the levels of SREBP1c mRNA (Fig. 5A), total and active NH2-terminal fragment of SREBP1c (SREBP1c CA) mature SREBP1 protein (Fig. 5B), and SREBP1c transcrip- (SREBP1c 1–403) (19). Infection with this virus caused a tion (Fig. 5C). However, in contrast to the regulation of the marked increase in ACCI mRNA levels in cells incubated ACCI gene (Fig. 1), exogenously added insulin had no for6hateither 3 or 30 mmol/l glucose (Fig. 4A). Similarly, effect on any of the above parameters (Fig. 5). Moreover, microinjection of single cells with a plasmid construct neither high [glucose] nor insulin (20 nmol/l) enhanced bearing SREBP1c CA caused a marked increase in ACCI SREBP1c processing (Fig. 5B). Finally, the effects of

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DIABETES, VOL. 51, AUGUST 2002 2541 ROLE OF SREBP1c IN PANCREATIC ISLET ␤-CELLS glucose on the SREBP1c promoter were unaltered by activation of the ACCI gene. Moreover, the failure of blockade of insulin secretion with verapamil or diazoxide elevated glucose concentrations to affect ACCI transcrip- (Fig. 5C), and neither exogenously added insulin nor the tion when insulin release was completely inhibited with insulin secretagogue KCl affected SREBP1c promoter ac- verapamil or diazoxide (Fig. 1B) suggests that the impact tivity (Fig. 5C). of intracellular metabolites of glucose may be limited to a To further explore the role of the induction of SREBP1c potentiation of the effects of released insulin. These find- or other transcription factors in the stimulation of the ings thus contrast results previously reported in INS1 cells ACCI gene by glucose, we tested the impact of inhibiting (3), where non-glucose nutrients and other secretagogues protein synthesis globally. Although incubation of cells for (KCl and forskolin) had no apparent effect on ACCI mRNA 6 h with cycloheximide led to a stimulation of ACCI mRNA levels. One possible explanation of this apparent discrep- levels at 3 mmol/l glucose, a significant further increase in ancy is that glucose regulates ACCI gene expression by ACCI [mRNA] in response to 30 mmol/l glucose was still distinct mechanisms in INS1 and MIN6 cells. However, we observed (ratios of ACCI: ␤-actin mRNA in the absence of have noted that incubation with KCl has deleterious effects cycloheximide 0.59 Ϯ 0.10 and 0.96 Ϯ 0.14 at 3 and 30 on MIN6 cell morphology and does not provoke the mmol/l glucose, respectively; corresponding values with accumulation of ACCI mRNA at the 24-h time point used 50 ␮g/ml cycloheximide were 1.08 Ϯ 0.02 and 1.68 Ϯ 0.19, by Brun et al. (3). Moreover, the effects of nutrient n ϭ 3 experiments in each case, P Ͻ 0.05 for the effect of secretagogues on the release of insulin were not examined 30 mmol/l glucose). by these authors. Thus, it would seem difficult to exclude Effects of glucose and insulin on SREBP1c binding a role for secreted insulin in these earlier studies, espe- DNA activity. The above data suggest that glucose acts to cially because the effects of blocking insulin release (while stimulate ACCI gene expression through a mechanism that preserving glucose metabolism) were not explored. requires preexisting SREBP1c protein but not the activa- Role of SREBP1c in the regulation of ACCI gene tion of SREBP1 gene expression or SREBP1c processing. expression by glucose and insulin. Consistent with a Therefore, to determine whether glucose or insulin en- mechanism involving the action of secreted insulin, the hances the DNA binding activity of preexisting SREBP1c, effects of glucose on ACCI gene expression reported here we used electrophoretic mobility shift assay. However, required the presence of nuclear SREBP1c (Figs. 2 and 3) binding to an oligonucleotide corresponding to the two (18). However, induction of SREBP1c gene expression by SRE half-sites delineated as the likely site of SREBP1c glucose should not be considered responsible for the binding by promoter deletion analysis (Fig. 2) was too effects of glucose on the ACCI gene at early time points weak to be detected in extracts from cells incubated at because ACCI mRNA was still induced by glucose when either 3 or 30 mmol/l glucose (not shown). the synthesis of new proteins was suppressed (see RE- SULTS). Moreover, high glucose concentrations stimulated DISCUSSION SREBP1c promoter activity independently of insulin re- Involvement of insulin release in the regulation of lease (Fig. 5C), whereas the latter apparently activated ACCI gene expression by glucose. Similar to preproin- ACCI transcription (Fig. 1). In this respect, the regulation sulin (13,38,47) and L-PK (12) gene expression, we show of the SREBP1c gene in ␤-cells is different from that in the here that, at relatively early time points (Յ6 h), ACCI gene liver, where insulin alone, but not glucose, induces expression in ␤-cells is probably stimulated by high glu- SREBP1c expression (19). cose concentrations through a mechanism principally in- Our data suggest instead that an insulin-stimulated volving stimulated insulin release (see Fig. 1 and RESULTS). modification of the NH2-terminal fragment of SREBP1c, Thus, addition of the hormone was as efficient as high and thus the transactivation or DNA binding capacity of [glucose] in increasing ACCI mRNA levels (Fig. 1A). this factor, may be responsible for the activation of the However, suggesting that intracellular metabolites of glu- ACCI gene in response to high glucose. Although the cose such as glucose-6-phosphate (3) may also play some nature of this modification is unknown, it is of note that role in transcriptional stimulation, added insulin (20 SREBP1c can be phosphorylated in vitro by mitogen- nmol/l) was less effective than high glucose in stimulating activated protein kinases (48,49), a family of enzymes ACCI promoter activity (Fig. 1B). Furthermore, 30 mmol/l strongly activated by glucose in ␤-cell lines (50,51). Fur- glucose still activated the ACCI promoter after expression ther studies of the phosphorylation state of SREBP1c in of active SREBP1c (Fig. 4A and B)—conditions under intact cells will be required to resolve these issues. The which glucose-stimulated insulin release was substan- mechanisms whereby glucose may regulate the ACCI and tially, although not completely, inhibited (Fig. 4C). How- other genes in the ␤-cell are summarized in Fig. 6. ever, under these conditions, where the ACCI promoter Lopez et al. (52) have previously shown that purified was “super-activated” by active SREBP1c, it is conceivable recombinant SREBP1c is capable of binding oligonucleo- that even the modest stimulation of insulin release by tides corresponding to the two half-site SREs in the region glucose (Fig. 4C) may contribute to further transcriptional of Ϫ285 to Ϫ247 with respect to the ACCI transcriptional

FIG. 4. Effect of overexpression of SREBP1c CA on ACCI gene expression, lipogenesis, and insulin secretion. A: ACCI mRNA levels were assessed after infection with the shown adenoviruses. B: ACCI (pII) promoter activity was assessed in cells co-microinjected with plasmids ACCIpII-

1082.LucFF and pCMV.RL, plus either empty pcDNA3 or pcDNA3.SREBP1c (1–403) (SREBP1c CA) before culture for6hateither 3 or 30 mmol/l ␮m. Mean data from three separate experiments are given (؎SE) in A and B.*P < 0.05, **P < 0.01, or ***P < 0.001 for 75 ؍ glucose. Scale bar the effect of 30 vs. 3 mmol/l glucose; #P < 0.05 and ##P < 0.01 for the effect of SREBP1c CA–expressing vs. null adenovirus. C: Adenoviral-mediated expression of SREBP1c (1–403) protein (left, upper panel) and the impact on lipid deposition (left, two lower panels) in cells incubated at 30 mmol/l glucose were assessed as described in RESEARCH DESIGN AND METHODS. Insulin secretion (right panel) was measured during perifusion with Krebs-Ringer bicarbonate medium at the indicated glucose concentrations. *P < 0.01 vs. null virus.

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FIG. 5. Regulation of SREBP1c gene expression by glucose and insulin. A: After incubation of cells at the indicated concentrations of glucose and added insulin, SREBP1c mRNA levels were assessed by semiquantitative RT-PCR, as given in RESEARCH DESIGN AND METHODS. Data (؎SE) are given from three separate experiments. B: SREBP1 protein was quantitated by Western analysis in cells incubated as in A, and the amounts of SREBP1 precursor (125 kDa) and mature SREBP1 (50 kDa) were quantitated by densitometric analysis of the immunoblots obtained in three separate experiments. C: SREBP1c promoter activity was quantitated by single-cell analysis (see RESEARCH DESIGN AND METHODS) after microinjection and incubation for6hateither 3 or 30 mmol/l glucose and in the presence of the indicated additions (see Fig. 1 legend). Means (؎SE) of data from 20–60 productively injected cells (three to five experiments) are given in the histogram. *P < 0.05 and ***P < 0.001 for the effects of 30 vs. 3 mmol/l glucose. verap, verapamil; diazo, diazoxide.

DIABETES, VOL. 51, AUGUST 2002 2543 ROLE OF SREBP1c IN PANCREATIC ISLET ␤-CELLS

search Council, and the European Union (CT 97.3011 to P.F., F.F., and G.A.R.). F.D. was supported by fellowships from Association de Langue Franc¸aise pour l’E´ tude du Diabe`te et des Maladies Me´taboliques, Institut Franc¸ais de la Nutrition (France), and the European Union (Marie Curie). We thank Dr. Isabelle Leclerc for helpful discussion.

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