The Transcription Factor SREBP-1c Is Instrumental in the Development of औ-Cell Dysfunction
2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês
10.1074/jbc.m212488200
ISSN1083-351X
AutoresHaiyan Wang, Pierre Maechler, Peter A. Antinozzi, Laura Herrero, Kerstin A. Hagenfeldt-Johansson, Anneli Björklund, Claes B. Wollheim,
Tópico(s)Pancreatic function and diabetes
ResumoAccumulation of lipids in non-adipose tissues is often associated with Type 2 diabetes and its complications. Elevated expression of the lipogenic transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), has been demonstrated in islets and liver of diabetic animals. To elucidate the molecular mechanisms underlying SREBP-1c-induced औ-cell dysfunction, we employed the Tet-On inducible system to achieve tightly controlled and conditional expression of the nuclear active form of SREBP-1c (naSREBP-1c) in INS-1 cells. Controlled expression of naSREBP-1c induced massive accumulation of lipid droplets and blunted nutrient-stimulated insulin secretion in INS-1 cells. K+-evoked insulin exocytosis was unaltered. Quantification of the gene expression profile in this INS-1 stable clone revealed that naSREBP-1c induced औ-cell dysfunction by targeting multiple genes dedicated to carbohydrate metabolism, lipid biosynthesis, cell growth, and apoptosis. naSREBP-1c elicits cell growth-arrest and eventually apoptosis. We also found that the SREBP-1c processing in औ-cells was irresponsive to acute stimulation of glucose and insulin, which was distinct from that in lipogenic tissues. However, 2-day exposure to these agents promoted SREBP-1c processing. Therefore, the SREBP-1c maturation could be implicated in the pathogenesis of औ-cell glucolipotoxicity. Accumulation of lipids in non-adipose tissues is often associated with Type 2 diabetes and its complications. Elevated expression of the lipogenic transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), has been demonstrated in islets and liver of diabetic animals. To elucidate the molecular mechanisms underlying SREBP-1c-induced औ-cell dysfunction, we employed the Tet-On inducible system to achieve tightly controlled and conditional expression of the nuclear active form of SREBP-1c (naSREBP-1c) in INS-1 cells. Controlled expression of naSREBP-1c induced massive accumulation of lipid droplets and blunted nutrient-stimulated insulin secretion in INS-1 cells. K+-evoked insulin exocytosis was unaltered. Quantification of the gene expression profile in this INS-1 stable clone revealed that naSREBP-1c induced औ-cell dysfunction by targeting multiple genes dedicated to carbohydrate metabolism, lipid biosynthesis, cell growth, and apoptosis. naSREBP-1c elicits cell growth-arrest and eventually apoptosis. We also found that the SREBP-1c processing in औ-cells was irresponsive to acute stimulation of glucose and insulin, which was distinct from that in lipogenic tissues. However, 2-day exposure to these agents promoted SREBP-1c processing. Therefore, the SREBP-1c maturation could be implicated in the pathogenesis of औ-cell glucolipotoxicity. sterol regulatory element-binding protein endoplasmic reticulum adipocyte determination and differentiation factor-1 peroxisome proliferator-activated receptor nuclear active form of SREBP-1c phosphate-buffered saline bovine serum albumin Krebs-Ringer-bicarbonate-HEPES buffer uncoupling protein carbonyl cyanide 4-trifluoromethoxyphenylhydrazone pancreas duodenum homeobox hepatocyte nuclear factor insulin receptor substrate tumor necrosis factor The lipogenic transcription factors, sterol regulatory element binding proteins (SREBPs)1are transmembrane proteins of the endoplasmic reticulum (ER). In response to low sterol and other unidentified factors, SREBP cleavage-activating protein escorts SREBPs from the ER to the Golgi, where SREBPs are sequentially cleaved by Site-1 protease and Site-2 protease. The processed mature SREBPs enter the nucleus and transactivate target genes (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar). Three SREBP isoforms have been identified: SREBP-1a and -1c (alternatively known as adipocyte determination and differentiation factor-1 (ADD1)) (2Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (536) Google Scholar), which are derived from the same gene through alternative splicing, and SREBP-2, which is encoded by a distinct gene (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar). SREBPs play an essential role in regulation of lipid homeostasis in animals and have been shown to directly activate the expression of more than 30 genes dedicated to the biosynthesis of cholesterol, fatty acids, triglycerides, and phospholipids (3Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 2002; 109: 1125-1131Crossref PubMed Scopus (3835) Google Scholar). SREBP-1 preferentially regulates genes implicated in fatty acid synthesis, whereas SREBP-2 preferentially activates genes involved in cholesterol synthesis (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar). In particular, SREBP-1c mediates insulin effects on lipogenic gene expression in both adipocytes and liver (4Kim J.B. Sarraf P. Wright M. Yao K.M. Mueller E. Solanes G. Lowell B.B. Spiegelman B.M. J. Clin. Invest. 1998; 101: 1-9Crossref PubMed Scopus (614) Google Scholar, 5Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (599) Google Scholar). Type 2 diabetes mellitus is a common disorder that affects ∼57 of the population worldwide, especially in industrialized countries (6Zimmet P. Alberti K.G. Shaw J. Nature. 2001; 414: 782-787Crossref PubMed Scopus (4594) Google Scholar). Affected patients are usually obese with accumulation of lipids in non-adipose tissues such as the pancreatic islets, liver, heart, skeletal muscle, and blood vessels (7Unger R.H. Zhou Y.T. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2327-2332Crossref PubMed Scopus (376) Google Scholar, 8Friedman J. Nature. 2002; 415: 268-269Crossref PubMed Scopus (102) Google Scholar, 9Unger R.H. Annu. Rev. Med. 2002; 53: 319-336Crossref PubMed Scopus (847) Google Scholar). This is associated with impaired glucose-stimulated insulin secretion, increased hepatic glucose production, peripheral insulin resistance, and late complications in various organs (10Cavaghan M.K. Ehrmann D.A. Polonsky K.S. J. Clin. Invest. 2000; 106: 329-333Crossref PubMed Scopus (307) Google Scholar, 11Saltiel A.R. J. Clin. Invest. 2000; 106: 163-164Crossref PubMed Scopus (103) Google Scholar). The ultimate precipitating process responsible for the development of Type 2 diabetes is the failure of the pancreatic औ-cells to compensate for insulin resistance (7Unger R.H. Zhou Y.T. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2327-2332Crossref PubMed Scopus (376) Google Scholar, 10Cavaghan M.K. Ehrmann D.A. Polonsky K.S. J. Clin. Invest. 2000; 106: 329-333Crossref PubMed Scopus (307) Google Scholar, 11Saltiel A.R. J. Clin. Invest. 2000; 106: 163-164Crossref PubMed Scopus (103) Google Scholar). However, the mechanism by which the औ-cells become unable to meet increased insulin demands remains to be established. Elevated expression of SREBP-1c has been demonstrated in islets or liver of diabetic animals, such as Zucker diabetic fatty rats,ob/ob mice, insulin receptor substrate-2-deficient mice, and a transgenic mouse model of lipodystrophy (7Unger R.H. Zhou Y.T. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2327-2332Crossref PubMed Scopus (376) Google Scholar, 12Kakuma T. 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On the other hand, preventing SREBP-1c overexpression is a common function of leptin, metformin, and PPARγ agonists, which is well correlated with their antidiabetic effects (7Unger R.H. Zhou Y.T. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2327-2332Crossref PubMed Scopus (376) Google Scholar,12Kakuma T. Lee Y. Higa M. Wang Z. Pan W. Shimomura I. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8536-8541Crossref PubMed Scopus (230) Google Scholar, 13Shimomura I. Hammer R.E. Ikemoto S. Brown M.S. Goldstein J.L. Nature. 1999; 401: 73-76Crossref PubMed Scopus (865) Google Scholar, 14Lin H.Z. Yang S.Q. Chuckaree C. Kuhajda F. Ronnet G. Diehl A.M. Nat. Med. 2000; 6: 998-1003Crossref PubMed Scopus (616) Google Scholar, 15Tobe K. Suzuki R. Aoyama M. Yamauchi T. Kamon J. Kubota N. Terauchi Y. Matsui J. Akanuma Y. Kimura S. Tanaka J. Abe M. Ohsumi J. Nagai R. Kadowaki T. J. Biol. Chem. 2001; 276: 38337-38340Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). An important function of leptin in the regulation of fatty acid homeostasis is to restrict the lipid storage in adipocytes and to limit lipid accumulation in non-adipocytes, thereby protecting them from lipotoxicity (7Unger R.H. Zhou Y.T. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2327-2332Crossref PubMed Scopus (376) Google Scholar, 9Unger R.H. Annu. Rev. Med. 2002; 53: 319-336Crossref PubMed Scopus (847) Google Scholar). Infusion of recombinant leptin reverses insulin resistance and hyperglycemia in the transgenic model of congenital generalized lipodystrophy and in ob/obmice (13Shimomura I. Hammer R.E. Ikemoto S. Brown M.S. Goldstein J.L. Nature. 1999; 401: 73-76Crossref PubMed Scopus (865) Google Scholar). Adenovirus-mediated hyperleptinemia also decreases the expression of SREBP-1c and lipogenic genes in liver and islets of wild-type fa/fa rats (12Kakuma T. Lee Y. Higa M. Wang Z. Pan W. Shimomura I. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8536-8541Crossref PubMed Scopus (230) Google Scholar). Leptin also prevents the SREBP-1c overexpression in insulin receptor substrate (IRS)-2-null mice (15Tobe K. Suzuki R. Aoyama M. Yamauchi T. Kamon J. Kubota N. Terauchi Y. Matsui J. Akanuma Y. Kimura S. Tanaka J. Abe M. Ohsumi J. Nagai R. Kadowaki T. J. Biol. Chem. 2001; 276: 38337-38340Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). It is noteworthy that metformin, an oral antihyperglycemic agent, also corrects fatty liver disease in ob/obmice by inhibition of obesity-related induction of SREBP-1c (14Lin H.Z. Yang S.Q. Chuckaree C. Kuhajda F. Ronnet G. Diehl A.M. Nat. Med. 2000; 6: 998-1003Crossref PubMed Scopus (616) Google Scholar). The inhibitory effects of metformin on hepatic SREBP-1c expression involves activation of AMP-activated protein kinase (17Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Invest. 2001; 108: 1167-1174Crossref PubMed Scopus (4471) Google Scholar). Similarly, Troglitazone, an antidiabetic agent and a high-affinity ligand for the PPARγ, prevents SREBP-1c overexpression in Zucker diabetic fatty rats (12Kakuma T. Lee Y. Higa M. Wang Z. Pan W. Shimomura I. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8536-8541Crossref PubMed Scopus (230) Google Scholar). In addition, it has recently been reported that adenovirus-mediated overexpression of SREBP-1c in MIN6 cells results in impaired glucose-stimulated insulin secretion (18Andreolas C. da Silva Xavier G. Diraison F. Zhao C. Varadi A. Lopez-Casillas F. Ferre P. Foufelle F. Rutter G.A. Diabetes. 2002; 51: 2536-2545Crossref PubMed Scopus (55) Google Scholar). Therefore, these studies suggest that overexpression of SREBP-1c may be the cause of both liver steatosis and islet औ-cell dysfunction. The present study was designed to evaluate the direct correlation between SREBP-1c overexpression and औ-cell dysfunction and to elucidate the underlying molecular mechanism. The Tet-On system was employed in INS-1 cells to achieve tightly controlled and inducible expression of a nuclear active form of SREBP-1c (naSREBP-1c; N-terminal 1–403 amino acids) (19Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (850) Google Scholar). Quantification of the gene expression profile in this INS-1 stable cell line revealed that naSREBP-1c induced औ-cell dysfunction by targeting multiple genes implicated in carbohydrate metabolism, lipid biosynthesis, cell growth, and eventually apoptosis. Rat insulinoma INS-1 cell-derived clones were cultured in RPMI 1640 containing 11.2 mm glucose (20Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Crossref PubMed Scopus (748) Google Scholar), unless otherwise indicated. The first step stable clone INS-rऔ cell line, which carries the reverse tetracycline/doxycycline-dependent transactivator (21Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2051) Google Scholar) was described previously (22Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (119) Google Scholar, 23Wang H. Maechler P. Ritz-Laser B. Hagenfeldt K.A. Ishihara H. Philippe J. Wollheim C.B. J. Biol. Chem. 2001; 276: 25279-25286Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The plasmid used in the secondary stable transfection were constructed by subcloning the cDNA encoding the nuclear active form of SREBP-1c (naSREBP-1c/ADD1-(1–403) (19Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (850) Google Scholar), kindly supplied by Dr. B. M. Spiegelman) into the expression vector PUHD10–3 (21Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2051) Google Scholar) (a generous gift from Dr. H. Bujard). The procedures for stable transfection, clone selection and screening were described previously (22Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (119) Google Scholar). For immunofluorescence, cells grown on polyornithine-treated glass coverslips were treated for 24 h with or without 500 ng/ml doxycycline. Cells were then washed, fixed in 47 paraformaldehyde, permeabilized with 0.17 Triton X-100 in phosphate-buffered saline containing 17 BSA (PBS-BSA). The preparation was then blocked with PBS-BSA before incubating with the first antibody, anti-SREBP-1 (Santa Cruz, Basel, Switzerland) (1:100 dilution), followed by the second antibody labeling. For Western blotting cells were cultured with 0, 75, 150, and 500 ng/ml doxycycline for 24 h. Rat islets were isolated by collagenase digestion as described (24Rubi B. Ishihara H. Hegardt F.G. Wollheim C.B. Maechler P. J. Biol. Chem. 2001; 276: 36391-36396Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and their nuclear proteins were extracted as previously reported (25Schreiber E. Matthias P. Muller M.M. Schaffner W. EMBO J. 1988; 7: 4221-4229Crossref PubMed Scopus (193) Google Scholar). Total cell proteins were prepared by lysis and sonication of naSREBP-1c#233 and INS-1E cells in buffer containing 20 mm Tris-HCl, pH 7.4, 2 mm EDTA, 2 mm EGTA, 17 Triton X-100, and 1 mmphenylmethylsulfonyl fluoride. Nuclear extracts and total cellular proteins were fractionated by 97 SDS-PAGE. The dilution for SREBP-1 antibody is 1:1,000. Immunoblotting procedures were performed as described previously (26Wang H. Maechler P. Hagenfeldt K.A. Wollheim C.B. EMBO J. 1998; 17: 6701-6713Crossref PubMed Google Scholar) using enhanced chemiluminescence (Pierce) for detection. Cells were cultured with 0, 75, 150, and 500 ng/ml doxycycline for 24 h. Cells were fixed and stained as previously reported (19Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (850) Google Scholar). Lipid droplets were visualized using phase-contrast microscopy (Nikon Diaphot). Cellular triglyceride content was determined using Triglyceride (GPO-TRINDER) kit (Sigma) according to the manufacturer's protocol. Insulin secretion in naSREBP-1c#233 cells was measured in 12-well plates over a period of 30 min, in Krebs-Ringer-bicarbonate-HEPES buffer (KRBH, 140 mm NaCl, 3.6 mm KCl, 0.5 mmNaH2PO4, 0.5 mm MgSO4, 1.5 mm CaCl2, 2 mmNaHCO3, 10 mm HEPES, 0.17 BSA) containing indicated concentrations of glucose. Insulin content was determined after extraction with acid ethanol following the procedures of Wanget al. (26Wang H. Maechler P. Hagenfeldt K.A. Wollheim C.B. EMBO J. 1998; 17: 6701-6713Crossref PubMed Google Scholar). Insulin was detected by radioimmunoassay using rat insulin as a standard (26Wang H. Maechler P. Hagenfeldt K.A. Wollheim C.B. EMBO J. 1998; 17: 6701-6713Crossref PubMed Google Scholar). Cells were cultured with or without 500 ng/ml doxycycline in 2.5 mmglucose medium for 16 h and then incubated for a further 8 h at 2.5, 6, 12, and 24 mm glucose concentrations. Alternatively, naSREBP-1c#233 cells were cultured with or without 75 ng/ml doxycycline in standard medium (11.2 mmglucose) for 0, 24, 48, and 96 h. Total RNA was extracted and blotted to nylon membranes as described previously (22Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (119) Google Scholar). The membrane was prehybridized and then hybridized to 32P-labeled random primer cDNA probes according to Wang and Iynedijian (22Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (119) Google Scholar). To ensure equal RNA loading and even transfer, all membranes were stripped and re-hybridized with a "housekeeping gene" probe cyclophilin. cDNA fragments used as probes for SREBP-1c, hepatocyte nuclear factor (HNF)-1α, HNF-4α, glucokinase, Glut-2, insulin, Sur1, Kir6.2, and pancreas duodenum homeobox (Pdx-1) mRNA detection were digested from the corresponding plasmids. cDNA probes for rat islet amyloid polypeptide, Nkx6.1, adenine nucleotide translocator 1 and 2, mitochondrial uncoupling protein 2 (UCP2), mitochondrial glutamate dehydrogenase, citrate synthase, glyceraldehydes-3 phosphate dehydrogenase, fatty acid synthase, acetyl-CoA carboxylase, glycerol-phosphate acyltransferase, carnitine palmitoyltransferase-1, acyl-CoA oxidase, 3-hydroxy-3-methylglutaryl-CoA reductase, P21WAF1/CIP1, P27KIP1, Bax, Bad, APO-1, Cdk-4, IRS-2, and glucagon-like peptide-1 receptor were prepared by reverse transcription-PCR and confirmed by sequencing. Cells seeded in 24-well plates were cultured with or without 500 ng/ml doxycycline in 11.2 mmglucose medium for 24, 48, and 96 h (day 1, day 2, and day 4, respectively). Cells were then maintained for 2 h in 2.5 mm glucose medium at 37 °C before loading with 10 ॖg/ml rhodamine-123 for 20 min at 37 °C in KRBH (28Maechler P. Kennedy E.D. Pozzan T. Wollheim C.B. EMBO J. 1997; 16: 3833-3841Crossref PubMed Scopus (164) Google Scholar). The Δψm was monitored in a plate-reader fluorimeter (Fluostar Optima, BMG Labtechnologies, Offenburg, Germany) with excitation and emission filters set at 485 and 520 nm, respectively, at 37 °C with automated injectors for glucose (addition of 13 mm on top of basal 2.5 mm) and carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP). Quantification of cell proliferation/viability was measured by a Quick Cell Proliferation Assay Kit (LabForce/MBL, Nunningen, Switzerland) according to the manufacturer's protocol. This assay is based on the reduction of a tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. Expansion in the number of viable cells results in an increase in the overall activity of the mitochondrial dehydrogenases and subsequently an augmentation in the amount of formazan dye formed. The formazan dye produced by viable cells was quantified with a multiwell spectrophotometer by measuring the absorbance at 440 nm. Experiments for DNA fragmentation were performed using a Quick Apoptosis DNA Ladder Detection Kit (LabForce/MBL) following the manufacturer's protocol. Detection of mitochondrial cytochromec release into cytosol was performed as described previously (29Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4422) Google Scholar). Briefly, cells in 15-cm dishes were harvested and suspended in Buffer A (20 mm HEPES-KOH, pH 7.5, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 250 mm sucrose, 1 mm phenylmethylsulfonyl fluoride) and homogenized by a Dounce homogenizer. Cell debris and nuclei were removed by centrifugation at 1000 × g for 10 min at 4 °C. The supernatant was further centrifuged at 10,000 ×g for 20 min. The cytosolic proteins (supernatant fractions) were separated by 157 SDS-PAGE and analyzed by Western blotting with a specific antibody against cytochrome c (LabForce/MBL). A rat insulinoma INS-1-derived stable cell line, INSrऔ (23Wang H. Maechler P. Ritz-Laser B. Hagenfeldt K.A. Ishihara H. Philippe J. Wollheim C.B. J. Biol. Chem. 2001; 276: 25279-25286Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), which expresses the reverse tetracycline-dependent activator (21Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2051) Google Scholar), was used for the secondary stable transfection with an expression vector, PUHD10–3 (21Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2051) Google Scholar), carrying the naSREBP-1c and a plasmid pTKhygro containing the hygromycin-resistant marker. Five-round stable transfection experiments were performed, and a total of 400 hygromycin-resistant clones were screened by Northern blotting for clones positively expressing naSREBP-1c after doxycycline induction. Our persistent effort was rewarded. Only one clone, termed naSREBP-1c#233, which actually represented undetectable background expression under non-induced conditions, showed remarkable expression of naSREBP-1c mRNA after doxycycline induction. From experience, we would have anticipated that 10–207 of hygromycin-resistant clones should positively express the transgene. The unexpected results indicate that expression of naSREBP-1c even at leakage level was sufficient to cause "औ-cell toxicity." The INS-1 stable cell line naSREBP-1c#233 provided a unique chance to study the impact of naSREBP-1c expression on औ-cell function. Immunofluorescence (Fig.1A) with an antibody against the N terminus of SREBP1c illustrated that nuclear localized naSREBP1c protein was induced homogeneously in naSREBP-1c#233 cells cultured with 500 ng/ml doxycycline for 24 h. Western blotting (Fig. 1B) with the same antibody demonstrated that naSREBP-1c#233 cells expressed the transgene-encoded protein in a doxycycline dose-dependent manner. As predicted, naSREBP-1c#233 cells did not show detectable expression of naSREBP-1c protein under non-induced conditions (Fig. 1). The protein levels of naSREBP-1c in cells cultured with 75, 150, and 500 ng/ml were ∼107, 257, and 1507, respectively, of endogenous level of SREBP-1c precursor protein (Fig. 1B). Oil Red O staining showed that induction of naSREBP-1c with 75, 150, and 500 ng/ml doxycycline for 24 h resulted in accumulation of lipid droplets in INS-1 cells cultured in standard medium (107 fetal calf serum) (Fig.2A). Cellular triglyceride content was increased by 397 (−Dox: 0.536 ± 0.08; +Dox: 0.745 ± 0.10 mg/mg protein, p < 0.001,n = 14) after 24 h of induction with 500 ng/ml doxycycline. Similar results were obtained in cells cultured in serum-free medium (data not shown), suggesting that the naSREBP-1c-induced lipid accumulation was not due to uptake of fatty acids by the cells. This contention was supported by results of quantitative Northern blotting. Induction of naSREBP-1c significantly increased the expression of genes dedicated to biosynthesis of fatty acids and cholesterol but did not alter the expression of genes involved in औ-oxidation of fatty acids (Fig. 2, B andC). The increased mRNA levels of fatty acid synthase, acetyl-CoA carboxylase, glycerol-phosphate acyltransferase, and 3-hydroxy-3-methylglutaryl-CoA reductase would explain the naSREBP-1c-induced lipid accumulation. In addition, the increased lipogenesis did not raise the transcript levels of carnitine palmitoyltransferase-1 and acyl-CoA oxidase, which are important for fatty acid metabolism. Induction of naSREBP1c with 500 ng/ml doxycycline for 24 h (Fig. 3A) or 75 ng/ml doxycycline for 96 h (Fig. 3B) caused blunted glucose- and leucine-stimulated but not K+-depolarization-induced insulin secretion. Glucose generates ATP and other metabolic-coupling factors important for insulin exocytosis through glycolysis and mitochondrial oxidation (30Wollheim C.B. Diabetologia. 2000; 43: 265-277Crossref PubMed Scopus (167) Google Scholar). Leucine stimulates insulin release through direct uptake and metabolism by the mitochondria, thereby providing substrates for the tricarboxylic acid cycle (30Wollheim C.B. Diabetologia. 2000; 43: 265-277Crossref PubMed Scopus (167) Google Scholar). K+ causes insulin secretion by depolarization of the औ-cell membrane, resulting in increased cytosolic Ca2+ (30Wollheim C.B. Diabetologia. 2000; 43: 265-277Crossref PubMed Scopus (167) Google Scholar). To explore the molecular targets of naSREBP-1c responsible for the impaired metabolism-secretion coupling, we examined the gene expression profile in naSREBP-1c#233 cells. Quantitative Northern blotting revealed that similar induction of naSREBP1c also caused marked down-regulation of glucokinase and Glut2 but up-regulation of mitochondrial UCP-2 (Fig. 4,A and B). In contrast, naSREBP-1c did not alter the mRNA levels of glyceraldehydes-3-phosphate dehydrogenase, mitochondrial adenine nucleotide translocator-1 and -2, glutamate dehydrogenase, citrate synthase, KATP channel subunits SUR1 and KIR6.2 (Fig. 4,A and B). Glucokinase is the rate-limiting enzyme for glycolysis and thereby determines औ-cell glucose sensing (22Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (119) Google Scholar,31Matschinsky F.M. Diabetes. 1996; 45: 223-241Crossref PubMed Scopus (0) Google Scholar). The effect of naSEBP-1c on औ-glucokinase expression is opposite to its action on liver glucokinase, which is transcribed from a distinct liver-specific promoter (5Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (599) Google Scholar, 32Magnuson M.A. Andreone T.L. Printz R.L. Koch S. Granner D.K. Proc. Natl. Acad. Sci. U. S. 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Induction of naSREBP1c also suppressed the expression of Pdx-1, HNF-4α, and CCAAT/enhancer-binding protein-औ in a dose- and time-dependent manner (Fig. 4, C andD). The HNF-1α expression was unaffected, whereas the mRNA level of a औ-cell transcription factor Nkx6.1 was increased by naSREBP-1c (Fig. 4, C and D). High-level induction of naSREBP-1c also decreased the transcript levels of insulin and islet amyloid polypeptide (Fig. 4C). Mitochondrial membrane potential (Δψm), reflecting electron transport chain activity, was measured in attached cells by monitoring rhodamine-123 fluorescence. In non-induced control cells, the addition of 13 mm glucose (15.5 mmfinal) hyperpolarized Δψm and 1 ॖm the protonophore FCCP deploarized Δψm. After induction of naSREBP1c, glucose-induced mitochondrial hyperpolarization was markedly inhibited as early as day 1 (24 h of induction) and completely abolished at days 2 and 4 (Fig. 5). Basal activity of the electron transport chain was progressively lost, as indicated by the dissipation of Δψm by FCCP, and completely abrogated at day 4 (Fig. 5). The mitochondrion is not only the powerhouse for cell growth and survival but also the arsenal for cell apoptosis (37Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6296) Google Scholar). Disruption of mitochondrial membrane potential is an essential event that commits a cell to undergo apoptosis (38Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3266) Google Scholar, 39Vander Heiden M.G. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1242) Google Scholar). Consistently, we also found that expression of naSREBP-1c even at leakage level was sufficient to cause "cell toxicity" during the screening procedure for naSREBP-1c-positive clones. To elucidate the molecular mechanism underlying naSREBP-1c-induced cell toxicity, we investigated the effect of naSREBP-1c on the expression of genes impo
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