The Transcription Factor SREBP-1c Is Instrumental in the Development of beta -Cell Dysfunction*

Haiyan WangDagger, Pierre Maechler§, Peter A. Antinozzi, Laura Herrero||, Kerstin A. Hagenfeldt-Johansson**, Anneli BjörklundDaggerDagger, and Claes B. Wollheim

From the Division of Clinical Biochemistry, Department of Internal Medicine, University Medical Centre, Geneva-4 CH-1211, Switzerland

Received for publication, December 9, 2002, and in revised form, February 21, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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 beta -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 beta -cell glucolipotoxicity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The lipogenic transcription factors, sterol regulatory element binding proteins (SREBPs)1 are 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 (1). Three SREBP isoforms have been identified: SREBP-1a and -1c (alternatively known as adipocyte determination and differentiation factor-1 (ADD1)) (2), which are derived from the same gene through alternative splicing, and SREBP-2, which is encoded by a distinct gene (1). 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 (3). SREBP-1 preferentially regulates genes implicated in fatty acid synthesis, whereas SREBP-2 preferentially activates genes involved in cholesterol synthesis (1). In particular, SREBP-1c mediates insulin effects on lipogenic gene expression in both adipocytes and liver (4, 5).

Type 2 diabetes mellitus is a common disorder that affects ~5% of the population worldwide, especially in industrialized countries (6). 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 (7-9). This is associated with impaired glucose-stimulated insulin secretion, increased hepatic glucose production, peripheral insulin resistance, and late complications in various organs (10, 11). The ultimate precipitating process responsible for the development of Type 2 diabetes is the failure of the pancreatic beta -cells to compensate for insulin resistance (7, 10, 11). However, the mechanism by which the beta -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 (7, 12-16). On the other hand, preventing SREBP-1c overexpression is a common function of leptin, metformin, and PPARgamma agonists, which is well correlated with their antidiabetic effects (7, 12-15). 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 (7, 9). Infusion of recombinant leptin reverses insulin resistance and hyperglycemia in the transgenic model of congenital generalized lipodystrophy and in ob/ob mice (13). Adenovirus-mediated hyperleptinemia also decreases the expression of SREBP-1c and lipogenic genes in liver and islets of wild-type fa/fa rats (12). Leptin also prevents the SREBP-1c overexpression in insulin receptor substrate (IRS)-2-null mice (15). It is noteworthy that metformin, an oral antihyperglycemic agent, also corrects fatty liver disease in ob/ob mice by inhibition of obesity-related induction of SREBP-1c (14). The inhibitory effects of metformin on hepatic SREBP-1c expression involves activation of AMP-activated protein kinase (17). Similarly, Troglitazone, an antidiabetic agent and a high-affinity ligand for the PPARgamma , prevents SREBP-1c overexpression in Zucker diabetic fatty rats (12). In addition, it has recently been reported that adenovirus-mediated overexpression of SREBP-1c in MIN6 cells results in impaired glucose-stimulated insulin secretion (18). Therefore, these studies suggest that overexpression of SREBP-1c may be the cause of both liver steatosis and islet beta -cell dysfunction.

The present study was designed to evaluate the direct correlation between SREBP-1c overexpression and beta -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) (19). Quantification of the gene expression profile in this INS-1 stable cell line revealed that naSREBP-1c induced beta -cell dysfunction by targeting multiple genes implicated in carbohydrate metabolism, lipid biosynthesis, cell growth, and eventually apoptosis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Establishment of INS-1 Cells Permitting Inducible Expression of naSREBP-1c-- Rat insulinoma INS-1 cell-derived clones were cultured in RPMI 1640 containing 11.2 mM glucose (20), unless otherwise indicated. The first step stable clone INS-rbeta cell line, which carries the reverse tetracycline/doxycycline-dependent transactivator (21) was described previously (22, 23). 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) (19), kindly supplied by Dr. B. M. Spiegelman) into the expression vector PUHD10-3 (21) (a generous gift from Dr. H. Bujard). The procedures for stable transfection, clone selection and screening were described previously (22).

Immunofluorescence and Western Blotting-- 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 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline containing 1% 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 (24), and their nuclear proteins were extracted as previously reported (25). 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, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride. Nuclear extracts and total cellular proteins were fractionated by 9% SDS-PAGE. The dilution for SREBP-1 antibody is 1:1,000. Immunoblotting procedures were performed as described previously (26) using enhanced chemiluminescence (Pierce) for detection.

Staining of Lipid Accumulation by Oil Red O and Measurements of Triglyceride Content-- Cells were cultured with 0, 75, 150, and 500 ng/ml doxycycline for 24 h. Cells were fixed and stained as previously reported (19). 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.

Measurements of Insulin Secretion and Cellular Insulin Content-- 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 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 2 mM NaHCO3, 10 mM HEPES, 0.1% BSA) containing indicated concentrations of glucose. Insulin content was determined after extraction with acid ethanol following the procedures of Wang et al. (26). Insulin was detected by radioimmunoassay using rat insulin as a standard (26).

Total RNA Isolation and Northern Blotting-- Cells were cultured with or without 500 ng/ml doxycycline in 2.5 mM glucose 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 mM glucose) for 0, 24, 48, and 96 h. Total RNA was extracted and blotted to nylon membranes as described previously (22). The membrane was prehybridized and then hybridized to 32P-labeled random primer cDNA probes according to Wang and Iynedijian (22). 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)-1alpha , HNF-4alpha , 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.

Mitochondrial Membrane Potential (Delta psi m)-- Cells seeded in 24-well plates were cultured with or without 500 ng/ml doxycycline in 11.2 mM glucose 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 µg/ml rhodamine-123 for 20 min at 37 °C in KRBH (28). The Delta psi m 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).

Cell Proliferation/Viability and Apoptosis-- 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 cytochrome c release into cytosol was performed as described previously (29). 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 15% SDS-PAGE and analyzed by Western blotting with a specific antibody against cytochrome c (LabForce/MBL).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Establishment of a Stable INS-1 Cell Line Permitting Inducible Expression of naSREBP-1c-- A rat insulinoma INS-1-derived stable cell line, INSrbeta (23), which expresses the reverse tetracycline-dependent activator (21), was used for the secondary stable transfection with an expression vector, PUHD10-3 (21), 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 10-20% 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 "beta -cell toxicity." The INS-1 stable cell line naSREBP-1c#233 provided a unique chance to study the impact of naSREBP-1c expression on beta -cell function.

All Cells Uniformly Express the Nuclear Localized naSREBP1c Protein in a Doxycycline Dose-dependent Manner-- 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 ~10%, 25%, and 150%, respectively, of endogenous level of SREBP-1c precursor protein (Fig. 1B).


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Fig. 1.   Induction of naSREBP-1c protein in a dose-dependent and an all-or-none manner. A, immunofluorescence staining with an antibody against the N terminus of SREBP-1c. The phase contrast image is shown in the upper panel. The naSREBP-1c#233 cells were cultured with (+Dox) or without (-Dox) 500 ng/ml doxycycline for 24 h. B, Western blotting of total cell extracts (100 µg) with the same antibody. Cells were cultured with 0, 75, 150, and 500 ng/ml doxycycline for 24 h.

Induction of naSREBP-1c Causes Rapid Accumulation of Lipid Droplets by Increasing Lipogenic Gene Expression-- 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 (10% fetal calf serum) (Fig. 2A). Cellular triglyceride content was increased by 39% (-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 beta -oxidation of fatty acids (Fig. 2, B and C). 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.


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Fig. 2.   Accumulation of lipid droplets and increased lipogenic gene expression are shown in INS-1 cells expressing naSREBP-1c. A, Oil Red O staining naSREBP-1c#233 cells cultured with 0, 75, 150, and 500 ng/ml doxycycline for 24 h. B, Northern blotting was performed using total RNA isolated from cells cultured with 500 ng/ml doxycycline in 2.5 mM glucose medium for 16 h and continued in 2.5, 6, 12, and 24 mM glucose medium for a further 8 h. The experiments were repeated two times with similar results. C, Northern blotting was conducted using total RNA isolated from cells cultured in standard glucose medium (11.2 mM glucose) with 75 ng/ml doxycycline for 0, 24, 48, and 96 h. Two independent experiments are shown side by side to demonstrate the consistency of the results.

Induction of naSREBP-1c Impairs Nutrient-stimulated Insulin Secretion-- 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 (30). Leucine stimulates insulin release through direct uptake and metabolism by the mitochondria, thereby providing substrates for the tricarboxylic acid cycle (30). K+ causes insulin secretion by depolarization of the beta -cell membrane, resulting in increased cytosolic Ca2+ (30). 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.


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Fig. 3.   Induction of naSREBP-1c results in impaired nutrient-stimulated insulin secretion. A, cells were cultured with (+Dox) or without (-Dox) 500 ng/ml doxycycline in standard medium (11.2 mM glucose) for 19 h and then equilibrated in 2.5 mM glucose medium for a further 5 h. B, cells were cultured with (+Dox) or without (-Dox) 75 ng/ml doxycycline in standard glucose medium (11.2 mM) for 91 h followed by an additional 5 h of equilibration in 2.5 mM glucose medium. Cellular insulin content in A and B was reduced, respectively, by 24.6 ± 4.8 (n = 6) and 21 ± 5.3 (n = 6) after naSREBP-1c induction. Insulin release from naSREBP-1c#233 cells stimulated by 21.5 mM glucose, 20 mM leucine, and 20 mM KCl in KRBH 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, 0.1% BSA) containing 2.5 mM glucose (Basal) was quantified by radio-immunoassay and normalized by cellular insulin content. Data represent mean ± S.E. of six independent experiments. **, p < 0.0001.

Induction of naSREBP-1c Alters the Expression of Genes Important for Glucose Metabolism and beta -Cell Function-- 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 beta -cell glucose sensing (22, 31). The effect of naSEBP-1c on beta -glucokinase expression is opposite to its action on liver glucokinase, which is transcribed from a distinct liver-specific promoter (5, 32). UCP-2 may act as a protonophore and dissipate the mitochondrial membrane potential, thereby uncoupling respiration from ATP synthesis (33-36).


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Fig. 4.   Induction of naSREBP-1c causes defective expression of genes implicated in glucose metabolism and beta -cell function. A and C, cells were cultured with or without 500 ng/ml doxycycline in 2.5 mM glucose medium for 16 h and then incubated for a further 8 h at indicated glucose concentrations. The experiments were repeated two times with similar results. B and D, cells were cultured with or without 75 ng/ml doxycycline in standard medium (11.2 mM glucose) for 0, 24, 48, and 96 h. Two independent experiments are shown side by side to demonstrate the consistency of the results. The gene expression patterns in naSREBP-1c#233 cells were quantified by Northern blotting. Total RNA samples (20 µg/lane) were analyzed by hybridizing with indicated cDNA probes.

Induction of naSREBP1c also suppressed the expression of Pdx-1, HNF-4alpha , and CCAAT/enhancer-binding protein-beta in a dose- and time-dependent manner (Fig. 4, C and D). The HNF-1alpha expression was unaffected, whereas the mRNA level of a beta -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).

Induction of naSREBP-1c Disrupts Mitochondrial Membrane Potential-- Mitochondrial membrane potential (Delta psi m), 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 mM final) hyperpolarized Delta psi m and 1 µM the protonophore FCCP deploarized Delta psi m. 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 Delta psi m 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 (37). Disruption of mitochondrial membrane potential is an essential event that commits a cell to undergo apoptosis (38, 39). 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 important for cell proliferation and apoptosis.


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Fig. 5.   Induction of naSREBP-1c dissipates mitochondrial membrane potential (Delta psi m) in naSREBP-1c#233 cells. Cells were cultured without (-Dox) or with (+Dox) 500 ng/ml doxycycline for 24, 48, and 96 h (day 1, day 2, and day 4, respectively). The Delta psi m was measured on attached cells in KRBH containing basal 2.5 mM glucose using rhodamine-123 fluorescence. Electron transport chain activation observed as hyperpolarization of Delta psi m was induced by the addition of 13 mM glucose (15.5 mM final), followed by the complete depolarization of Delta psi m using 1 µM of the uncoupler FCCP. Values are means plus standard deviations (n = 4) of one of a total of four to six independent experiments.

SREBP-1c Targets Multiple Genes Implicated in beta -Cell Growth and Survival-- As shown in Fig. 6, induction of naSREBP-1c decreased the expression of cyclin-dependent kinase 4 and IRS-2 in a dose- and time-dependent manner. In contrast, the mRNA level of glucagon-like polypeptide-1 receptor was barely changed (Fig. 6). Both cyclin-dependent kinase 4 and IRS-2 have been reported to promote beta -cell development, proliferation, or survival (40, 41). In addition, the mRNA level of P21WAF1/CIP1 was dramatically increased by naSREBP-1c induction, whereas the P27KIP1 remained constant (Fig. 6). The increased expression of the P21WAF1/CIP1, a cyclin-dependent kinase inhibitor, could also lead to INS-1 cell growth arrest. Furthermore, as demonstrated in Fig. 6, naSREBP-1c up-regulated the expression of proapoptotic genes, APO-1/Fas/CD95 and Bax (37, 38), but did not alter the mRNA levels of Bad and tumor necrosis factor (TNF)-1alpha . The up-regulation of APO-1 and Bax could contribute to the naSREBP-1c-induced INS-1 cell apoptosis (see below).


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Fig. 6.   Induction of naSREBP-1c alters the expression of genes important for beta -cell growth and survival. A, cells were cultured with or without 500 ng/ml doxycycline in 2.5 mM glucose medium for 16 h and then incubated for a further 8 h at indicated glucose concentrations. The experiments were repeated two times with similar results. B, cells were cultured with or without 75 ng/ml doxycycline in standard medium (11.2 mM glucose) for 0, 24, 48, and 96 h. Two separate experiments are demonstrated in parallel. The gene expression profile in naSREBP-1c#233 cells was quantified by Northern blotting. Total RNA samples (20 µg/lane) were analyzed by hybridizing with indicated cDNA probes.

Induction of naSREBP-1c Results in Cell Growth Arrest and Apoptosis in INS-1 Cells-- To assess the impact of naSREBP-1c on INS-1 cell growth and viability, we performed the WST-1 assay. 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. Induction of naSREBP-1c with 500 ng/ml doxycycline for 24 h or with 75 ng/ml doxycycline for 4 days in naSREBP-1c#233 cells significantly inhibited INS-1 cell growth/viability. The measurements of optical density at 440 nM were reduced by, respectively, 31.6 ± 4.2% and 36.3 ± 5.5% relative to non-induced cells (n = 4, p < 0.001). Mitochondrial cytochrome c release and DNA fragmentation are characteristic hallmarks of cells undergoing apoptosis (37, 42). Extensive DNA fragmentation was observed in naSREBP-1c#233 cells cultured with 500 ng/ml doxycycline for 48 h or with 75 ng/ml doxycycline for a week (Fig. 7A). Consistently, similar induction of naSREBP-1c also induced mitochondrial cytochrome c release (Fig. 7B). These results suggest that naSREBP-1c induces INS-1 cell apoptosis in a dose- and time-dependent manner.


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Fig. 7.   Apoptosis in INS-1 cells induced by naSREBP-1c and maturation of SREBP-1 protein in INS-1E cells regulated by factors causing beta -cell dysfunction. A, DNA fragmentation in naSREBP-1c#233 cells cultured either with 500 ng/ml doxycycline for 1, 2, and 4 days (left panel) or with 75 ng/ml doxycycline for 1, 4, and 7 days (right panel). B, cytochrome c released in cytosolic fractions isolated from naSREBP-1c#233 cells cultured either with 500 ng/ml doxycycline for 1, 2, and 4 days (left panel) or with 75 ng/ml doxycycline for 1, 4, and 7 days (right panel). Cytosolic proteins (100 mg/lane) were separated with 15% SDS-PAGE and analyzed by immunoblotting with an anti-cytochrome c antibody. C, immunoblotting with antibody against the N terminus of SREBP-1. Total cell extracts (100 µg of protein/lane) from INS-1E cells (lanes 1-8) or nuclear proteins (50 µg of protein) from freshly isolated rat islets (lane 9) were separated by 9% SDS-PAGE. INS-1E cells were treated for either 6 h (lanes 1 and 2) or 48 h (lanes 3 and 4) with 30 mM glucose or 100 nM exogenous insulin as indicated. Lanes 5-7 represent, respectively, total cell extracts from INS-1E cultured for 48 h in standard medium (11.2 mM glucose) containing 1.5 mM long-chain free fatty acids (2:1 oleate/palmitate), 10 ng/ml TNF-alpha , and 10 µM H2O2. Total cell lysates from INS-1E cells under standard culture conditions are shown in lane 8 as control.

SREBP-1c Processing in beta -Cells Is Distinct from Lipogenic Tissues-- Unlike liver and adipose tissue, pancreatic beta -cells have evolved to secrete insulin rather than store energy in response to rising blood glucose and nutrient levels. The released insulin subsequently promotes the lipogenic gene expression in liver and adipocytes through up-regulation of SREBP-1 expression and increase of its processing (4, 5, 43). The molecular mechanism by which beta -cells restrict lipogenesis in response to physiological glucose and insulin stimulation remains undefined. We performed Western blotting using an antibody specific for the N terminus of SREBP-1, which should recognize both precursor and mature SREBP-1. As shown in Figure 7C, the mature nuclear form of SREBP-1 was not detectable in nuclear extracts from freshly isolated rat islets. Consistently, only the precursor but not the mature nuclear form of SREBP-1 protein was detected in total cell extracts from native INS-1E cells (Fig. 7C). Similar results were obtained in INS-1E cells treated for 6 h with 30 mM glucose and 100 nM exogenous insulin (Fig. 7C).

Low sterol-mediated cleavage of SREBP precursor proteins is not the only mechanism for the processing of SREBPs. It has been suggested that insulin and cytokines stimulate phosphorylation and transcriptional activity of SREBPs via the mitogen-activated protein kinase cascade (44-48). Caspase-3/CPP32, a cysteine protease and mediator of apoptosis, also induces cleavage of SREBPs and release of their transactive N-terminal fragments (42). In addition, TNF-alpha stimulates the maturation of SREBP-1c in human hepatocytes (49). Furthermore, hyperglycemia in animal models of diabetes causes increased SREBP-1 maturation and renal lipid accumulation leading to diabetic nephropathy (50). Moreover, ethanol, ER stress, shear stress, and cytokines have been reported to up-regulate the lipogenic gene expression in hepatocytes and/or vascular endothelial cells by activation of SREBPs (14, 49, 51-54).

Hyperglycemia, hyperlipidemia, elevated TNF-alpha , and increased oxidative stress have been linked to beta -cell dysfunction in Type 2 diabetes (9, 55-63). We therefore studied the SREBP-1 maturation in native INS-1E cells, the most differentiated INS-1 cell clone (64). The cells were exposed for 48 h to 30 mM glucose, 100 nM insulin, 1.5 mM long-chain free fatty acids (2:1 oleate/palmitate), 10 ng/ml TNF-alpha , and 10 µM H2O2 (Fig. 7C). The mature nuclear form of SREBP-1 was detected in INS-1E cells treated for 48 h with glucose, insulin, TNF-alpha , or H2O2 (Fig. 7C). In contrast, free fatty acids were without effects (Fig. 7C). The level of the mature form of SREBP-1 in INS-1E cells treated with high glucose for 48 h is ~10% of that of the endogenous precursor protein (Fig. 7C). This corresponds to the induction observed with 75 ng/ml doxycycline in naSREBP-1c#233 cells (Fig. 1B).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intracellular lipid accumulation, termed lipotoxicity (metabolic syndrome X), in skeletal muscle, myocardium, blood vessels, kidney, liver, and pancreatic islets has been speculated to cause, respectively, insulin resistance, cardiac dysfunction, vascular complications, nephropathy, fatty liver (hepatic steatosis), and beta -cell dysfunction in Type 2 diabetes (9, 50). However the molecular mechanism underlying the pathogenesis of lipotoxicity in pancreatic beta -cells and other non-adipose tissues remains undefined. In fact, lipotoxicity is a confusing concept. Some investigators refer to lipotoxicity as the consequence of increased circulating free fatty acids and cellular lipid accumulation (7-9), whereas others suggest that glucotoxicity is the prerequisite for lipotoxicity, at least in beta -cells (58, 59, 65-68). The present study and other observations2 support the latter (see bellow). In creased expression of SREBP-1c has been demonstrated in islets and/or liver in several animal models of diabetes (7, 12-16). Overexpression of SREBP-1 has been suggested to be the cause of hepatic steatosis (14, 16, 51, 53, 69, 70) and diabetic nephropathy (50). The present study provides direct and strong evidence proving that the transcriptional activity of SREBP-1c also mediates beta -cell dysfunction.

Conditional expression of naSREBP-1c, a nuclear localized N-terminal form of SREBP-1c with intact transcriptional activity, results in massive accumulation of lipid-droplets in INS-1 cells, a clonal beta -cell line. This could be explained by marked increases in lipogenic gene expression and unaltered mRNA levels of genes involved in fatty acid beta -oxidation. The lipogenic transcription factor naSREBP-1c also induces impairment of nutrient-induced insulin secretion, suggesting defective glucose metabolism. This could be caused by the down-regulation of glucokinase, up-regulation of UCP-2, and disrupted mitochondrial membrane potential observed in these INS-1 cells expressing naSREBP-1c. In addition, we also show that naSREBP-1c suppresses the expression of insulin, Pdx-1, and HNF-4alpha . Induction of naSREBP-1c also leads to INS-1 cell growth arrest and apoptosis possibly by both suppressing the expression of cyclin-dependent kinase 4 and IRS-2 and promoting the expression of P21WAF1/CIP1, APO-1/Fas/CD95, and Bax. Since the SREBP-1c precursor is a known substrate for caspase 3, we postulate that SREBP-1c may function as a proapoptotic gene in beta -cells. Similarly, lipid accumulation, impaired glucose-stimulated insulin secretion, defective beta -cell gene expression (insulin, Pdx-1, glucokinase, and Glut-2), disorganized mitochondrial ultrastructure, and "lipoapoptosis" have been reported in islet beta -cells of diabetic animals (7, 71-73). We have, therefore, not only established an in vitro cellular model for beta -cell glucolipotoxicity but also elucidated the underlying mechanism.

In addition, the present study also suggests that, in contrast to hepatocytes and adipocytes, islet beta -cells have evolved to limit the lipogenic gene expression in response to acute stimulation of glucose and insulin by restricting the SREBP-1c processing. Our data are in disagreement with a previous study, claiming that SREBP-1c maturation in MIN-6 cells is acutely regulated by glucose (18). Whereas 6 h of stimulation with glucose had no effect in the present study, we could indeed detect the mature form of SREBP-1c in INS-1E cells after 48 h of exposure to high concentrations of glucose or insulin. In contrast, 48 h of treatment with free fatty acids did not increase the levels of the active form of SREBP-1c. Glucotoxicity, a phenomenon occurring after prolonged exposure to high concentrations of glucose, has been reported to induce lipid accumulation in INS-1 cells by raising lipogenic gene expression (58). Our study may have established a link between glucotoxicity and lipotoxicity and provided an explanation for the predominant role of high glucose in beta -cell dysfunction (59, 65). A similar effect may account for the increased apoptosis and subsequent decreased beta -cell mass in Type 2 diabetic patients compared with obese subjects (27). We also found that a 48-h culture of INS-1E cells with 30 mM glucose caused cell apoptosis and defective gene expression similar to naSREBP-1c induction, whereas 72 h of incubation with 1.5 mM free fatty acids (2:1 oleate/palmitate) did not have such deleterious effects.2 Therefore, naSREBP-1c may induce beta -cell dysfunction independent of lipid accumulation. Hyperglycemia and hyperinsulinemia are characteristics of Type 2 diabetes. We hypothesize that as a result of the persistent hyperglycemia and hyperinsulinemia present in diabetes, SREBP-1c protein is processed and modified to an active form. Activated SREBP-1c alters the expression of various target genes that contribute to beta -cell dysfunction and therefore exacerbates the progression of Type 2 diabetes. Furthermore, our results suggest that the SREBP-1c maturation regulated by cytokines and oxidative stress may also play a role in beta -cell dysfunction in Type 2 diabetes. It should be noted that the present work was performed in a rat insulinoma cell line and therefore may not entirely reflect the situation in native beta -cells, necessitating caution in the interpretation.

We hypothesize that SREBPs have evolved to allow animals adapting to starvation by mediating insulin action on energy storage and promoting cell survival by maintaining constant lipid composition of membranes. Overnutrition and sedentary life-style in industrialized modern society may lead to lipotoxicity in non-adipose tissue. The important contribution of our study is that the transcription factor SREBP-1c is instrumental in the pathogenesis of beta -cell lipotoxicity by targeting multiple genes implicated in carbohydrate metabolism, lipid biosynthesis, cell growth, and apoptosis. Similar mechanisms may also apply to the lipotoxicity in other non-adipose tissues. Development of chemical compounds acting like leptin, PPARgamma agonists, and metformin through suppression of SREBP-1c function should have therapeutic potential in the treatment of Type 2 diabetes and its complications.

    ACKNOWLEDGEMENTS

We are grateful to D. Cornut-Harry, D. Nappey, Y. Dupré, S. Polti, and V. Calvo for expert technical assistance. We are indebted to Drs. B. M. Spiegelman (SREBP-1c/ADD1-(1-403) cDNA), H. Bujard (PUHD10-3 vector), and N. Quintrell (pTKhygro plasmid).

    FOOTNOTES

* This work was supported by Swiss National Science Foundation Grant 32-49755.96 (to C. B. W.) and the Leenaards Foundation (to P. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 41-22-702-5548; Fax: 41-22-702-5543; E-mail: Haiyan.Wang@medicine.unige.ch.

§ Fellow of the Dr. Max Cloetta Foundation.

Present address: Cellular Biochemistry and Biophysics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 251, New York, NY 10021.

|| Present address: Dept. of Biochemistry, School of Pharmacy, University of Barcelona, E-08028 Barcelona, Spain.

** Present address: Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges, Switzerland.

Dagger Dagger Present address: Rolf Luft Center of Diabetes Research, Endocrine and Diabetes Unit, Dept. of Molecular Medicine, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden.

Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M212488200

2 H. Wang and C. B. Wollheim, unpublished data.

    ABBREVIATIONS

The abbreviations used are: SREBP, sterol regulatory element-binding protein; ER, endoplasmic reticulum; ADD1, adipocyte determination and differentiation factor-1; PPAR, peroxisome proliferator-activated receptor; naSREBP, nuclear active form of SREBP-1c; PBS, phosphate-buffered saline; BSA, bovine serum albumin; KRBH, Krebs-Ringer-bicarbonate-HEPES buffer; UCP, uncoupling protein; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; Pdx, pancreas duodenum homeobox; HNF, hepatocyte nuclear factor; IRS, insulin receptor substrate; TNF, tumor necrosis factor.

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