Julien Véret

Nicolas Coant

Evgeny V Berdyshev

Anastasia Skobeleva

Nicole Therville

Danielle Bailbé

Irina Gorshkova

Viswanathan Natarajan

Bernard Portha

Herve Le Stunff
email: herve.le-stunff@univ-paris-diderot.fr

Hervé Le Stunff

Dr Hervé Le Stunff

Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in gluco-lipotoxicity-induced apoptosis of INS-1 β-cells

Keywords: CPT-1, carnitine palmitoyl-transferase, Cer : ceramide, CerS : ceramide synthase, Dihydroceramide, FB1, fumonisin-B1, Type 2 diabetes, pancreatic β-cells, gluco-lipotoxicity, ceramides, apoptosis, ceramide synthases, lipidomic

may come

Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in gluco-lipotoxicity-induced apoptosis of INS-1 β-cells.

Introduction

Type 2 diabetes mellitus is a disease characterized by progressive impaired insulin secretion and peripheral insulin resistance [START_REF] Kahn | Banting Lecture. Insulin action, diabetogenes, and the cause of type II diabetes[END_REF][START_REF] Poitout | Glucolipotoxicity: fuel excess and beta-cell dysfunction[END_REF][START_REF] Kahn | The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of Type 2 diabetes[END_REF]. The pathogenesis of β-cell failure is due to both β-cell dysfunction and reduced β-cell mass. The reduction in insulin secretory capacity and β-cell mass observed during type 2 diabetes is thought to be caused by chronic hyperglycaemia, a phenomenon that has been termed "glucotoxicity" [START_REF] Butler | Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes[END_REF]. In addition to hyperglycaemia, accumulated evidence suggests that type 2 diabetes is often associated with abnormalities in lipid metabolism and excessive circulating lipids levels [START_REF] Kahn | The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of Type 2 diabetes[END_REF][START_REF] Carey | Abdominal fat and insulin resistance in normal and overweight women: Direct measurements reveal a strong relationship in subjects at both low and high risk of NIDDM[END_REF]. Free fatty acids (FFA) are important physiological fuel for islets, and act as supplemental nutrient able to potentiate insulin secretion in response to glucose [START_REF] Crespin | Stimulation of insulin secretion by infusion of free fatty acids[END_REF][START_REF] Stein | The insulinotropic potency of fatty acids is influenced profoundly by their chain length and degree of saturation[END_REF]. However, chronically elevated levels of FFA in the circulation have been postulated to cause peripheral insulin resistance and impairment of insulin secretion from β-cells, a phenomenon that has been termed "lipotoxicity" [START_REF] Poitout | Glucolipotoxicity: fuel excess and beta-cell dysfunction[END_REF][START_REF] Boden | Free fatty acids in obesity and type 2 diabetes: defining their role in the development of insulin resistance and beta-cell dysfunction[END_REF]. Indeed, palmitate, one of the most abundant FFA in plasma, has detrimental effects on β-cell function, including impairment of glucose-induced insulin release [START_REF] Sako | A 48-hour lipid infusion in the rat time-dependently inhibits glucose-induced insulin secretion and B cell oxidation through a process likely coupled to fatty acid oxidation[END_REF][START_REF] Zhou | Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle[END_REF], defective insulin gene expression [11][12][13], and induction of β-cell apoptosis [14][15][START_REF] Maedler | Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function[END_REF][17]. More recently, Marchetti and colleagues have provided evidence that prolonged exposure to FFA induced apoptosis in isolated human islets [18].

The chronic adverse effects of FFA on β-cell function and viability are potentiated by the presence of hyperglycaemia, a phenomenon that has been termed "gluco-lipotoxicity" [START_REF] Prentki | Malonyl-CoA signaling, lipid partitioning, and glucolipotoxicity: role in beta-cell adaptation and failure in the etiology of diabetes[END_REF]20]. Indeed, inhibition of insulin gene expression by long term treatment of β-cells with palmitate is observed only in the presence of elevated glucose concentrations [START_REF] Poitout | Glucolipotoxicity: fuel excess and beta-cell dysfunction[END_REF]12]. Prentki and colleagues have shown that saturated FFA exhibit low toxicity at low glucose concentrations but FFA synergize with elevated glucose concentrations to promote β-cell apoptosis [17]. The molecular mechanisms underlying the pathogenesis of (gluco)lipotoxicity in pancreatic β-cells are not completely understood. At low glucose levels, FFA are readily degraded by β-oxidation and therefore are non-toxic to β-cells. In contrast, when glucose and FFA are simultaneously elevated, glucose inhibits FFA oxidation, raising the levels of long chain acyl-CoA (LC-CoA), which serve for complex lipid synthesis [START_REF] Poitout | Glucolipotoxicity: fuel excess and beta-cell dysfunction[END_REF]21]. Indeed, LC-CoA can be esterified into phospholipids and triglycerides [22][23][24][25] which promote β-cell dysfunction and lipo-apoptosis [12,23]. Among these lipids, ceramides have been suggested to be important mediators of FFA-induced β-cell dysfunction and apoptosis.

Ceramides are produced either by de novo biosynthesis or turnover of complex sphingolipids [24,25]. De novo synthesis of ceramides occurs at the cytosolic face of the endoplasmic reticulum and is initiated by the condensation of serine and palmitoyl-CoA catalysed by serine palmitoyltransferase (SPT, Fig. 1). The 3-keto-sphinganine formed is rapidly reduced to dihydrosphingosine, which is subsequently N-acylated by (dihydro)ceramide synthases to form dihydroceramides [26,27]. Finally, a trans double bond at C4-C5 is introduced by dihydroceramide desaturase to produce ceramides [24,25]. Previous studies showed that cell permeable analogues of ceramide impaired insulin production in pancreatic β-cells [28]. Using pharmacological approaches, de novo ceramide synthesis has been implicated in inhibition of glucose-stimulated insulin gene expression by palmitate in rodent islets [12]. In addition, fumonisin-B1 (FB1), a ceramide synthase inhibitor blocked palmitateinduced apoptosis in rodent and human islets [START_REF] Maedler | Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function[END_REF]18]. Moreover, Unger and colleagues have shown that ceramides are increased in islets of obese Zucker diabetic fatty (ZDF) rats and that FFA-induced apoptosis is blocked by inhibitors of ceramide synthesis [14,15].

Ceramide species are distinguishable by the length and/or saturation of their N-acyl chains [24,27]. In β-cells, despite such evidence for a role of ceramides in their dysfunction/apoptosis, nothing is known about the ceramide species produced in response to (gluco)-lipotoxicity. Recent studies have reported distinct cellular functions for ceramides with specific N-acyl chain length [26,27]. Determining which ceramide species are produced A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

under these conditions may shed light on the mechanisms involved in the ceramide-dependent β-cell gluco-lipotoxicity. Therefore, in the present study, we used a LC/MS/MS methodology to examine the impact of FFA oversupply on ceramide profiles in pancreatic β-cells. Due to the need for relatively large amount of biological material to perform sphingolipidomic analyses, we use the β-cell line, INS-1 which can be expanded to quantities sufficient for diverse experimentation. These data revealed that high glucose levels potentiate palmitateinduced de novo ceramide synthesis. Importantly, we found that gluco-lipotoxicity induced the expression of the ceramide synthase 4 isoform in β-cells. This induction was associated with the formation of specific ceramide species such as C18:0, C22:0 and C24:1, which contributed to the apoptotic effect of gluco-lipotoxicity on β-cells. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Materials and Methods

Materials

Tissue culture media was from Lonza.  32 PATP was purchased from Perkin Elmer. Palmitate, fatty acid-free BSA and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were from Sigma-Aldrich. FB1 and D,L-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1propanol (PPMP) were from Biomol. Diacylglycerol kinase was from Calbiochem. Apo-ONE homogenous caspase-3/7 assay was from Promega. All solvents were from Merck Eurolab or Fisher Scientific. Ceramides, dihydroceramides, sphingosine, dihydro-sphingosine, C17ceramide and C17-sphingosine were from Avanti polar lipids. Anti-HA and anti-CerS4 antibodies were from Covance and Abcam, respectively. Mission®siRNA against CerS4 and control siRNA were from Sigma. Lipofectamine LTX and RNAiMAX were purchased from Invitrogen.

Cell culture conditions

Rat Insulinoma INS-1 cells (clone 368), kindly provided by Merck-Serono, were grown in RPMI 1640 medium buffered with 10 mM HEPES containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 50 M 2--mercaptoethanol and 100 U/ml penicillin/streptomycin. Prior to each experiment, cells were plated for 48 h in 96-well plates (22x10 3 cells/well) for MTT assay and caspase-3/7 activity, in 6-well plates (1x10 6 cells/well) for ceramide levels, in 12-well plates (1x10 6 cells/well) for insulin secretion and in 50 mm plates (5x10 6 cells/plate) for analysis of sphingolipids by LC/MS/MS. Palmitate was administered to the cells as a conjugate with fatty acid-free BSA. Briefly, dried aliquots of palmitate in ethanol were dissolved in PBS containing 5% BSA to obtain a 4 mM stock solution. The molar ratio of FFA to BSA was 5:1. The FFA stock solutions were diluted in RPMI supplemented with 1% FBS (described hereafter as incubation medium) to obtain a 0.4 mM final concentration at a fixed concentration of 0.5% BSA.

Cell transfection

The cDNA encoding human CerS4 [27] was amplified by PCR using a sequence specific sense primer (5'-cgggatccagcagagaatgctgtccagt-3') containing the BamHI restriction site and the reverse primer (5'-cggaattcctaagcgtaatctggaacatcgtatgggtatgtggctgttgtgtgcctg-3') containing the HA sequence tag. The amplification product was subcloned in the Zero Blunt TOPO vector PCR Cloning kit (Invitrogen). Then, the CerS4 cDNA was cloned into the pcDNA5-TO vector (Invitrogen) at the level of the BamHI restriction site and its sequence was verified. INS-1 cells were transiently transfected with the empty pcDNA5-TO vector or the pcDNA5-TO containing HA-tagged CerS4 construct using Lipofectamine LTX (Invitrogen). Transfection efficiencies were typically 40-70% for INS-1 cells. In some experiments, cells were transfected with 50 or 100 nmol of sequence-specific siRNA against CerS4 and control siRNA using Lipofectamine RNAiMAX (Invitrogen).

Western Blotting

Equal amounts of proteins were separated by 10% SDS-PAGE and then transblotted to nitrocellulose. Blots were probed with either a polyclonal anti-CerS4 antibody or a monoclonal anti-HA antibody, stripped, and re-probed with a polyclonal anti-actin antibody. Immunoreactive bands were visualized by enhanced chemiluminescence with appropriate horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) and SuperSignal West Pico Chemiluminescent Substrate (Pierce). At the end of each treatment, cells were treated with MTT (1mg/ml) for 4 hr. Supernatants were discarded and DMSO was added. Absorbance was measured at 560 nm using a microplate reader (Dynex-MRX). Each experimental condition tested was performed in triplicate.

Measurement of MTT cell viability

Measurement of caspase-3/7 activity

Caspase-3/7 activity assays were performed with the Promega Apo-ONE Homogeneous Caspase-3/7 Assay kit. Briefly, lysis buffer containing the fluorogenic Z-DEVD-R110 substrate was added to each well and fluorescence was measured every 6 min over a 120 min period using a Fluostar plate reader set at 37°C (excitation at 485 nm and emission at 530 nm). Caspase-3/7-specific activity was expressed as the slope of the kinetic in arbitrary units. Each experimental condition tested was performed in triplicate.

Quantitative PCR

Total RNA was isolated from INS-1 cells using the RNeasy mini kit (Qiagen). Total RNA (4 µg) from each sample was reverse transcribed with 40 U of M-MLV Reverse Transcriptase (Invitrogen) using random hexamer primers. The primers used for PCR were derived from rat sequences and designed using OLIGO6 (the primer sets used are described in supplemental table 1). Real-time quantitative PCR amplification reactions were carried out in a LightCycler 1.5 detection system (Roche) using the LightCycler FastStart DNA Master plus SYBR Green I kit (Roche). Reverse transcribed RNA (10 ng) was used as the template for each reaction. All reactions were run in duplicate with no template control. The PCR conditions were: 95°C for 10 min, followed by 40 cycles at 95°C for 10 s, 60°C for 10 s and 72°C for 10 s. mRNA transcript levels of 4 housekeeping genes (rpL19, Tbp, cyclophilin a, 18S) were assayed. Since similar results were obtained with the 4 housekeeping genes, only Tbp was retained for normalization of other transcripts.

Enzymatic measurement of ceramide levels

Ceramide levels in cellular extracts were measured by the diacylglycerol (DAG) kinase enzymatic method as previously described [29]. Briefly, aliquots of the chloroform phases from cellular lipid extracts were resuspended in 7.5% (w/v) octyl--D-glucopyranoside/5 mM cardiolipin in 1 mM DETPAC/10 mM imidazole (pH 6.6). The enzymatic reaction was started by the addition of 20 mM DTT, 0.88 U/ml E. coli DAG kinase, 5 Ci/10 mM [-32 P]ATP and the reaction buffer (100 mM imidazole (pH 6.6), 100 mM NaCl, 25 mM MgCl2, and 2 mM EGTA). After incubation for 1 h at room temperature, lipids were extracted with chloroform/methanol/HCl (100:100:1, v/v) and 1 M KCl. [-32 P]-ceramide phosphate was resolved by TLC with chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1, v/v) and quantified with a phosphorimager (Storm, Amersham). Known amounts of bovine ceramide standards were included in each assay. Ceramide levels are expressed as fmol by nmol of phospholipid (PL) levels. Each measurement was done in duplicate.

Measurement of total cellular phospholipids

Total phospholipids present in cellular lipid extracts used for ceramide analysis were quantified as described previously [30] with minor modifications. Briefly, a mixture of 10N H 2 SO 4 /70% perchloric acid (3:1, v/v) was added to lipid extracts which were incubated for 30 min at 210°C. After cooling, water and 4.2% ammonium molybdate in 4 N HCl/0.045% malachite green (1:3 v/v) was added. Samples were incubated at 37°C for 30 min, and absorbance was measured at 660 nm. Cellular lipids were extracted by modified Bligh and Dyer procedure [31] with the use of 0.1 N HCl for phase separation. C17-Cer (30 pmol) and C17-shingosine (30 pmol) employed as internal standards, were added during the initial step of lipid extraction. The extracted lipids were dissolved in methanol/chloroform (4:1, v/v) and aliquots were taken out to determine total phospholipid content. Samples were concentrated under a stream of nitrogen, redissolved in methanol, transferred into autosampler vials and subjected to consecutive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis.

Lipid extraction and sample preparation for LC/MS/MS

Analysis of sphingoid bases and ceramides by LC/MS/MS

Analyses of sphingolipids were performed by LC/MS/MS as described previously [32,33]. Instrumentation employed was an API4000 triple quadrupole mass spectrometer (Applied Biosystems) interfaced with an automated Agilent 1100 series liquid chromatograph and autosampler (Agilent Technologies). Briefly, sphingolipids were ionized via electrospray ionization (ESI) with detection via multiple reaction monitoring (MRM) in positive ions mode. The resolution of sphingoid bases was achieved with a Discovery C18 column (2.1 x 50 mm, 5 µm particle size, Supelco, Bellefonte, PA) and a gradient from methanol/water/formic acid (61:38:1, v/v) with 5 mM ammonium formate to methanol/acetonitrile/formic acid (39:60:1, v/v) with 5 mM ammonium formate at a flow rate of 0.5 ml/min. The MRM transitions employed for detection of sphingoid bases were as follows: m/z 286>268 (C17-Sph, internal standard); m/z 300>282 (Sph); and m/z 302>284 (DHSph).

Ceramide molecular species were resolved using a 3 x 100 mm X-Terra XDB-C8 column (3.5 µm particle size, Waters, Milford, MA) and a gradient from methanol/water/formic acid (61:39:0.5, v/v) with 5 mM ammonium formate to acetonitrile/chloroform/water/formic acid (90:10:0.5:0.5, v/v) with 5 mM ammonium formate at a flow rate of 0.5 ml/min. MRM transitions monitored for the elution of ceramide molecular species were as follows: m/z 510>264, 14:0-Cer; m/z 538>264, 16:0-Cer; m/z 540>284, 16:0-DHCer; m/z 552>264, 17:0-Cer (internal standard); m/z 564>264, 18:1-Cer; m/z 566>284, 18:1-DHCer; m/z 566>264, 18:0-Cer; m/z 568>284, 18:0-DHCer; m/z 594>264, 20:0-Cer; m/z 596>284, 20:0-DHCer; m/z 624>284, 22:0-DHCer; m/z 650>264, 24:1-Cer; m/z 652>284, 24:1-DHCer; m/z 652>264, 24:0-Cer; m/z 654>284, 24:0-DHCer; m/z 680>264, 26:1-Cer; m/z 682>264, 26:0-Cer; m/z 708>264, 28:1-Cer; m/z 710>264, 28:0-Cer. Standard curves for each of the sphingoid bases or ceramide molecular species were constructed via the addition of increasing concentrations of the individual analyte to 30 pmol of the structural analogs of the sphingolipid classes used as the internal standards. Linearity and the correlation coefficients of the standard curves were obtained via a linear regression analysis. The standard curves were linear over the range of 0.0 -300 pmol of each of the sphingolipid analytes with correlation coefficients (R 2 ) >0.98. Parameters of declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP) were determined for each individual analyte by the infusion of the corresponding standards. Turbo-V ion source was operated at 550°C, GS1=40, GS2=50, and Curtain Gas=20. Correction for ion suppression by the matrix was controlled by creating standard curves in the presence of total lipid extract from human pulmonary artery endothelial cells (4 nmol total lipid phosphorus).

Insulin secretion

INS-1 cells were seeded in 12-well plates and treated for 24 h with 0.4 mM palmitate in the presence of various glucose concentrations. Cells were then pre-incubated in KRBH containing 0.2% fatty-acid free BSA and 5.5 mM glucose for 1 h. Insulin secretion was measured following a 60 min incubation in KRBH containing 0.2% defatted BSA with 5.5 mM glucose or 16.7 mM glucose. The insulin concentration in the medium was determined by radio-immunoassay as described previously [34].

Statistical analysis

Data are expressed as means ± S.E.M. Significance was assessed by the Student's t test unpaired and two-tailed. P values less than 0.05 were considered as significant.

Results

Palmitate and high glucose elicit ceramide accumulation

Athough palmitate has been shown to mediate part of its lipotoxic effects on β-cells through ceramide synthesis [12,14], the chain length and degree of saturation of ceramides have not been determined. Therefore, we tested the effect of 0.4 mM palmitate, in the presence of various concentrations of glucose, on ceramide levels in INS-1 cells. In a first step, using the DAG kinase assay, which determined ceramide levels after phosphorylation to ceramide-1phosphate [START_REF] Perry | Quantitative determination of ceramide using diglyceride kinase Methods[END_REF], we found that palmitate induced a 2.2-fold increase of ceramide levels at low glucose concentrations in INS-1 cells (Fig 1B). Surprisingly, we found that glucose also raised the levels of ceramides in INS-1 cells (Fig 1B). Next, we examined the levels of ceramide molecular species by LC/MS/MS. The most abundant ceramide molecular species in INS-1 cells were very long chain (VLC) ceramides such as C24:0 and C24:1 followed by long chain (LC) ceramides C22:0, C18:0 and C16:0 (supplemental table 2). As observed with the DAG kinase assay, LC/MS/MS analysis showed that high glucose concentrations increased ceramide levels in INS-1 cells in a time-dependent manner (Fig. 2A andB; supplemental 1). Indeed, increasing the concentration of glucose to 10 mM compared to low glucose levels, induced the accumulation of ceramides C20:0, C24:0 and C26:0 (Fig. 2A andB) at 24 h. Thirty mM glucose did not further increase the levels of these ceramide species, but rather slightly increased ceramides C16:0 and C18:0 levels in INS-1 cells. At 5 mM glucose, palmitate was able to increase ceramide levels as soon as 12 h after the treatment and with a greater efficiency than high glucose levels (Fig. 2C andD). Palmitate increased both LC-and VLC-ceramides at 12 h, most obviously enhancing the ceramides C16:0, C18:0, C20:0 and C22:0 species (3-fold increase). After 24 h of treatment, palmitate only increased the levels of LC-ceramides (supplemental 1 C and D). Altogether, these data show that both palmitate and high glucose concentrations triggered ceramide accumulation in INS-1 cells.

Palmitate and glucose induce dihydroceramide accumulation

In the de novo pathway, ceramides are produced by desaturation of dihydroceramides (Fig. 1A). However, ceramides could also result from catabolism of complex sphingolipids such as sphingomyelins and glycospingolipids [24]. Therefore, differences between dihydroceramide and ceramide profiles following palmitate or glucose treatment might indicate which pathway of ceramide production is regulated by these compounds. Increasing the concentrations of glucose induced a large increase of total dihydroceramides in INS-1 cells in a dose-dependent manner (Fig. 3A; supplemental 2). Interestingly, high glucose levels increased dihydroceramide species bearing N-acyl chain length similar to those incorporated into ceramides (Fig. 3B). Thirty mM glucose preferentially increased the levels of VLCdihydroceramide species such as DH24:0 (Fig. 3B). Adding 0.4 mM palmitate in the presence of 5 mM glucose also induced an increase in the overall level of dihydroceramides after 12 h in INS-1 cells, which decreased by 24 h (Fig. 3C). Looking at the dihydroceramide molecular species, we found that palmitate increased the levels of DH16:0, DH18:0 and DH24:0 after 12 h of treatment (Fig. 3D). At 24 h, similarly to what was observed in ceramides, we found that palmitate only increased the levels of LC-dihydroceramides in INS-1 cells (supplemental 2). The N-acyl chain length pattern of dihydroceramides was comparable to that of ceramides, supporting the idea that palmitate induced de novo synthesis of ceramide in INS-1 cells.

High glucose potentiates the accumulation of dihydrosphingosine induced by palmitate

Increased de novo biosynthesis of ceramide is accompanied by a substantial accumulation of their precursors, dihydrosphingosine and dihydroceramides. As shown in Fig. 4A, high glucose concentrations induced an increase in dihydrosphingosine levels at 12 h. Dihydrosphingosine levels increased before the accumulation of dihydroceramides, supporting the observation that high glucose by itself up-regulates the de novo pathway of ceramide formation. Palmitate also increased dihydrosphingosine levels by 3-fold at low glucose concentrations after 12 h of treatment. Interestingly, we found that 30 mM glucose potentiated the production of dihydrosphingosine induced by palmitate at 12 h and 24 h (Fig. 4A; supplemental 3A). Because dihydrosphingosine could potentially be formed from the hydrolysis of dihydroceramides by ceramidases [START_REF] Mao | Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate[END_REF], we analyzed the levels of another sphingoid base, i.e., sphingosine, which is produced by de-acylation of ceramide pools in mammals. LC/MS/MS measurements of sphingosine indicated that neither palmitate nor high glucose alone, or together modulated the levels of this compound after 12 h and 24 h of treatment (Fig. 4B; supplemental 3B). Altogether, these data suggest that high glucose concentrations stimulate the entry of palmitate in the de novo ceramide synthesis at the level of dihydrosphingosine production in INS-1 cells.

High glucose potentiates palmitate-induced ceramide accumulation

As shown in Fig. 5A andB, 30 mM glucose increased the accumulation of dihydroceramides induced by palmitate after 12 h of treatment. However, the effect of glucose appear to be selective as it potentiated by 2 to 3-fold the palmitate-induced production of only DH18:0, DH22:0, DH24:0 and DH24:1 species. After 24 h of treatment, 30 mM glucose still amplified the accumulation of DH18:0 induced by palmitate (supplemental 4). Then, we examined whether this specific production of dihydroceramides has an impact on the accumulation of ceramide species induced by palmitate with high glucose levels. Using the DAG kinase assay, we found that 30 mM glucose potentiated the production of ceramides induced by palmitate (Fig. 1B). Interestingly, LC/MS/MS analysis showed that the N-acyl chain lengths of ceramide species induced by palmitate with 30 mM glucose were similar to those incorporated into dihydroceramides (Fig. 5 C and D). Indeed, accumulation of C18:0, C22:0 and C24:1 induced by palmitate at 12 h was potentiated in the presence of 30 mM glucose compared to 5 mM glucose. In contrast, accumulation of C16:0 and C20:0 was not affected in the presence of high glucose concentrations (Fig. 5C andD). The effect of high glucose was transient since it was unable to amplify the accumulation of these ceramide species after 24 h of palmitate treatment (supplemental 4). Altogether, these data suggest that gluco-lipotoxicity could mediate its apoptotic effect through the production of specific ceramide species rather than an overall increase of ceramide in INS-1 cells.

Palmitate with high glucose induces caspase-3/7 activation through ceramide accumulation At 5 mM glucose, palmitate exerted no toxicity towards INS-1 cells (Fig. 6A) and did not alter 16.7 mM glucose-induced insulin secretion (supplemental 5). In contrast, incubation of INS-1 cells with palmitate in the presence of 10 mM and 30 mM glucose, reduced cell viability by 28% and 51%, respectively (Fig. 6A) and drastically decreased insulin secretion induced by 16.7 mM of glucose (supplemental 5). A similar effect of palmitate on cell viability was observed when assessing the ability of cells to exclude trypan blue (data not shown). To confirm that the reduction of cell number was part of an apoptotic process, we analyzed the effect of palmitate on caspase-3/7 activation at various concentrations of glucose. At 5 mM glucose, palmitate did not alter caspase activity. In contrast, palmitate increased caspase-3/7 activity by 2-and 7-fold in the presence of 10 mM and 30 mM glucose, respectively (Fig. 6B). To confirm that caspase activation was dependent on ceramide biosynthesis, we treated the cells with FB1, the inhibitor of CoA-dependent ceramide synthases. Twenty five M FB1 reduced ceramide accumulation and caspase-3/7 activation induced by palmitate in the presence of 30 mM glucose, by 47 and 55%, respectively (Fig. 6C and7A). Then, we used D,L-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, a potent inhibitor of glucosyl-ceramide synthase to block the conversion of ceramide into glucosyl-ceramide in the Golgi apparatus. Ten M of PPMP induced accumulation of several (dihydro)-ceramide species (Fig. 7B-D) which increased caspase3/7 activity at low and high glucose concentrations (Fig. 6D). Moreover, PPMP potentiated the accumulation of long chain (dihydro)-ceramide species induced by palmitate with high glucose (Fig. 7B-D). Interestingly, PPMP potentiated the accumulation of long chain ceramide in response to gluco-lipotixicity. The increased accumulation of ceramide in the presence of PPMP potentiated palmitate induced-caspase-3/7 activation by 14-and 53-fold at low and high glucose concentrations, respectively (Fig. 6D). Altogether, these data support the idea that ceramides rather than glucosyl-ceramides mediate INS-1 cell apoptosis induced by glucolipotoxicity.

Palmitate with high glucose induces ceramide synthase 4 expression

The structural variety of ceramides in mammals relies on the existence of a family of enzymes, the ceramide synthases (CerS) [26,27]. Six CerS have been identified and possess a characteristic substrate preference for particular fatty acyl-CoA [26,27]. Quantitative realtime PCR experiments showed that INS-1 cells expressed CerS1-5 (Fig. 8A). Whereas no change in CerS expression was detected in β-cells after incubation with palmitate at 5 mM glucose (Fig. 8A), palmitate increased the levels of CerS4 mRNA by 3-fold in the presence of 30 mM glucose (Fig. 8B). Western blot analysis revealed that palmitate with low and high glucose concentrations increased in a time-dependent manner the protein levels of CerS4 in INS-1 cells (Fig. 8C). We over-expressed HA-CerS4 in INS-1 cells to determine the role of CerS4 in ceramide accumulation induced by palmitate in the presence of high glucose concentrations. Western blot analysis with anti-HA mAb or anti-CerS4 antibodies revealed HA-tagged CerS4 over-expressed in INS-1 cells (Fig. 9A). INS-1 cells transiently expressing HA-CerS4 for 24 h where incubated for an additional 24 h with palmitate in the presence of 30 mM glucose. Over-expressed CerS4 potentiated accumulation of ceramide levels induced by palmitate in the presence of high glucose concentrations (Fig. 9B). Looking at the ceramide molecular species, we found that transient over-expressed HA-CerS4 increased by 20% the levels of ceramide C18:0 and C22:0 but had no effect on the accumulation of ceramide C16:0 and C24:0 (Fig. 9C). Altogether, these results suggest that CerS4 plays a central role in the accumulation of selective ceramide species induced by gluco-lipotoxicity in INS-1 cells.

Ceramide synthase 4 potentiates palmitate-induced β-cell apoptosis

Over-expressed HA-CerS4 was unable to significantly increase caspase3/7 activation induced by palmitate at low glucose levels (Fig. 10A). In contrast, the increase of ceramide levels induced by HA-CerS4 was associated with a 2-fold increase of caspase3/7 activity induced by palmitate in the presence of 30 mM glucose (Fig. 10A). Moreover, siRNA against CerS4 reduced significantly the induction of CerS4 induction by palmitate in the presence of high glucose concentrations (Fig. 10C). Interestingly, down-regulation of endogenous CerS4 by siRNA inhibited by 39% caspase3/7 activation induced by palmitate in the presence of high glucose concentrations (Fig. 10D). CerS4 is an enzyme which show preference for fatty acids such as stearic, lignoceric, and nervonic acids [26,27]. In agreement, with this observation, over-expressed of HA-CerS4 potentiated ceramide accumulation induced by stearate (Fig. 9B), a C18:0 saturated fatty acid, which is particularly toxic for β-cells [START_REF] Maedler | Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function[END_REF]17]. This ceramide accumulation exacerbated caspase3/7 activation accumulation induced by stearate with 30 mM glucose (Fig. 10B). Altogether, these results suggest that CerS4 might play a critical role in β-cell apoptosis induced by gluco-lipotoxicity through the synthesis of specific ceramide species such as C18:0. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Discussion

The biochemical basis of β-cell gluco-lipotoxicity is not completely understood, although it requires the metabolism of glucose and lipids [START_REF] Poitout | Glucolipotoxicity: fuel excess and beta-cell dysfunction[END_REF][START_REF] Prentki | Malonyl-CoA signaling, lipid partitioning, and glucolipotoxicity: role in beta-cell adaptation and failure in the etiology of diabetes[END_REF]. Previous studies have shown that de novo synthesis of ceramides is involved in β-cell apoptosis induced by glucolipotoxicity [14,[START_REF] Maedler | Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function[END_REF]. In the present study, we found that palmitate at low glucose concentrations induces ceramide accumulation in β-cells, without inducing β-cell death. These results suggest that this production of ceramides is not qualitatively and/or quantitatively sufficient to trigger apoptosis. Another possibility is that palmitate concomitantly activates signalling pathways that counteract the apoptotic action of ceramide. Indeed, recent studies have shown that palmitate-induced stimulation of purinergic P2X7 receptors [START_REF] Glas | Purinergic P2X7 receptors regulate secretion of interleukin-1 receptor antagonist and beta cell function and survival[END_REF] and autophagy [START_REF] Choi | Protective role of autophagy in palmitate-induced INS-1 beta-cell death[END_REF] reduces β-cell apoptosis triggered by the fatty acid. Moreover, El-Assad et al. ( 2010) have recently shown that gluco-lipotoxicity induced stearoyl-CoA desaturase-1 (SCD-1) in INS-1 β-cells. This enzyme participates in the conversion of stearate to oleate, a fatty acid which does not induce β-cell apoptosis but rather protects β-cells from the deleterious effect of palmitate [START_REF] Maedler | Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function[END_REF][START_REF] Flowers | Loss of stearoyl-CoA desaturase-1 improves insulin sensitivity in lean mice but worsens diabetes in leptin-deficient obese mice[END_REF].

In agreement with previous studies [17, 23], we found that palmitate was more toxic to β-cells in the presence of elevated glucose concentrations. Caspase-3/7 activation confirmed that gluco-lipotoxicity induced apoptosis leading to β-cell death. High glucose concentrations potentiated ceramide accumulation induced by palmitate. Interestingly, we found that exposing INS-1 cells to high glucose alone increased ceramide production. It is known that glucose metabolism following an increase of its level in β-cells leads to the production of malonyl-CoA, a potent inhibitor of fatty acid oxidation [START_REF] Poitout | Glucolipotoxicity: fuel excess and beta-cell dysfunction[END_REF]21]. Therefore, chronic hyperglycaemia may channel both endogenous fatty acyl-CoA and exogenous palmitate into lipid synthesis, such as ceramide, which in turn affect β-cell fate. Accordingly, we found that both palmitate and high glucose concentrations increased the levels of dihydrosphingosine and dihydroceramide, the precursors for the de novo biosynthesis of ceramide. Importantly, high glucose concentrations potentiated palmitate-induced production of dihydrosphingosine and dihydroceramides in β-cells. Recently, the "salvage pathway", a novel pathway for ceramide production has been described, in which hydrolysis of complex sphingolipids produces ceramide and eventually sphingosine which is then re-acylated to form ceramide with specific N-acyl chains [40]. In this study, dihydrosphingosine levels accumulated whereas sphingosine levels remained unchanged in β-cells in response to gluco-lipotoxicity, suggesting that ceramide accumulation induced by both compounds does not involve the salvage pathway but is mainly due to de novo ceramide biosynthesis. Moreover, these results suggest that chronic hyperglycaemia induced incorporation of endogenous fatty acyl-CoA and added palmitate at least at the level of dihydrosphingosine synthesis, which in turn induced βcell apoptosis by increasing ceramide levels.

Using FB1, a potent inhibitor of ceramide synthases, we confirmed that de novo ceramide production partially contributed to the apoptotic effect of gluco-lipotoxicity on βcells [14, [START_REF] Maedler | Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function[END_REF]18]. Glycosphingolipid metabolites of ceramides have been proposed to play a role in the development of insulin resistance in vitro and in Zucker diabetic fatty acid (ZDF) rats [41]. Interestingly, inhibition of glycosphingolipid synthesis prevented the loss of pancreatic β-cells in ZDF rats [START_REF] Zhao | Inhibiting glycosphingolipid synthesis improves glycemic control and insulin sensitivity in animal models of type 2 diabetes[END_REF] suggesting a potent role of these lipids on lipo-apoptosis. Here, we show that an inhibitor of glucosyl-ceramide synthase, an enzyme which convert ceramide into glucosyl-ceramide, potentiated the accumulation of long chain ceramides and caspase-3/7 activation by palmitate in INS-1 cells at high glucose levels. Thus, at variance to glycosphingolipid-induced insulin resistance, it seems that ceramides rather than glycosphingolipids are responsible for the toxic effect of palmitate on β-cells. Our results are in agreement with a recent study showing that over-expression of glucosyl-ceramide synthase in β-cells reduced apoptosis induced by palmitate at low glucose concentrations [START_REF] Boslem | A lipidomic screen of palmitatetreated MIN6 beta-cells links sphingolipid metabolites with endoplasmic reticulum (ER) stress and impaired protein trafficking[END_REF]. Although a high glucose concentration on its own increased ceramide levels, 24 h of treatment was not associated with β-cell death, in agreement with previous studies [17,[START_REF] Choi | Protective role of autophagy in palmitate-induced INS-1 beta-cell death[END_REF]. Recent studies have reported distinct cellular functions for ceramide with specific N-acyl chain length [44,[START_REF] Senkal | Role of human longevity assurance gene 1 and C18-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas[END_REF]. Therefore, we examined whether palmitate and high glucose concentrations alone or together induced specific ceramide species in these cells, which would affect differentially β-cell fate. LC/MS/MS analysis showed that high glucose concentrations increased the mass of all (dihydro)-ceramide acyl chain species, most obviously enhancing C24:0, the predominant species in INS-1 cells. In agreement, it has been shown that high glucose concentrations induced de novo lipid synthesis in INS-1 cells [46] which will provide the necessary fatty acids for ceramide synthesis. Lipidomic analysis revealed that palmitate at low glucose increased all ceramide species, most notably enhancing C16:0, C18:0, C20:0 and C22:0. Interestingly, have shown that FFA increases SPT expression in islets from normal and diabetic ZDF rats, suggesting that ceramide accumulation in β-cells relies on up-regulation of the de novo synthesis pathway. Altogether, these results suggest that palmitate and high glucose concentrations taken separately do not favour a specific ceramide profile in β-cells but rather force the entry of available fatty acyl-CoA into ceramide synthesis.

Lipidomic analysis also showed that high glucose concentrations exhibit a certain selectivity on the production of (dihydro)-ceramides induced by palmitate. Indeed, the (dihydro)-ceramide species induced by palmitate plus high glucose concentrations were specifically C18:0, C22:0 and C24:1. Accumulation of these ceramide species strongly suggest that they play a critical role in β-cell apoptosis induced by palmitate with high glucose. Recently, Prentki and co-workers have shown that gluco-lipotoxicity induced early changes in lipid partitioning in order to induce β-cell dysfunction and apoptosis [47]. Indeed, gluco-lipotoxicity induced the expression of proteins which favoured fatty acid esterification such as SCD-1 through desaturation of fatty acids, and decreased the expression of enzymes involved in lipid oxidation such as the  subunit of AMP-kinase. However, modulation of enzymes involved in ceramide metabolism was not reported in this study. In mammals, the variety of ceramide species relies on the existence of a family of enzymes, ceramide synthases (CerS) [26,27]. Six CerS have been identified and possess a characteristic substrate preference for particular fatty acyl-CoA [26,27]. Our data revealed that in the presence of high glucose concentrations palmitate led to increased CerS4 mRNA and protein levels. Surprisingly, palmitate also increased CerS4 protein levels at low glucose concentrations. Recent data suggest that the level of another ceramide synthase, CerS1, is regulated via ubiquitination and proteasome by diverse stresses [START_REF] Sridevi | Ceramide synthase 1 is regulated by proteasomal mediated turnover[END_REF], suggesting that palmitate could also increase CerS4 protein levels by altering its turnover in β-cells. Over-expression of CerS4 in INS-1 potentiated ceramide production induced by gluco-lipotoxicity. Interestingly, transient over-expression of CerS4 selectively potentiated accumulation of C18:0 and C22:0 ceramide species, without affecting the levels of C16:0 ceramide or very long chain ceramides in response to gluco-lipotoxicity. Altogether, these results support the idea that induction of CerS4 in β-cells contributes to the selective pattern of ceramide species produced by glucolipotoxicity.

Over-expressed CerS4 was unable to increase significantly apoptosis induced by palmitate at low glucose levels. This result is probably related to the elevated -oxidation pathway at low glucose concentrations in β-cells, which do not favour fatty acid esterification into ceramide. In contrast, ceramide accumulation induced in INS-1 over-expressing CerS4 was associated with an enhanced apoptosis induced by gluco-lipotoxicity. Moreover, siRNA against CerS4 decreased partially the induction of CerS4 and caspase3/7 activity induced by gluco-lipotoxicity. The slight effect of siCerS4 could be related to its efficacy or the higher degree of redundancy and inter-regulation between CerS. Indeed, a recent study have shown that down regulation of specific CerS is associated with the maintenance of ceramide levels at the expense of glycosphingolipids and up-regulation of non-targeted CerS [START_REF] Mullen | Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism[END_REF]. Whereas CerS4 did not affect the sensitivity of cells to chemotherapeutic drugs [50], our data show for the first time that CerS4 could affect the sensitivity of β-cells to gluco-lipotoxicity. In ZDF rats, the leptin unresponsiveness is associated with an increase of palmitate-induced SPT mRNA in islets, which contributes to accumulation of ceramide and β-cell apoptosis [14,15]. Interestingly, recent data have shown that leptin-induced decreased ceramide levels are associated with a reduction of CerS2 and CerS4 mRNA expression in white adipose tissue [51]. Whether up-regulation of CerS4 expression by hyperlipidemia occurs and contributes to apoptosis of islets from ZDF rats is a possibility that needs to be examined.

Among the ceramide species produced by gluco-lipotoxicity, the C18:0 species contains stearate, a saturated fatty acid particularly toxic for β-cells [START_REF] Maedler | Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function[END_REF]17]. Interestingly, we found that ceramide production and caspase-3/7 activation induced by stearate was drastically increased by over-expressed CerS4 in INS-1 cells. Recent studies provided evidence that fatty acid elongase 6 (Elovl-6) mRNA levels are increased by hyperglycaemia in INS-1 [52] and are decreased in islets of Langerhans from diabetic ZDF rats [53]. Down-regulation of Elovl-6 in INS-1 cells attenuated palmitate induced-apoptosis whereas over-expression of Elovl-6 potentiated apoptosis [53]. Importantly, activity of Elovl-6 is mostly involved in elongation of de novo-synthesized palmitate to produce stearate. Therefore, our results suggest that glucolipotoxicity could mediate β-cell apoptosis at least through ceramide C18:0 by a concomitant up-regulation of CerS4 and Elovl-6 gene. Our results are in agreement with previous studies showing the pro-apoptotic role for ceramide C18:0. Indeed, ceramide C18:0 has been shown to contribute to chemotherapeutic drug-induced apoptosis of chronic meyloid leukaemia cells [START_REF] Senkal | Role of human longevity assurance gene 1 and C18-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas[END_REF] whereas it was down-regulated in carcinoma tissue compared to adjacent normal tissues [54]. However, we cannot exclude a role for the other ceramide species induced by glucolipotoxicity in β-cell apoptosis. Moreover, despite any change in their expression, other CerS may play a role in the induction of β-cell apoptosis, especially CerS1 and CerS3 which can synthesize ceramide C18:0 [26, 27] and are regulated by allosteric modifications in the case of CerS1 [START_REF] Sridevi | Ceramide synthase 1 is regulated by proteasomal mediated turnover[END_REF]. Finally, our data do not support a role for ceramide C16:0, a ceramide specie considered to have pro-apoptotic properties under a broad spectrum of stimuli [44,55], in βcell apoptosis since its levels was not increased by gluco-lipotoxicity.

In conclusion, the present study demonstrates that gluco-lipotoxicity can induce apoptosis by a dual mechanism involving the rate-limiting step in the de novo ceramide biosynthesis (i.e., generation of 3-keto-dihydrosphingosine and dihydrosphingosine) and the formation of ceramides with specific N-acyl chain lengths. The latter mechanism involved the induction of CerS4 by gluco-lipotoxicity, an enzyme which potentiated β-cell apoptosis at least through the production of Cer-18:0. These results suggest that downstream the lipid partitioning induced by gluco-lipotoxicity, synthesis of specific ceramide species could play a critical role in β-cell apoptosis. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure Legends Fig. 1. Palmitate and high concentrations of glucose induce ceramide accumulation in INS-1 cells. (A)

De novo ceramide synthesis in mammals. The de novo synthesis of ceramide starts in the endoplasmic reticulum with the condensation of serine and palmitoyl-CoA, followed by reduction of 3-keto-dihydro-sphingosine to dihydro-sphingosine. The latter compound is subsequently N-acylated by a family of ceramide synthases to form dihydroceramides. Finally, a trans double bond at C4-C5 is introduced by dihydro-ceramide desaturase to produce ceramides. Ceramides are then utilized for formation of more complex sphingolipids. (B) Cells were incubated for 24 h without or with 0.4 mM palmitate in the presence of 5 mM (G5), 10 mM (G10) or 30 mM (G30) of glucose. Ceramide levels were measured by the diacylglycerol kinase assay and expressed as a percent of ceramide levels at 5 mM glucose. Data are means ± SEM of 3-4 independent experiments performed in duplicate. * G30 vs G5 and G10 vs G5, p < 0.05. ** G10 plus palmitate vs G10 and G30 plus palmitate vs G30, p <0.05. Fig. 2. Chain-length specificity of ceramide production in response to palmitate and high concentrations of glucose in INS-1 cells. Cells were incubated in the presence of various concentrations of glucose for 24 h (A and B) or 0.4 mM palmitate with 5 mM glucose for 12 h (C and D). Levels of N-acyl chain lengths of ceramides were determined by LC-MS/MS measurement as described in Materials and Methods. Values are expressed as a fold increase over 5 mM glucose treatment. Data are means ± SEM of 3 independent experiments. * G30 vs G5 and G10 vs G5, Palmitate vs None, p < 0.05. G5, 5 mM glucose; G10, 10 mM glucose; G30, 30 mM glucose. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 19 May 2011 as manuscript BJ20101386 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101386

Biochemical

  Journal Immediate Publication. Published on 19 May 2011 as manuscript BJ20101386 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101386A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 19 May 2011 as manuscript BJ20101386 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101386A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 19 May 2011 as manuscript BJ20101386 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101386 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

  Biochemical Journal Immediate Publication. Published on 19 May 2011 as manuscript BJ20101386 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101386A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Biochemical Journal Immediate Publication. Published on 19 May 2011 as manuscript BJ20101386 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101386A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 19 May 2011 as manuscript BJ20101386 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101386

Biochemical

  Journal Immediate Publication. Published on 19 May 2011 as manuscript BJ20101386 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101386 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 19 May 2011 as manuscript BJ20101386 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101386

Fig. 3 .

 3 Fig. 3. Palmitate and high concentrations of glucose increase dihydro-ceramide production in INS-1 cells. Cells were incubated in the presence of various concentrations of glucose for 24 h (A and B) or 0.4 mM palmitate with 5 mM glucose for 12 h (C and D). Levels of total (A and C) and N-acyl chain length of dihydro-ceramide (B and D) were determined by LC-MS/MS measurement as described in Materials and Methods. Values are expressed as fmol/nmol of PL (A and C) or as a fold increase over 5 mM glucose treatment (B and D). Data are means ± SEM of 3 independent experiments. * G30 vs G5 and G10 vs G5, Palmitate vs None, p < 0.05. G5, 5 mM glucose; G10, 10 mM glucose; G30, 30 mM glucose.

Fig. 4 .

 4 Fig. 4. Palmitate and high concentrations of glucose increase dihydro-sphingosine production in INS-1 cells. Cells were incubated with 0.4 mM palmitate in the presence of 5 mM (G5) or 30 mM (G30) of glucose for 12 h. Levels of dihydro-sphingosine (A) and sphingosine (B) were determined by LC-MS/MS measurement as described in Materials and Methods. Values are expressed as fmol/nmol of PL. Data are means ± SEM of 3 independent experiments. * G5+ palmitate vs G5 and G30 + palmitate vs G30, p < 0.05. ** G30 + palmitate vs G5, p <0.05.

Fig. 5 .

 5 Fig. 5. High concentrations of glucose potentiate dihydro-ceramide and ceramide production induced by palmitate in INS-1 cells. Cells were incubated for 12 h with 0.4 mM palmitate in the presence of 5 mM (G5) or 30 mM (G30). Levels of N-acyl chain lengths of dihydroceramides (A and B) and ceramides (C and D) were determined by LC-MS/MS measurement as described in Materials and Methods. Values are expressed as a fold increase over 5 mM glucose treatment. Data are means ± SEM of 3 independent experiments. * G5 + palmitate vs G5 and G30 + palmitate vs G5, p <0.05. ** G30 + palmitate vs G5 + palmitate, p <0.05. G5, 5 mM glucose, G5P4, 5 mM glucose + 0.4 mM palmitate, G30, 30 mM glucose, G30P4, 30 mM glucose + 0.4 mM palmitate.

Fig. 6 .

 6 Fig. 6. Gluco-lipotoxicity activates caspase-3/7 through ceramide accumulation in INS-1 cells. Cells were incubated for 24 h with 0.4 mM palmitate in the presence of various concentrations of glucose. Cell viability (A) and caspase-3/7 activity (B) were determined by MTT assay the Apo-ONE Homogeneous Caspase-3/7 Assay kit, respectively. Values are expressed as a percent of living cells at 5 mM glucose for cell viability and as arbitrary units (AU) for caspase activity. Data are mean ± SEM of 4-5 independent experiments performed in triplicate. . * G30 vs G5 and G10 vs G5, p < 0.05. ** G10 + palmitate vs G10 and G30 + palmitate vs G30, p <0.05. Cells were pre-incubated without or with 25 M FB1 (C) or 10 M PPMP (D), then incubated for 24 h with 0.4 mM palmitate in the presence of 5 or 30 mM glucose. Caspase 3/7 activity was determined as described above. Data are means ± SEM of 3 independent experiments performed in triplicate. * G5 + PPMP vs G5, G30 + PPMP vs G30 and G30 + palmitate vs G30, p < 0.05. ** G30 + palmitate + FB1 vs G30 + palmitate, G5 + palmitate + PPMP vs G5 +palmitate and G30 + palmitate + PPMP vs G30 + palmitate, p< 0.05. G5, 5 mM glucose; G10, 10 mM glucose; G30, 30 mM glucose.

Fig. 7 .

 7 Fig. 7. Fumonisin B1 and PPMP inversely regulate ceramide accumulation induced by gluco-lipotoxicity in INS-1 cells. Cells were pre-incubated or not with 25 M FB1 (A) or 10 M PPMP (B, C and D), then incubated for 24 h with 0.4 mM palmitate in the presence of 30 mM glucose. (A) Ceramide levels were measured by the diacylglycerol kinase assay and expressed as a percent of ceramide levels at 5 mM glucose. Data are means ± SEM of 3 independent experiments performed in duplicate. Levels of N-acyl chain length of dihydroceramides (B) and ceramides (C and D) were determined by LC-MS/MS measurement as described in Materials and Methods. Values are expressed as a fold increase over 30 mM glucose treatment (B and D).* G30 + palmitate vs G30 and G30 + PPMP vs G30, p < 0.05. ** G30 + PPMP vs G30 and G30 + palmitate + PPMP or FB1 vs G30 + palmitate, p< 0.05. G30, 30 mM glucose, G30P4, 30 mM glucose + 0.4 mM palmitate.

Fig. 8 .

 8 Fig. 8. Palmitate with high glucose levels induces ceramide synthase 4 expression in INS-1 cells. Cells were incubated for different times with 0.4 mM palmitate in the presence of 5 mM or 30 mM glucose. (A and B) Ceramide synthases (CerS) mRNA expression was determined after 12 h of treatment by quantitative real-time PCR. Data are means ± SEM of 3 independent experiments performed in duplicate. * G30 + palmitate vs G30, p < 0.05. (C) CerS4 protein levels were determined by western-blot using an anti-CerS4 antibody.

Fig. 9 :

 9 Fig.9: Over-expression of ceramide synthase 4 enhances ceramide production induced by gluco-lipotoxicity in INS-1 cells. Cells were transiently transfected with the empty vector alone or the HA-tagged CerS4 pcDNA5-TO construct. (A) Over-expressed CerS4 was determined by western blot using an anti-HA or an CerS4 antibody. INS-1/vector and INS-1/CerS4 cells were incubated for 24 h with 0.4 mM palmitate or 0.4 mM stearate in the presence of 30 mM glucose. Levels of ceramides (B) and N-acyl chain length of ceramides (C) were determined by the diacylglycerol kinase assay and LC-MS/MS measurement, respectively. Values are expressed as expressed as fmol of ceramide by nmol of PL (B) and a fold increase over 30 mM glucose treatment (C). Data are means ± SEM of 3 independent experiments. * G30 + fatty acids vs G30 in vector cells, p < 0.05; ** G30 + fatty acids in CerS4 cells vs G30 + fatty acids in vector cells, p < 0.05. G30, 30 mM glucose; G30P4, 30 glucose + 0.4 mM palmitate; G30St, 30 mM glucose + stearate.

Fig. 10 :

 10 Fig. 10 : Modulation of ceramide synthase 4 expression regulates caspase-3/7 activation induced by gluco-lipotoxicity in INS-1 cells. Cells were transiently transfected with the

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  Biochemical Journal Immediate Publication. Published on 19 May 2011 as manuscript BJ20101386 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101386

  Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

		Biochemical Journal Immediate Publication. Published on 19 May 2011 as manuscript BJ20101386
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101386	A c c e p t e d M a n u s c r i p t

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Acknowledgments

This project was supported by grants from Centre National de la Recherche Scientifique (CNRS) and Agence Nationale de la Recherche (ANR-06-JCJC-0040). Julien Veret received a doctoral fellowship from the French Ministry of Higher Education (Physiology and Pathophysiology Doctoral School ED n°394). Dr. Evgeny Berdyshev and Prof. Viswanathan Natarajan were supported in part by the University of Chicago, Department of Medicine through the support to the Lipidomics facility. INS-1 cells (clone 368) were a kind gift of Dr. Christine Charon, Merck Serono. We thank Prof. Thierry Levade for his review of the manuscript and his helpful comments. We thank Sylvie Coursol and Carl K.-Y. Ng for critically reading the manuscript. Biochemical Journal Immediate Publication. Published on 19 May 2011 as manuscript BJ20101386 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101386

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.


Rongxi Yang
email: r.yang@dkfz.de

Bettina Schlehe

Kari Hemminki

Christian Sutter

Peter Bugert

Barbara Wappenschmidt

Juliane Volkmann

Raymonda Varon

Bernhard H F Weber

Dieter Niederacher

Norbert Arnold

Alfons Meindl

Claus R Bartram

Rita K Schmutzler

Barbara Burwinkel

Á B Burwinkel

Á B Schlehe

Á C R Bartram

Á R K Schmutzler

Á A Meindl

A genetic variant in the pre-miR-27a oncogene is associated with a reduced familial breast cancer risk

Keywords: Breast cancer risk, MicroRNA, SNP, Case-control study

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

MicroRNAs (miRNAs) are an abundant class of small endogenous noncoding RNAs (20-22 nt) that negatively regulate the target gene transcription through hybridization to incomplete complementary sequences in the 3 0 untranslated region of their target mRNAs. It results in either the degradation of target messenger RNAs or repression of their translation [START_REF] Bartel | MicroRNAs: genomics, biogenesis, mechanism, and function[END_REF][START_REF]The functions of animal microRNAs[END_REF]. MicroRNAs are initially transcribed from genomic DNA to long primary transcripts (pri-miRNAs) and then are cleaved by nuclear Drosha into 60-70 nt hairpin-shaped precursor RNAs (pre-miRNAs) [START_REF] Lee | MicroRNA maturation: stepwise processing and subcellular localization[END_REF][START_REF] Lee | The nuclear RNase III Drosha initiates microRNA processing[END_REF]. Pre-miRNAs are exported to the cytoplasm by Exportin-5 and are further processed into *22 nt mature miRNA duplexes by the cleavage of Dicer [START_REF] Lund | Nuclear export of microRNA precursors[END_REF][START_REF] Hutvagner | A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA[END_REF][START_REF] Ketting | Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans[END_REF]. In association with RNA-induced silencing complex (RISC), one strand of the miRNA duplex matches target mRNA sequence in the 3 0 un-translated regions. This binding finally leads to the degradation of the mRNAs or the inhibition of their translation and consequently down-regulates the expression of the protein [START_REF] Martinez | Single-stranded antisense siRNAs guide target RNA cleavage in RNAi[END_REF][START_REF] Schwarz | Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways[END_REF]. As miRNAs can regulate multiple target mRNAs, they have been identified to be crucial in the process of immune system regulation, hematopoiesis, angiogenesis, cell development, cell proliferation, differentiation, and apoptosis [START_REF] Baltimore | MicroRNAs: new regulators of immune cell development and function[END_REF][START_REF] Brennecke | Bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila[END_REF][START_REF] Chang | Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis[END_REF][START_REF] Calin | MiR-15a and miR-16-1 cluster functions in human leukemia[END_REF][START_REF] Dews | Augmentation of tumor angiogenesis by a Myc-activated micr-oRNA cluster[END_REF][START_REF] Xiao | MicroRNA control in the immune system: basic principles[END_REF]. Elevated or decreased expression of miRNAs has been found in various tumor types. Some miRNAs thereby have been considered as tumor suppressors or oncogenes [START_REF] Johnson | RAS is regulated by the let-7 microRNA family[END_REF][START_REF] He | A microRNA polycistron as a potential human oncogene[END_REF][START_REF] O'donnell | c-Myc-regulated microRNAs modulate E2F1 expression[END_REF]. They are shown to be involved in cancer initiation and progression by suppression and the expression of cancerrelated genes [START_REF] Esquela-Kerscher | Oncomirs-microRNAs with a role in cancer[END_REF]. Furthermore, miRNA expression profiles can be molecular markers for cancer diagnosis and therapy [START_REF] Calin | MicroRNA signatures in human cancers[END_REF][START_REF] Lu | MicroRNA expression profiles classify human cancers[END_REF].

Breast cancer is the leading cause of cancer-related death in women and next to lung cancer, the second most common cancer in the world [START_REF] Parkin | Global cancer statistics, 2002[END_REF]. Up to 10% of women, who are diagnosed with breast cancer, report a family history [START_REF] Hopper | Genetic epidemiology of female breast cancer[END_REF][START_REF] Narod | Modifiers of risk of hereditary breast and ovarian cancer[END_REF]. According to the polygenic model of inherited breast cancer, unfavorable combinations of polymorphic genetic variants in low-penetrance susceptibility genes contribute to the excess familial breast cancer risk. Most of these susceptibility genes have not been discovered yet [25,[START_REF] Pharoah | Polygenic susceptibility to breast cancer and implications for prevention[END_REF].

It is suggested that miRNA mutations or mis-expressions correlate with various human cancer risks [START_REF] Esquela-Kerscher | Oncomirs-microRNAs with a role in cancer[END_REF]. Although the single nucleotide polymorphisms (SNPs) in miRNAs target sites have been studied [START_REF] Tchatchou | A variant affecting a putative miRNA target site in estrogen receptor (ESR) 1 is associated with breast cancer risk in premenopausal women[END_REF][START_REF] Landi | Polymorphisms within micro-RNA-binding sites and risk of sporadic colorectal cancer[END_REF][START_REF] Kapeller | First evidence for an association of a functional variant in the microRNA-510 target site of the serotonin receptortype 3E gene with diarrhea predominant irritable bowel syndrome[END_REF], the effects of SNPs in miRNAs remain largely unknown. It motivated us to explore polymorphisms in the breast cancer-related miRNA genes and investigate their associations with familial breast cancer risk.

In our study, we selected 11 miRNAs from the literature (miR-373, miR-520c, miR-126, miR-335, miR-10b, let-7a-1, let-7a-2, let-7a-3, miR-27a, miR-21, and miR-145). All the 11 miRNAs have been reported to be associated with breast cancer initiation, progression, and metastasis and with known mechanisms [START_REF] Tavazoie | Endogenous human microRNAs that suppress breast cancer metastasis[END_REF][START_REF] Ma | Tumour invasion and metastasis initiated by microRNA-10b in breast cancer[END_REF][START_REF] Yu | let-7 regulates self renewal and tumorigenicity of breast cancer cells[END_REF][START_REF] Huang | The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis[END_REF][START_REF] Lu | Hypermethylation of let-7a-3 in epithelial ovarian cancer is associated with low insulin-like growth factor-II expression and favorable prognosis[END_REF][START_REF] Sempere | Altered microRNA expression confined to specific epithelial cell subpopulations in breast cancer[END_REF][START_REF] Mertens-Talcott | The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and the G2-M checkpoint in MDA-MB-231 breast cancer cells[END_REF][START_REF] Frankel | Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells[END_REF]. We systematically scanned for SNPs located in these breast cancer-relevant miRNA genes (including pre-miRNAs and about ±200 bp flanking regions) by sequencing these regions, and then investigated the four most promising SNPs which were selected according to the defined criteria in a large German familial study cohort. Our analysis revealed that the [G] allele of SNP rs895819, located in pre-miR-27a oncogene, has a significant association with a reduced familial breast cancer risk.

Materials and methods

Study population

Our study cohort contains 1,217 German familial breast cancer (BC) patients and 1,422 unrelated healthy German women. The breast cancer index cases were tested BRCA1/2 mutation-negative by applying mutational screening on all exons with denaturing high performance liquid chromatography (DHPLC), and followed by direct sequencing of conspicuous exons [START_REF] Meindl | Comprehensive analysis of 989 patients with breast or ovarian cancer provides BRCA1 and BRCA2 mutation profiles and frequencies for the German population[END_REF]. All the 1,217 familial breast cancer cases are BRCA1 and BRCA2 mutation negative. In the case group, 115 patients had bilateral breast cancer and were considered as a subgroup. The BC samples were collected during the years 1997-2007 by seven centers of the German Consortium for Hereditary Breast and Ovarian Cancer (centres of Heidelberg, Wu ¨rzburg, Cologne, Kiel, Du ¨sseldorf, Munich, and Berlin, see authors' affiliations). German index patients were first diagnosed with breast cancer and then referred to a family registry. The informed consent for the study was given to all the breast cancer patients. About 3% of patients refused to donate their blood samples for research purposes.

The control population included unrelated healthy female blood donors collected by the German Red Cross Blood Service of Baden-Wuerttemberg-Hessia and Institute of Transfusion Medicine and Immunology (Mannheim), sharing the same ethnic background with the breast cancer patients (Caucasian/German population). All of the control population are healthy when they donated their blood, and none of them has a reported familial history of breast cancer. Age distributions in controls and breast cancer cases were similar (controls: mean age 45.8 years, median age 49 years, range from 18 to 68 years; cases: mean age 46.2 years, median age 46 years, range from 19 to 87 years). The controls were randomly selected during the years 2004-2007 for this study, and no further inclusion criteria were applied during recruitment. This study was approved by the Ethics Committee of the University of Heidelberg (Heidelberg, Germany). According to the German guidelines for blood donation, all blood donors were examined by a standard questionnaire. The informed consent for this study was given to all the participants. About 5% of blood donors refused to use their blood samples for research purposes.

SNP verification and sequencing

Breast cancer-related 11 miRNA genes were selected from the literature. In order to verify annotated single SNPs from the SNP database (NCBI) and to identify potential new SNPs, 11 miRNA genes, including pre-miRNAs and about ±200 bp flanking regions, were amplified by the standard PCR (in order to unfold the secondary structure of miR-NAs, 5% DMSO was added to the PCR buffer). Owing to high homology with other genomic regions, specific primer design was not possible for miR-520c. Thus, miR-520c was not further analyzed. PCR products were purified with the ExoSAP-IT purification kit (USB Corp.) and were then sequenced in one of the directions by the 3130XL Genetic Analyzer (Applied Biosystems). Sequencing results were analyzed with Sequencing Analysis 5.2 software (Applied Biosystems). As a result, 10 SNPs were verified in these segments. Sequences of primers are available upon request.

Genotyping

According to our defined criteria, five out of 10 SNPs (rs12983273 located 127 bp upstream of pre-miR-373, rs4636297 located 12 bp downstream of miR-126, rs7310 85 located 63 bp downstream of pre-let-7a-3, rs3807348 located 62 bp downstream of pre-miR-335, and rs895819 in the pre-miR-27a, Table 1) were selected for further investigation for their putative influence on breast cancer risk. The three SNPs, rs12883273, rs731085, and rs3807348 were analyzed by the method of TaqMan allelic discrimination. However, the TaqMan assay performance of rs4636297 in miR-126 could not be improved even when adding DMSO, formamide, or alternating the temperature profile. The SNP rs895819 could not be analyzed by TagMan assay due to failed primer and probe design. As rs895819 is the only SNP located within the pre-miRNA, which we were especially interested in, we genotyped this SNP by sequencing as describe above. Primers and Taq-Man MGB probes were purchased from Applied Biosystems (Foster City, CA). Genomic DNA of 5 ng was used for each reaction. PCR cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min, and 45-50 cycles of (92°C, 15 s, and 60°C, 60 s). Samples were analyzed with SDS 1.2 software (Applied Biosystems) in ABI Prism 7900HT detection system. The TaqMan SNP assay results were validated by regenotyping 10% of all the samples. The concordance rate was 100%. Sequences of primers and probes are available upon request.

Statistical analysis

Hardy-Weinberg equilibrium test was undertaken using the chi-square ''goodness-of-fit'' test by a tool from the Institute of Human Genetics, Technical University Munich, Munich, Germany (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). Genotypespecific odds ratios (OR), 95% confidence intervals (CI), and P values were computed by unconditional logistic regression with SAS version 9.1 (SAS Institute Inc., Cary, NC). Age, treated as a continuous variable was included in the regression as covariate. P values were calculated using two-sided chi-square test. The power (a = 0.05) was calculated using the power and sample size calculation software PS version 2.1.31 (http://www.mc.vanderbilt.edu/prev med/ps/index.htm) [START_REF] Dupont | Power and sample size calculations for studies involving linear regression[END_REF]. SNPs linked with all the 10 SNPs (Table 1) with r 2 C 0.8 and block definition were identified using HaploView version 3.32 (http://www.broad.mit.edu/ mpg/haploview). The linked SNPs' breast cancer associations were further verified whether they had been analyzed by the Cancer Genetic Markers of Susceptibility genome wide association study (CGEMS) (https://caintegrator.nci. nih.gov/cgems/browseSetup.do).

Results

We performed a systematic literature and database search in NCBI and found 11 miRNAs (miR-373, miR-520c, miR-126, miR-335, miR-10b, let-7a-1, let-7a-2, let-7a-3, miR-27a, miR-21, miR-145) to be involved in breast cancer with known mechanisms [START_REF] Tavazoie | Endogenous human microRNAs that suppress breast cancer metastasis[END_REF][START_REF] Ma | Tumour invasion and metastasis initiated by microRNA-10b in breast cancer[END_REF][START_REF] Yu | let-7 regulates self renewal and tumorigenicity of breast cancer cells[END_REF][START_REF] Huang | The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis[END_REF][START_REF] Lu | Hypermethylation of let-7a-3 in epithelial ovarian cancer is associated with low insulin-like growth factor-II expression and favorable prognosis[END_REF][START_REF] Sempere | Altered microRNA expression confined to specific epithelial cell subpopulations in breast cancer[END_REF][START_REF] Mertens-Talcott | The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and the G2-M checkpoint in MDA-MB-231 breast cancer cells[END_REF][START_REF] Frankel | Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells[END_REF]. In order to identify potential new SNPs as well as to verify the annotated SNPs from the NCBI SNP database in these segments, we sequenced these 10 pre-miRNA genes in a sample set of more than 100 randomly chosen familial breast cancer cases. We found 10 SNPs in six pre-miRNAs or their flanking regions (miR-373, miR-226, miR-335, miR-10b, let-7a-3, and miR-27a, Table 1); evaluation of miR-520c failed because of high homology with other genomic regions making specific primer design impossible. No SNPs were found in the other four pre-miRNAs or their flanking regions (miR-21, miR-145, let-7a-1, and let-7a-2, Table 1).

In order to determine the most interesting candidates for the investigation of breast cancer risk, we analyzed 200 kb flanking regions of all the 10 SNPs, including the haplotype blocks of these SNPs by HaploView. We further investigated whether the SNPs themselves and their linkage SNPs (r 2 C 0.8) had been analyzed, so far, by the Cancer CGEMS. The CGEMS is a whole-genome association study conducted by the National Cancer Institute (NCI) enterprise to identify breast cancer susceptibility genes using Illumina HumanHap550 assays on approximately 1,200 breast cancer patients and 1,200 control subjects. If the SNPs or their linkage SNPs (r 2 C 0.8) have been analyzed in CGEMS and didn't show any indication for a possible association with breast cancer (P value of allele or genotype comparison between cases and controls is larger than 0.2), they were excluded from further analysis. This was the case for SNP rs1867863. Furthermore, SNPs with a rare homozygote frequency of less than 1% were also excluded due to the limited power of the study. Base on these criteria, 5 SNPs (rs12983273 located 127 bp upstream of pre-miR-373, rs4636297 located 12 bp downstream of miR-126, rs731085 located 63 bp downstream of pre-let-7a-3, rs3807348 located 62 bp downstream of pre-miR-335 and rs895819 in the pre-miR-27a) were identified (Table 1).

Genotype analysis of these SNPs was performed first on genomic DNA of a subset of our study cohort, which contains BRCA1/2 mutation-negative index patients from 797 German breast cancer families with a mean age of 45.8 years, and 758 unrelated German control individuals with a mean age of 48 years. The TaqMan assay performance of rs4636297 in miR-126 failed and could not be improved even with adding DMSO, formamide or alternating the temperature profile, and thus, was not considered for further investigation. All of the samples sequenced previously were genotyped again by the TaqMan assay and attained concordance rates of 100%. As rs895819 showed a significant association with familial breast cancer, this SNP was further analyzed on an enlarged sample set (in total 1,217 familial breast cancer cases versus 1,422 controls; see materials and methods). In the investigation of rs895819 on the whole sample set, we reached a 97.7% callrate in cases (1,189) and a 99.5% callrate in controls [START_REF] Bartel | MicroRNAs: genomics, biogenesis, mechanism, and function[END_REF]416). Genotype distributions in controls and cases were consistent with the Hardy-Weinberg equilibrium (HWE). In all the SNPs analyzed, the P value is larger than 0.05. The TaqMan assay results were validated by regenotyping 10% of all the samples attaining concordance rates of more than 99.5% for all the investigated SNPs.

Genotype frequencies of rs12983273, rs731085, and rs3807348 were similar between familial breast cancer cases and control samples, showing no association with familial breast cancer (Table 2). The analysis of the polymorphism rs895819, which is the only SNP located in the pre-miRNA, revealed a significant association with familial breast cancer. The rare G-variant allele was less frequent in cases than in controls ([G] vs. [A]: OR = 0.88, 95% CI 0.78-0.99, P = 0.0287; P trend = 0.0288, Table 2).

The age stratification (age \ 50 years and C50 years) showed that the protective effect of the [G] allele of rs895819 was mainly in the younger age group (\50 years of age) with slightly more decreased risk ([G] vs. [A]: OR = 0.83, 95% CI 0.70-0.98, P = 0.0314; P trend = 0.0336, Table 3). In contrast, in the older age group (C50 years of age), the genotype frequencies of rs895819 showed no significant association with familial breast cancer (Table 3). In the subgroup of bilateral familial breast cancer cases, the protective effect was even slightly stronger ([G] vs. [A]: OR = 0.70, 95% CI 0.52-0.95, P = 0.0238; P trend = 0.0215, Table 3).

Discussion

In recent studies, increasing evidences suggest that miR-NAs are crucial factors in cancer progress as oncogenes or tumor suppressors by degradation of the target mRNAs and/or inhibition of their translation [START_REF] Zhang | MicroRNAs as oncogenes and tumor suppressors[END_REF]. MicroRNAs are also considered to be potential biomarkers and therapeutic targets in human cancers [START_REF] Osaki | MicroRNAs as biomarkers and therapeutic drugs in human cancer[END_REF]. Variations in expression of miRNAs have been reported to be related to many tumors including breast cancer [START_REF] Calin | MicroRNA signatures in human cancers[END_REF][START_REF] Lu | MicroRNA expression profiles classify human cancers[END_REF], and miRNAs are also involved in breast cancer progress [START_REF] Verghese | Small is beautiful: microRNAs and breast cancer-where are we now?[END_REF]. Differential miR-NA expression could be caused by sequence variations, such as mutations and SNPs. So far, SNPs in the miRNA target sites have been widely studied [START_REF] Tchatchou | A variant affecting a putative miRNA target site in estrogen receptor (ESR) 1 is associated with breast cancer risk in premenopausal women[END_REF][START_REF] Landi | Polymorphisms within micro-RNA-binding sites and risk of sporadic colorectal cancer[END_REF][START_REF] Kapeller | First evidence for an association of a functional variant in the microRNA-510 target site of the serotonin receptortype 3E gene with diarrhea predominant irritable bowel syndrome[END_REF], but the association between SNPs in miRNAs and familial breast cancer risk remains still largely unknown.

Our study is focused on the SNPs in the breast cancerrelated miRNAs. In addition to affecting the target hybridization [START_REF] Tchatchou | A variant affecting a putative miRNA target site in estrogen receptor (ESR) 1 is associated with breast cancer risk in premenopausal women[END_REF], a mutation or a SNP in a miRNA gene may also have an impact on the transcription and/or procession of the pri-and pre-miRNAs [START_REF] Wu | Genetic variations of microRNAs in human cancer and their effects on the expression of miRNAs[END_REF]. Thus, we took the pre-miRNAs and their flanking regions (about ±200 bp) into consideration. Furthermore, as the mutations in BRCA1 and BRCA2 account for approximately 30% of familial breast cancer cases in Germany [START_REF] Meindl | Comprehensive analysis of 989 patients with breast or ovarian cancer provides BRCA1 and BRCA2 mutation profiles and frequencies for the German population[END_REF], only BRCA1/2 mutation-negative familial breast cancer cases were included in our study to rule out the influence of the disease-associated mutations. Given our sample size, we obtained a power of 80% (a = 0.05) to detect an OR of 0.80 for SNP rs895819, not considering the usage of familial cases. The power of an association study based on familial cases was even about two times higher than that of unselected cases [START_REF] Houlston | The future of association studies of common cancers[END_REF][START_REF] Antoniou | Polygenic inheritance of breast cancer: implications for design of association studies[END_REF].

The three SNPs located in the pre-miRNA flanking regions (rs12983273 located 127 bp upstream of pre-miR-373, rs731085 located 63 bp downstream of pre-let-7a-3, rs3807348 located 62 bp downstream of pre-miR-335, rs895819 in the pre-miR-27a, Table 1), did not show a significant association with familial breast cancer. However, the [G] allele of rs895819 in the pre-miR-27a oncogene was associated with a decreased breast cancer risk. This effect was even stronger in the subgroup of \50 years of age and in bilateral familial breast cancer cases with slightly decreased respective ORs, whereas no significant effect was observed in the C50 years of age group. The potential age-or hormone-related effect is discussed later in this section. In bilateral breast cancer cases (a second primary tumor in the contralateral breast), it is believed that the underlying ''mutation'' is not limited to the epithelial cells of one breast but rather systemic variation [START_REF] Weitzel | A comparison of bilateral breast cancers in BRCA carriers[END_REF]. Bilateral breast cancer is considered to have a strong genetic background [START_REF] Greene | Genetics of breast cancer[END_REF]. Thus, the SNP effect is expected to be stronger in this subgroup. However, due to the limited samples size of bilateral cases, it is also possible that the association of rs895819 with reduced bilateral breast cancer risk was detected by chance. The SNP rs895819 is located in the loop of pre-miR-27a. In order to investigate whether this variation might alter the secondary structure of the pre-miR-27a, we used the RNAfold program [START_REF] Markham | DINAMelt web server for nucleic acid melting prediction[END_REF] (http://www.bioinfo.rpi.edu/ applications/hybrid/) to predict the most stable secondary structure of both wild-type and variant. According to the prediction of the RNAfold program, the [G] variant of rs895819 affected neither the pre-miR-27a's conformation nor the free energy (data not shown). It has been suggested that a mutation causing structural change in the crucial region of miRNA could affect the maturation and the process of miRNA [START_REF] Jazdzewski | Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma[END_REF][START_REF] Hu | Genetic variants of miRNA sequences and non-small cell lung cancer survival[END_REF][START_REF] Duan | Single nucleotide polymorphism associated with mature miR-125a alters the processing of pri-miRNA[END_REF]. Furthermore, Wu et al. found that even some SNPs with no influence on miRNA secondary structure can affect the expression of miRNA through some unclear mechanism, suggesting that the processing and maturation of miRNA is more complex and subtle than predicted [START_REF] Wu | Genetic variations of microRNAs in human cancer and their effects on the expression of miRNAs[END_REF]. Zeng et al. suggested that Drosha selectively cleaves RNA hairpins bearing a large terminal loop. When the loop is shortened by mutation or deletions, the maturation process will be harmed [START_REF] Zeng | Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha[END_REF]. In their study, an A-G mutation was introduced to the terminal loop of miR-27a. As a result, reduced productions of both pre-miRNA-27a and mature miRNA-27a were observed when the size of the terminal loop was reduced by the new bonds formed by mutations [START_REF] Zeng | Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha[END_REF]. According to the predicted structure of miR-27a, rs895819 is located in the center of the terminal loop. The [A]-[G] variant of rs895819 may result in an additional C-G binding with a [C] allele in the loop, which will probably reduce the size of the loop. Consequently the cleavage by Drosha would be blocked, and the maturation process is thereby inhibited. Piskounova et al. showed that some Drosha inhibitors such as Lin28, selectively bind to the terminal loop region of miRNA precursors and mediate the miRNA procession [START_REF] Piskounova | Determinants of microRNA processing inhibition by the developmentally regulated RNAbinding protein Lin28[END_REF][START_REF] Newman | Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing[END_REF]. Thus, it is also possible that the [A]-[G] variant changes the binding affinity of some Drosha inhibitors and consequently affects the processing of miR-27a. In conclusion, we hypothesize that rs895819 may play an important role in the maturation of miR-27a. However, this hypothesis needs to be tested.

By means of the mRNA target gene prediction database (http://microrna.sanger.ac.uk/), we found more than 1,000 target genes of miR-27a, including some cancer-related genes, such as aspartate-specific cysteine protease (CASP8) [START_REF] Sun | A six-nucleotide insertion-deletion polymorphism in the CASP8 promoter is associated with susceptibility to multiple cancers[END_REF], mutL homolog 1(MLH1), vascular endothelial growth factor C (VEGFC), and BCL2-associated X protein (BAX). Scott et al. found that the transcription of ZBTB10 is upregulated after miR-27a antisense transfection [START_REF] Scott | Rapid alteration of microRNA levels by histone deacetylase inhibition[END_REF]. In consistence with this result, Mertens-Talcott et al. [START_REF] Mertens-Talcott | The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and the G2-M checkpoint in MDA-MB-231 breast cancer cells[END_REF] suggested that transfection of antisense miR-27a results in increased expression of ZBTB10 mRNA (a SP1 suppressor) and consequently decreases the expression of specificity proteins, SP1, SP3 and SP4, which are overexpressed in tumors and contribute to the proliferative and angiogenic phenotype of cancer cells. Moreover, the transfection of antisense miR-27a was also accompanied by decreased expression of Sp-dependent survival and angiogenic genes, such as survivin, vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1) [START_REF] Mertens-Talcott | The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and the G2-M checkpoint in MDA-MB-231 breast cancer cells[END_REF]. Mertens-Talcott et al. [START_REF] Mertens-Talcott | The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and the G2-M checkpoint in MDA-MB-231 breast cancer cells[END_REF] also found the transfection of antisense miR-27a increases Myt-1 mRNA levels and enhances phosphorylation of cdc2. Thus, the percentage of cells in G 2 -M was consequently increased. Other studies also found that the suppression of miR-27a can inhibit gastric cancer cell growth [START_REF] Liu | MicroRNA-27a functions as an oncogene in gastric adenocarcinoma by targeting prohibitin[END_REF] and overexpression of miR-27a increases the fat accumulation and cell proliferation of Hepatic stellate cells [START_REF] Ji | Overexpressed microRNA-27a and 27b influence fat accumulation and cell proliferation during rat hepatic stellate cell activation[END_REF]. Therefore, miR-27a is considered as an oncogene. Our results consistently, showed that the wild genotype [AA] had higher frequency in cases than in controls, whereas the variant [G] allele reduced the risk of miR-27a. We propose that the [G] allele of rs895819 attenuates the maturation process of oncogenic miR-27a.

Several reports demonstrated that SP1 and other Sp proteins play an important role in regulation of 17b-estradiol (E 2 )-dependent genes in breast cancer cell lines and in many other cell-types [START_REF] Safe | Sp transcription factor family and its role in cancer[END_REF][START_REF] Safe | Nuclear receptor-mediated transactivation through interaction with Sp proteins[END_REF]. The 17b-estradiol (E 2 )dependent interaction of estrogen receptor (ER) and Sp proteins modulates the activation of a cluster of genes [START_REF] Stoner | Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor alpha and SP proteins[END_REF][START_REF] Porter | Functional synergy between the transcription factor Sp1 and the estrogen receptor[END_REF], for example, c-fos, IGF-binding protein 4 (IGFBP-4), B-cell lymphoma 2 (Bcl2), E2F transcription factor 1 (E2F1), vascular endothelial growth factor (VEGF) and cyclin D1 [START_REF] Safe | Non-classical genomic estrogen receptor (ER)/specificity protein and ER/activating protein-1 signaling pathways[END_REF]. A recent study reported that Sp proteins are involved in the estrogen-induced transcription of BRCA1 [START_REF] Hockings | Involvement of a specificity proteins-binding element in regulation of basal and estrogen-induced transcription activity of the BRCA1 gene[END_REF]. As the expression of Sp proteins is regulated by miR-27a [START_REF] Mertens-Talcott | The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and the G2-M checkpoint in MDA-MB-231 breast cancer cells[END_REF], it may explain why the protective impact of the rs895819 variant in pre-miR-27a was predominantly observed in premenopausal woman.

Furthermore, the expression of P-glycoprotein and MDR1 mRNA is decreased by the treatment of multidrug resistant (MDR) cancer cell lines with the antago-miRs of miR-27a, which is accompanied by enhanced intracellular accumulation of cytotoxic drugs that are transported by P-glycoprotein [START_REF] Zhu | Role of microRNA miR-27a and miR-451 in the regulation of MDR1/P-glycoprotein expression in human cancer cells[END_REF]. It suggests that the rs895819 variant might influence the therapeutic outcome. A report from Japan found the frequency of [G] allele of rs895819 to be increased in males with peptic ulcer or severe mucosal atrophy compared to control individuals [START_REF] Arisawa | A polymorphism of microRNA 27a genome region is associated with the development of gastric mucosal atrophy in Japanese male subjects[END_REF], which is the opposite direction we have observed in our study. The study showed no significant association in the whole study population and female subgroup, whereas a borderline significant was found in the male subgroup [START_REF] Arisawa | A polymorphism of microRNA 27a genome region is associated with the development of gastric mucosal atrophy in Japanese male subjects[END_REF].As their investigated sample set was small (23 male controls and 91 male cases), their detected genotype frequency might not be representative. On the contrary, their result suggested a difference between male and female and indicated the possible gender specific effect of rs895819. It is the same as we hypothesized that miR-27a might be involved in the estrogen dependent pathway.

Our study systematically focused on SNPs located in breast cancer-related miRNAs. Recent studies have found more miRNAs involved in progression of breast cancer [START_REF] Wu | Suppression of cell growth and invasion by miR-205 in breast cancer[END_REF][START_REF] Shen | Novel genetic variants in microRNA genes and familial breast cancer[END_REF]. Under strong selections, hundreds of miRNA genes have been found in various species, and many miRNA genes are highly conserved [START_REF]The functions of animal microRNAs[END_REF]. Thus, SNPs, especially SNPs with allele frequency larger than 5% are underrepresented in miRNAs. Because of the limited sample size of our study, we only investigated SNPs with an allele frequency [ 5%. As the miRNAs have important biological functions, it would be of great relevance to investigate the SNPs in the miRNAs. Although some of the very rare mutations or SNPs are more likely to be already deleterious in earlier disease, it would be also interesting to investigate all SNPs and mutations within miRNAs, especially the ones in the miRNA 'seed' regions, for a putative effect on cancer risk. Owing to the rare frequency of these variants within miRNAs, very large study populations are required, which can only be achieved in multicenter collaborations.

In summary, we found that the [G] allele of rs895819, located in the terminal loop of in pre-miR-27a oncogene, is associated with reduced familial breast cancer risk. However, large studies from multicenter studies will be necessary to verify the association.

Table 1

 1 Searching for SNPs in breast cancer-related miRNAs in German familial breast cancer cases by sequencing

	SNP a	Genotypes Sequencing	Distance to the	Genotype	CGEMS data d	Linkage SNPs
			results (%) b	pre-miRNA (bp)	frequencies		in 200 kb
					in NCBI c		flanking region
	rs12983273 miR-373 GG	Not sequenced -127	GG = 0.76	Not investigated No linkage SNPs
		GA			GA = 0.37		with r 2 [ 0.8
		AA			AA = 0.08
	rs10425222 miR-373 CC	116 (93.5)	?172	CC = 0.93	Not investigated rs4619513, r 2 = 1.0
		AC AA	8 (6.5) 0 (0)		AC = 0.07		not been investigated by CGEMS
	New SNP miR-373	CC	115 (92.7)	?114	*	Not investigated *
		CT	9 (7.3)
		TT	0 (0)
	rs4636297 miR-126	GG	50 (43.9)	?12	*	Not investigated *
		GA	45 (39.5)
		AA	19 (16.7)
	rs3807348 miR-335	AA	60 (40.3)	?62	*	Not investigated *
		AG	57 (38.3)
		GG	32 (21.5)
	rs41272366 miR-335 TT	145 (97.3)	?20	*	Not investigated *
		TA	4 (2.7)
		AA	0 (0)
	rs1867863 miR-10b	GG	51 (36.7)	-61	GG = 0.41	Not investigated rs4972806, r 2 = 0.89,
		GT	66 (47.5)		GT = 0.47		P = 0.31 by CGEMS
		TT	22 (15.8)		TT = 0.12
	rs731085 let-7a-3	CC	45 (39.1)	?63	No Caucasian data Not investigated *
		CG	53 (46.1)
		GG	17 (14.8)
	rs895819 miR-27a	AA	53 (49.5)	In pre-miRNA-27a GG = 0.55	Not investigated *
		AG	40 (37.4)		AG = 0.37
		GG	14 (13.1)		AA = 0.08
	rs11671784 miR-27a	CC	102 (95.3)	In pre-miRNA-27a *	Not investigated *
		CT	5 (4.7)
		TT	0
	miR-21	*	100	*	*	*	*
	miR-145	*	117	*	*	*	*
	let-7a-1	*	144	*	*	*	*
	let-7a-2	*	97	*	*	*	*

Table 2

 2 Genotype frequencies of SNPs in selected miRNAs in German study population

	SNP	Genotypes	Case (%)	Control (%)	OR	95% CI	P
	miR-373 rs12983273	CC	566 (73.7)	540 (73.3)	1.000
		CT	184 (24.0)	175 (23.7)	1.008	0.794-1.280	0.9477
		TT	18 (2.3)	22 (3.0)	0.854	0.451-1.619	0.6295
		[T] vs. [C]			0.947	0.795-1.193	0.7969
							P trend = 0.8009 a
	miR-335 rs3807348	AA	257 (32.9)	234 (31.8)	1.000
		AG	373 (47.8)	368 (50.0)	0.917	0.729-1.153	0.4564
		GG	150 (19.2)	134 (18.2)	1.015	0.756-1.361	0.9224
		[G] vs. [A]			0.994	0.860-1.149	0.9372
							P trend = 0.9374 a
	let 7a-3 rs731085	GG	306 (39.2)	308 (42.2)	1.000
		GC	365 (46.7)	324 (44.4)	1.125	0.904-1.400	0.2911
		CC	110 (14.1)	97 (13.3)	1.152	0.839-1.583	0.3827
		[C] vs. [G]			1.087	0.936-1.261	0.2741
							P trend = 0.2780 a
	miR-27a rs895919	AA	576 (48.4)	605 (42.7)	1.000
		AG	486 (40.9)	660 (46.6)	0.774	0.657-0.911	0.0021
		GG	127 (10.7)	151 (10.7)	0.883	0.680-1.148	0.3543
		[G] vs. [A]			0.878	0.781-0.987	0.0287
							P trend = 0.0288

a Adjusted for age; all analyses done with SAS Version 9.1 Proc Logistic a Chi-square test for trend

Table 3

 3 Genotype frequencies of rs895819 within pre-miRNA-27a in familial breast cancer subgroups

	SNP	Genotypes	Case (%)	Control (%)	OR	95% CI	P
	miR-27a rs895919 age C 50	AA	173 (45.4)	302 (43.5)	1.000
		AG	167 (43.8)	319 (46.0)	0.915	0.701-1.193	0.5106
		GG	41 (10.8)	73 (10.5)	0.984	0.642-1.507	0.9397
		[G] vs. [A]			0.965	0.799-1.165	0.7099
							P trend = 0.7072 a
	miR-27a rs895919 age \ 50	AA	403 (49.9)	303 (42.0)	1.000
		AG	319 (39.5)	341 (47.2)	0.735	0.579-0.932	0.0109
		GG	86 (10.6)	78 (10.8)	0.785	0.536-1.149	0.2132
		[G] vs. [A]			0.830	0.701-0.984	0.0314
							P trend = 0.0336 a
	miR-27a rs895919 bilateral	AA	61 (54.0)	605 (42.7)	1.000
		AG	44 (38.9)	660 (46.6)	0.662	0.442-0.991	0.0448
		GG	8 (7.1)	151 (10.7)	0.525	0.246-1.121	0.0961
		[G] vs. [A]			0.703	0.518-0.954	0.0238
							P trend = 0.0215 a
	Adjusted for age; all analyses done with SAS Version 9.1 Proc Logistic
	a Chi-square test for trend

a All SNPs located in breast cancer-related miRNA or their flanking regions (±200 bp). The SNPs used in further investigation shown in bold letters b The miRNA genes (including the pre-miRNA and ±200 bp flanking regions) were sequenced in a sample set of more than 100 familial breast cancer cases (the number of each genotype is given and percentage is given in bracket) c All the genotype frequencies listed here are Caucasian data from NCBI (http://www.ncbi.nlm.nih.gov/) d Data from cancer genetic markers of susceptibility genome wide association study (CGEMS) (https://caintegrator.nci.nih.gov/cgems/ browseSetup.do)

Breast Cancer Res Treat (2010) 121:693-702

Acknowledgments We thank Ludwig Heesen, Michelle Dick, Ying Wang, and Anja Schwaeger for their help in genotyping. We are grateful to Bowang Chen for statistical analysis as well as to Sandrine Tchatchou for the technical support. The German breast cancer samples were collected as a part of a project funded by the Deutsche Krebshilfe (Grant number: 107054). This study was supported by the Helmholtz society, the German Cancer Research Center (DKFZ), EU, LSHC-CT-2004-503465, and the Dietmar-Hopp Foundation.