Free Radical Biology & Medicine 49 (2010) 1522–1533

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Free Radical Biology & Medicine

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Original Contribution

Carbonyl reductase 1 protects pancreatic β-cells against oxidative stress-inducedapoptosis in glucotoxicity and glucolipotoxicity

M.A. Rashid a, Seonmin Lee a, Eunyoung Tak a, Jisun Lee a, Tae Gyu Choi a, Joo-Won Lee b, Jae Bum Kim b,Jang H. Youn a,c, Insug Kang a, Joohun Ha a, Sung Soo Kim a,⁎a Medical Science and Engineering Research Center for Bioreaction to Reactive Oxygen Species and Biomedical Science Institute (BK-21), Department of Biochemistry and MolecularBiology, School of Medicine, Kyung Hee University, Seoul 130-701, Koreab Department of Biological Sciences, Seoul National University, Kwanak-Gu, Seoul, Koreac Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, Los Angeles, CA 90089, USA

Abbreviations: ACC, acetyl-CoA carboxylase; ABCA1,aldehyde dehydrogenase; AR, aldose reductase; Bip/Grprotein/glucose-regulated protein-78; CBR1, carbonydichlorodihydrofluorescein diacetate; FAS, fatty acid synglucose-stimulated insulin secretion; HNE, 4-hydroxyno2-en-4-one; MDA, malondialdehyde; MTT, 3-(4,5-dimettetrazolium bromide; NAC, N-acetyl-L-cysteine; ONA, 4-oenal; PDI, protein disulfide isomerase; ROS, reactive oxregulatory element binding protein (SREBP) 1c; TG, trig⁎ Corresponding author. Fax: +82 2 959 8168.

E-mail address: sgskim@khu.ac.kr (S.S. Kim).

0891-5849/$ – see front matter © 2010 Elsevier Inc. Adoi:10.1016/j.freeradbiomed.2010.08.015

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 February 2010Revised 5 August 2010Accepted 12 August 2010Available online 19 August 2010

Keywords:Carbonyl reductase 1Reactive oxygen speciesLipogenesisLipid peroxidationER stressPancreatic β-cell failureType 2 diabetesFree radicals

Carbonyl reductase 1 (CBR1) plays an important role in the detoxification of reactive lipid aldehydes.Oxidative stress has been implicated in the pathogenesis of pancreatic β-cell failure. However, the functionalrole of CBR1 in pancreatic β-cell failure has not been studied yet. Therefore, we investigated the role of CBR1in pancreatic β-cell failure under glucotoxic and glucolipotoxic conditions. Under both conditions,knockdown of CBR1 by specific siRNA increased β-cell apoptosis, expression of lipogenic enzymes (suchas ACC, FAS, and ABCA1), intracellular lipid accumulation, oxidative stress, ER stress, and nuclear SREBP1c,but decreased glucose-stimulated insulin secretion. In contrast, overexpression of CBR1 showed the oppositeeffects. The antioxidants N-acetyl-L-cysteine and Tiron, as well as the FAS inhibitor cerulenin, reversed theeffects of CBR1 knockdown. Interestingly, the expression level and enzyme activity of CBR1 weresignificantly decreased in pancreatic islets of db/db mice, compared with those of wild-type mice. Inconclusion, CBR1 protects pancreatic β-cells against oxidative stress and promotes their survival inglucotoxicity and glucolipotoxicity.

cholesterol transporter; ALDH,p78, immunoglobulin-bindingl reductase 1; DCF-DA, 2′,7′-thase; FFA, free fatty acid; GSIS,n-2-enal; HNO, 1-hydroxynon-hylthiazol-2-yl)-2,5-diphenyl-xononanal; ONE, 4-oxonon-2-ygen species; SREBP1c, sterollyceride.

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© 2010 Elsevier Inc. All rights reserved.

Pancreaticβ-cell failure plays a key role in the pathogenesis of type 2diabetes. Although the exact mechanism underlying β-cell destructionis not known, it has been suggested that both hyperglycemia andhyperlipidemia contribute to β-cell destruction. Both glucotoxicity andlipotoxicity are important in thepathogenesis of type2diabetes becausethey lead to interference with insulin signal transduction and thusinsulin resistance on the one hand and β-cell destruction on the other[1,2]. The functional consequences of glucotoxicity and glucolipotoxicityfor β-cells include inhibition of glucose-stimulated insulin secretion(GSIS), impairment of insulin gene expression, and induction of celldeath by apoptosis.

Hyperglycemia is one of the major factors contributing to oxidativestress in pancreatic β-cells. Under normal glucose concentrations,pancreatic β-cells metabolize glucose easily via the glycolysis pathwayand tricarboxylic acid (TCA) cycle, during which production of reactiveoxygen species (ROS) from mitochondria remains manageable. How-ever, under high glucose concentrations, mitochondria produce exces-sive amounts of ROS as they utilize alternative glucose-metabolizingpathways prone to inductionof oxidative stress. Thesepathways includeglyceraldehyde autoxidation to methylglyoxal and glycation, enedioland α-ketoaldehyde formation, glucosamine and hexosamine metabo-lism, and sorbitol metabolism [3,4]. In addition to increasing theproduction of ROS by mitochondria, glucose is known to induce anincrease in ROS generated by NADPH oxidase in the cell membrane.The administration of glucose to normal subjects results in a markedincrease in ROS generation even without an increase in glucose tosupranormal levels. Thus, glucose is a powerful inducer of oxidativestress [5]. Both sources of ROS, the mitochondria and NADPHoxidase, are likely to induce a marked increase in the oxidative stressto theβ-cells [6,7]. As far as lipids are concerned, an increase in free fattyacid concentrations is known to occur in insulin-resistant states such asobesity and the metabolic syndrome. When free fatty acid (FFA)concentrations are increased in normal subjects to the levels found inthe obese, marked increases in ROS generation and inflammatory stress

http://dx.doi.org/10.1016/j.freeradbiomed.2010.08.015mailto:sgskim@khu.ac.krhttp://dx.doi.org/10.1016/j.freeradbiomed.2010.08.015http://www.sciencedirect.com/science/journal/08915849

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are induced [8]. Indeed, obesity is associated with increased lipolysisand FFA concentrations, chronic oxidative stress, inflammation, andinsulin resistance [9–11]. This statemay also contribute to oxidative andinflammatory stress in theβ-cells. In addition, pancreatic β-cells are notwell equipped with antioxidative defense mechanisms, and thusare easily overwhelmed by redox imbalance arising from the overpro-duction of ROS. Eventually, when hyperglycemia occurs, it provokesfurther excessive ROSproduction and oxidative damage of nucleic acids,proteins, and membrane lipids [12–14]. These processes damagepancreatic β-cells and may eventually lead to marked losses of β-cellfunction in type 2 diabetes.

Carbonyl reductase 1 (CBR1) is a NADPH-dependent, monomeric,and cytosolic enzyme belonging to a family of short-chain dehydro-genases/reductases. The enzyme consists of 277 amino acid residuesand is widely distributed in human tissues such as liver, epidermis,stomach, small intestine, kidney, neuronal cells, and smooth musclefibers [15]. The best substrates of CBR1 are quinones, includingubiquinone-1 and tocopherolquinone (vitamin E). Ubiquinones(coenzyme Q) are constitutive parts of the respiratory chain, andtocopherolquinone protects lipids of biological membranes againstlipid peroxidation, indicating that CBR1 may play an importantrole as an oxidation–reduction catalyst in biological processes [16].Furthermore, CBR1 inactivates highly reactive lipid aldehydes, such as4-oxonon-2-enal (ONE), 4-hydroxynon-2-enal (HNE), and acrolein,which are able to modify protein and DNA damage within cells [17]. Amutation in the gene encoding a homologue of CBR1 causes oxidativestress-induced neurodegeneration in Drosophila melanogaster [18],and overexpression of the human CBR1 in NIH3T3 cells protects fromROS-induced cellular damage [19], both supporting CBR1 as a majorcontributor to the control of oxidative stress.

Although ROS have been implicated in the pathogenesis ofpancreatic β-cell failure and HNE–albumin adducts were shown tobe increased in the serum of type 2 diabetes patients [20], the role ofCBR1 has never been explored in type 2 diabetes. Therefore, wehypothesized that (i) CBR1 exerts a beneficial role in protectingpancreatic β-cells from oxidative stress under glucotoxic andglucolipotoxic conditions, in vitro, and (ii) pancreatic islets from thediabetic db/db mice show low levels of CBR1 expression and enzymeactivity.

Materials and methods

Materials

RPMI 1640 medium and fetal bovine serum were purchased fromLonza (Walkersville, MD, USA) and G418 from Duchefa (St. Louis, MO,USA). T0901317 was purchased from Calbiochem (San Diego, CA, USA).Cerulenin,N-acetyl-L-cysteine (NAC), Tiron, 2′,7′-dichlorodihydrofluor-escein diacetate (DCF-DA), Hoechst 33342, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and oil red O wereacquired fromSigma (St. Louis,MO,USA). CBR1 antibodywaspurchasedfrom Abnova (Taipei, Taiwan), acetyl-CoA carboxylase (ACC) fromUpstate Biotechnology (Lake Placid, NY, USA), and cholesterol trans-porter (ABCA1) from Novus Biologicals (Littleton, CO, USA). Antibodiesspecific to fatty acid synthase (FAS), HNE, andmalondialdehyde (MDA)were purchased from Abcam (Cambridge, UK). Antibodies to aldosereductase (AR), aldehyde dehydrogenase (ALDH), actin, immunoglob-ulin-binding protein/glucose-regulated protein-78 (Bip/Grp78), insu-lin, SREBP1c, and lamin B were acquired from Santa Cruz Biotechnology(Santa Cruz, CA, USA). Antibodies against C/EBP-homologous protein(CHOP), phosphorylationof the eukaryotic initiation factor2α (p-eIF2α),poly(ADP ribose) polymerase (PARP), and caspase 3 were purchasedfrom Cell Signaling Technology (Danvers, MA, USA), and antibodyagainst protein disulfide isomerase (PDI) was obtained from Stressgen(Assay Designs, Ann Arbor, MI, USA).

Cell culture and stable transfection

RINm5F and HIT-T15 cells were grown in RPMI 1640 mediumsupplemented with 10% fetal bovine serum, 100 units/ml penicillin,and 100 μg/ml streptomycin sulfate (Invitrogen, San Diego, CA, USA).pcDNA3.0 (3 μg) used as mock and pcDNA3-CBR1/WT (3 μg) used asCBR1/WT were transfected into RINm5F and HIT-T15 cells usingGenePORTER (Genlantis, San Diego, CA, USA). Cells used for stabletransfection were cultured in the selective medium with 600 mg/mlG418 for a month. Then, drug-resistant individual clones wereisolated and transferred to a six-well plate for further amplificationin the presence of selective medium.

Small interfering RNA

Small interfering RNAs (siRNAs) specific to either CBR1 (CBR1siRNA) or control sequence (scrambled siRNA)were prepared by Sigma.siRNA (0.5 μg) was transfected into cells using Lipofectamine2000 transfection reagent (Invitrogen). CBR1siRNA target sequenceswere as follows: sense, 5′-GAAAGGAGUCCAUGCGAAAdT-3′; antisense,5′-UUCGCAUGGACUCCUUUCdT-3′. The universal negative control wasused as scrambled siRNA. The efficiency of siRNA-based interference ofCBR1 was monitored by Western blot analysis.

MTT assay

Cell viability was assessed using the MTT conversion assay in a 12-well plate. The culture medium was replaced by 1 ml of mediumcontaining0.5 mg/mlMTT, and cellswere incubated for 30 min at 37 °C.The blue-colored tetrazolium crystals resulting from mitochondrialenzymatic activity on MTT substrate were solubilized with 200 μl ofdimethyl sulfoxide. The absorbance was read at 595 nm in a microplatereader (Bio-Rad, Hercules, CA, USA). Cell survival was expressed as thepercentage of absorbance relative to that of the untreated cells.

Hoechst 33342 staining

Equal numbers of cells were treated with or without T0901317(10 μmol/L) for 2 days under high-glucose conditions, incubated for30 min with Hoechst 33342 loading dye, fixed for 20 min in 4%formaldehyde, and then washed three times in ice-cold phosphate-buffered saline (PBS). The stained cellsweremonitoredusinganLSM510confocal lasermicroscope (Carl Zeiss, Oberkochen, Germany). Apoptoticcells were identified by nuclear condensation and fragmentation.

Measurements of cellular triglycerides, free fatty acids, and cholesterol

The cellular contents of triglycerides and cholesterol in RINm5Fcells were measured using triglyceride and cholesterol assay kits(ThermoDMA, Louisville, CO, USA). The amount of FFAs was determinedusing a nonesterified fatty acid assay kit (Roche, Indianapolis, IN, USA).Eachanalysiswasperformedaccording to themanufacturer's instruction.

Oil red O staining

Equal numbers of RINm5F cells were seeded in a six-wellmicroplate and grown to 80% confluency in RPMI medium. Cellswere then treated with or without T0901317 (10 μmol/L) for 3 daysand fixed for 1 h in 4% formaldehyde. After being fixed, the cells werewashed four times with sterile water. Oil red O solution (3 mg/ml in60% isopropanol) was added to each well and incubated at roomtemperature for 15 min. After being dyed, the cells were washed withsterile water and observed under a microscope. Dye was extractedfrom cell culture dishes with isopropanol (1 ml), and the absorbancewas measured spectrophotometrically at 510 nm using methodspreviously described [21,22].

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Analysis of cellular ROS levels

Cellular ROS levels were estimated via DCF fluorescence as anindicator using the following two methods [23].

Confocal microscopyEqual numbers of RINm5F cells were seeded on glass coverslips in a

12-well microplate and grown to 80% confluency in RPMI medium andincubatedwith orwithout T0901317 (10 μmol/L) for 2 days under high-glucose (31.2 mmol/L) conditions. The cells were then loadedwith DCF-DA(10 μmol/L) at 37 °C for 30 minandwashedfive times in ice-coldPBS.After beingwashed in PBS, the cells on the glass coverslips were coveredon the slide with histological mounting medium (National Diagnostics,Atlanta, GA, USA). Fluorescence was evaluated using an LSM510confocal laser microscope (Carl Zeiss). The excitation and emissionwavelengths for the DCF-DA were 488 and 525 nm, respectively.

Flow cytometryEqual numbers of RINm5F cells were seeded in a six-well

microplate and grown to 80% confluency in RPMI medium andincubated with or without T0901317 (10 μmol/L) for 2 days underhigh-glucose (31.2 mmol/L) conditions. Cells were then loaded withDCF-DA (10 μmol/L) at 37 °C for 30 min. Cells were washed with PBS,harvested in 1 ml of trypsin, and then suspended in PBS. Thesupernatant was removed by centrifugation at 1000 rpm for 5 min.The cell pellet was resuspended in 0.5 ml of PBS. Fluorescencewas assessed via flow cytometry (FACSCalibur; Becton–Dickinson,Franklin Lakes, NJ, USA). The mean DCF fluorescence intensity wasassessed with an excitation wavelength of 488 nm and an emissionwavelength of 525 nm. The untreated cells were used as a referencefor the ROS levels.

Preparation of nuclear extracts

Equal numbers of HIT-T15 cells were seeded in a 100-mm dish andgrown to 80% confluency in RPMImedium. The cells were then treatedwith or without T0901317 (10 μmol/L) for 36 h under low-glucose orhigh-glucose conditions. The cells were washed with PBS, harvestedin 1 ml of trypsin, and then suspended in PBS. The supernatant wasremoved by centrifugation at 1000 rpm for 5 min. The cell pellet wasresuspended in cytosolic Buffer A (10 mmol/L Hepes, pH 7.9,10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol (DTT),and 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF)), adjusted to1% Nonidet P-40, and homogenized. The homogenate was centrifugedat 4500 rpm for 5 min. The supernatant was retained for cytosolprotein analysis. The nuclear cell pellet was resuspended in nuclearBuffer B (20 mmol/L Hepes, pH 7.9, 0.4 mol/L NaCl, 1 mmol/L DTT,1 mmol/L PMSF, 1% Nonidet P-40) and centrifuged at 14,000 rpm for15 min. The supernatant was used for analysis of nuclear protein.

Measurement of insulin secretion

Equal numbers of HIT-T15 cells were seeded in a 12-wellmicroplate and grown to 80% confluency in RPMI medium. The cellswere then treated with or without T0901317 (10 μmol/L) for 36 hunder low-glucose or high-glucose conditions and incubated inKrebs–Ringer–bicarbonate–Hepes buffer (KRBH; 140 mmol/L NaCl,3.6 mmol/L KCl, 0.5 mmol/L NaH2PO4, 0.5 mmol/L MgSO4, 1.5 mmol/L CaCl2, 2 mmol/L NaHCO3, 10 mmol/L Hepes) supplemented with2.8 mmol/L glucose and 0.1% bovine serum albumin (BSA) at 37 °C for1 h under 95% O2/5% CO2 atmosphere for preincubation. Theincubation of the cells was performed with KRBH containing 2.8 or25 mmol/L glucose for 1 h at 37 °C, and then the medium wascollected for detection of insulin secretion using methods previouslydescribed [24]. Insulin secretion was measured using an enzymeimmunoassay (ALPCO Diagnostics, Salem, NH, USA).

Pancreatic islet isolation

Pancreatic islets were isolated from db/db mice (males, 12 weeksof age) and wild-type mice by collagenase XI (Sigma–Aldrich)digestion and were purified using Ficoll gradient solutions (29, 24,and 15% (wt/vol) in Hanks’ balanced salt solution). After centrifugationfor 30 min, the isletswere removed from the layer between the 24% andthe15% layers andwashed twicewith coldHanks’ balanced salt solutionusing methods previously described [25]. The isolated islets werecultured in suspension in RPMI 1460 medium supplemented with 10%fetal bovine serum (GIBCO, Grand Island, NY, USA). Tomake cell lysates,150–200 islets were lysed in 80 mmol/L Tris, pH 6.8, 5% SDS, 5 mmol/LEDTA, 2 mmol/L N-ethylmaleimide, and 2 mmol/L PMSF and sonicatedfor 1.5 min in the cup of a sonicator usingmethods previously described[26]. Protein concentrations were determined with an assay based onBradford's technique (Bio-Rad). Forty micrograms of protein was takenfor detection of CBR1 expression byWestern blot analysis, and 200 μg ofprotein was taken for measurement of CBR1 activity.

Real-time quantitative RT-PCR

Expression of mRNA for CBR1 was measured via a TaqMan-basedreal-time RT-PCR assay. The primer and probe sequences designed bySigma–Proligo (The Woodlands, TX, USA) were as follows:CBR1 forward, 5′-AGTGGTGAATGTGTCCAGCA-3′; CBR1 reverse, 5′-CAGGACTGTCACCCCAATCT-3′; PDX-1 forward, 5′-GGTATAGCCGGAGA-GATGC-3′; PDX-1 reverse, 5′-CTGGTCCGTATTGGAACG-3′; preproinsulinforward, 5′-GCTCTGTACCTGGTGTGTGG-3′; preproinsulin reverse, 5′-GTGCCAAGGTCTGAAGATCC-3′; cyclophilin A forward, 5′-CTTGCTGCA-GACATGGTCAAC-3′; cyclophilin A reverse, 5′-GCCATTATGGCGTGT-GAAGTC-3′. Real-time PCR amplification was conducted in a 96-welloptical tray with a final reaction volume of 20 μl containing 10 μl ofTaqMan Universal master mix (PE Applied Biosystems, Branchburg, NJ,USA), 0.2 μl of forward (20 pmol/L) and reverse (20 pmol/L) primers,1 μl of probe (1 μmol/L), and 20 ng of reverse-transcribed total RNA. RT-PCR was conducted in an ABI Prism 7300 (PE Applied Biosystems). Therelative expression of mRNA was calculated after normalization tocyclophilin A mRNA.

Measurement of CBR1 activity

CBR1 activity was measured in pancreatic islets isolated from 12-week-oldwild-type anddb/dbmice (n=4)using the specificNAD(P)H:quinoneoxidoreductase (NQO1) inhibitordicoumarol in thepresenceofthe substrate menadione and the NADPH cofactor using methodspreviously described [27]. Typical incubationmixtures (1 ml) containedsodium phosphate buffer (0.1 mol/L, pH 7.4), 200 μmol/L NADPH,200 μmol/L menadione, and 5 μmol/L dicoumarol. Mixtures wereequilibrated for 2 min at 37 °C after the addition of pancreatic islets(200 μg). The rates ofNADPHoxidationwere recorded for 4 min at 37 °Cin a Cary Varian Bio 300 UV–visible spectrophotometer (Palo Alto, CA,USA). Enzymatic velocities were automatically calculated by linearregression of the Δabs/Δtime points (2400 readings) and expressed asμmol/min ·mg. Protein concentrations were determined with an assaybased on Bradford's technique (Bio-Rad).

Western blot analysis

Cell lysates (20 μg protein) were separated via SDS–PAGE andelectroblotted onto a polyvinylidene difluoride (PVDF) membrane for1 h at100 Vat 4 °C. Themembraneswere blockedwith 3%bovine serumalbumin for 1 h in TBST (20 mmol/L Tris–HCl, pH 7.5, 50 mmol/L NaCl,0.1% Tween 20). After being blocked, the membranes were incubatedovernight at 4 °C with primary antibodies diluted (1:1000) in TBST.After being washed in TBST buffer, the membranes were incubated for1 h in anti-rabbit, anti-goat, or anti-mouse IgG–horseradish peroxidase

Fig. 1. Regulation of pancreatic β-cell survival by CBR1. (A) CBR1 expression level was monitored by Western blot analysis in RINm5F cells transfected with scrambled siRNA, CBR1siRNA, mock, and CBR1/WT. The data are expressed as themeans±SE of at least three independent experiments. *, #Pb0.01 comparedwith scrambled-siRNA- andmock-transfectedcontrols. (B) MTT assay. The transfected cells were treated with or without T0901317 (10 μmol/L) for 36 h in medium containing low glucose (11.2 mmol/L) or high glucose(31.2 mmol/L). The data are expressed as the means±SE of at least three independent experiments. *, #Pb0.01 compared with scrambled-siRNA- and mock-transfected controls.(C) Apoptotic cell death was monitored by Western blot analysis of processed PARP and caspase 3 cleavage. (D) Cells incubated for 2 days in the above medium containing highglucose (31.2 mmol/L) were subjected to Hoechst 33342 staining, and visualized under confocal microscopy. Arrowheads indicate chromosomal DNA fragmentation of the cell. SC,scrambled; M, mock.

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(Santa Cruz Biotechnology, Santa Cruz, CA, USA) secondary antibodiesdiluted (1:2000) in TBST, and the labeled proteins were detected withchemiluminescence reagents as described by the manufacturer (Santa

Cruz Biotechnology). Unless specified, actin was immunoblotted tostandardize the quantity of sample proteins for all Western blottinganalyses. The detection of insulin protein was accomplished according

Fig. 2. Regulation of lipogenesis by CBR1 in pancreatic β-cells. (A) Expression levels of ACC, FAS, and ABCA1 were measured byWestern blot analysis. RINm5F cells transfected withscrambled siRNA, CBR1 siRNA, mock, and CBR1/WT were treated with or without T0901317 (10 μmol/L) for 36 h in medium containing low glucose (11.2 mmol/L) or high glucose(31.2 mmol/L). The data are expressed as the means±SE of at least three independent experiments. *, #Pb0.05 compared with scrambled-siRNA- and mock-transfected controls.(B and C) Cells incubated for 3 days in the above medium containing high glucose (31.2 mmol/L) were subjected to oil red O staining. The data are expressed as the means±SE of atleast three independent experiments. *, #Pb0.05 compared with scrambled-siRNA- and mock-transfected controls. Arrowheads indicate lipid accumulation within the cell. SC,scrambled; M, Mock.

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to previous methods with minor modification [28]. In brief, cell lysates(30 μg)were separated by SDS–PAGE using 12% gels. The proteinsweretransferred to a PVDF membrane, and the membranes were blockedwith 3% BSA for 3 h in TBST. After being blocked, the membranes wereincubated overnight at 4 °C with insulin primary antibody diluted inTBST (1:1000). After beingwashed in TBST buffer, themembraneswereincubated for 1 h in horseradish peroxidase-conjugated secondary anti-rabbit polyclonal antibody (1:5000) and visualized by enhanced

chemiluminescence. The SeeBlue prestained molecular weight markerstandards (Invitrogen) were used in our experiment.

Densitometry and statistical analysis

Thedensity of bands in theWesternblotting analysiswas determinedvia densitometry, using Quantity One software (Bio-Rad). The results areexpressed as means±SE and are from at least three independent

image of Fig.�2

Fig. 3. CBR1 attenuates lipid accumulation in pancreaticβ-cells. (A andD) Cellular triglycerides, (B and E) FFAs, and (C and F) cholesterol levelswere determined in the scrambled-siRNA-,CBR1-siRNA-,mock-, andCBR1/WT-transfected cells incubated for 3 days inmediumcontaininghighglucose (31.2 mmol/L) and in the presenceor absenceof T0901317 (10 μmol/L). Thedata are expressed as the means±SE of at least three independent experiments. *, #Pb0.05 compared with scrambled-siRNA- and mock-transfected controls. SC, scrambled.

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experiments. Statistical analyses were conducted via Student's t test.Unless otherwise indicated, a P value of b0.05was considered significant.

Results

Carbonyl reductase 1 regulates pancreatic β-cell survival in glucotoxicityand glucolipotoxicity

It was recently reported that chronic activation of liver X receptorby a synthetic ligand, T0901317, under high-glucose conditionscontributes to severe glucolipotoxicity-induced β-cell apoptosis[25]. Therefore, we investigated the role of CBR1 in RINm5F pancreatic

β-cell apoptosis under glucotoxic and glucolipotoxic conditions usingT0901317, as previously described [25]. RINm5F cells were transientlytransfected with CBR1 siRNA or stably transfected with mock andCBR1/WT. CBR1 expression levels were assessed by Western blotanalysis. Expression of CBR1 was almost completely suppressed byspecific siRNA interference, whereas addition of exogenous CBR1showed an almost threefold increase over basal expression levels(Fig. 1A). Next, we tested cell survival using the MTT assay, Westernblot analysis for detection of PARP and caspase 3 cleavage,and Hoechst 33342 staining for detection of DNA fragmentation.Compared with scrambled-siRNA transfectants, MTT assays showedthat siRNA-based CBR1 knockdown significantly lowers cell survival

image of Fig.�3

Fig. 4. CBR1 attenuates lipid peroxidation in pancreatic β-cells. RINm5F cellstransfected with (A) scrambled siRNA or CBR1 siRNA or (B) mock or CBR1/WT weretreated with or without T0901317 (10 μmol/L) for 36 h in medium containing lowglucose (11.2 mmol/L) or high glucose (31.2 mmol/L). The levels of lipid peroxidationproduct (HNE and MDA) and carbonyl-reducing enzymes (AR and ALDH) weredetected by Western blot analysis. The data are expressed as the means±SE of at leastthree independent experiments. *, #Pb0.05 compared with scrambled-siRNA- andmock-transfected controls. SC, scrambled; M, mock.

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rates under low-glucose/low-fat conditions and that this effectis enhanced under high-glucose/high-fat conditions. In contrast,overexpression of CBR1 significantly increased cell survival ratescompared to mock transfectants under all conditions (Fig. 1B).Consistently, apoptotic markers such as cleaved PARP and cleavedcaspase 3 were distinctly elevated in cells with CBR1 knockdown, butdiminished in CBR1-overexpressing cells (Fig. 1C). Hoechst 33342staining also showed similar patterns of apoptosis (Fig. 1D). Theseresults suggest that CBR1 is a key protective molecule in regulatingpancreatic β-cell survival in glucotoxicity and glucolipotoxicity.

Carbonyl reductase 1 regulates lipogenesis in pancreatic β-cells

Hyperglycemia induces lipogenic gene expression and promotes denovo synthesis of FFAs and triglycerides in β-cells [29,30]. To examine

the effects of CBR1 on lipogenesis in pancreatic β-cells, CBR1-knockdown or -overexpressing cells were incubated under the sameconditions as described above. The expression levels of lipogenicenzymes, such as ACC, FAS, and ABCA1, were significantly increased inCBR1-knockdown cells (Fig. 2A), but decreased in CBR1-overexpressingcells (Fig. 2A). Oil red O staining was performed using the cells culturedunder high-glucose conditions, with or without T0901317, to examinehowCBR1 affects lipid accumulation. Results showed lipid accumulationpatterns similar to the expression patterns of the lipogenic enzymes, i.e.,higher lipid accumulation in the cells with CBR1 knockdown (Figs. 2Band C), but lower lipid accumulation in CBR1-overexpressing cells(Figs. 2B and C). To further test the effects of CBR1 on lipid accumulationinpancreaticβ-cells,wemeasured the levels of intracellular triglycerides(TGs), FFAs, and cholesterol after exposure to T0901317 and high-glucose conditions. Results showed that the levels of cellular TGs andFFAs are significantly increased by CBR1 knockdown (Figs. 3A and B). Incontrast, overexpressionof CBR1decreased intracellularaccumulationofTG and FFAs (Figs. 3D and E). Interestingly, the cholesterol level was notchanged by either CBR1 knockdown or CBR1 overexpression, indicatingthat cholesterolmetabolism inβ-cells is not affected by redox status andplays a minimal role in β-cell dysfunction (Figs. 3C and F).

Carbonyl reductase 1 attenuates lipid peroxidation in pancreatic β-cells

Lipid peroxidation by ROS plays a central role in the pathogenesisof oxidative stress [31]. Lipid peroxidation products comprise highlyreactive lipid aldehydes, such ONE, HNE, MDA, and acrolein, which areable to cause protein and DNA damage within cells. CBR1 metabolizesreactive lipid metabolites to less toxic or nontoxic products [17]. Also,CBR1 diminishes cellular oxidative stress by reducing oxidizedquinones produced in the mitochondrial electron transport chain, aswell as oxidized vitamin E [16]. Therefore, we investigated the effectsof CBR1 on lipid peroxidation in pancreatic β-cells by Western blotanalysis. Cells with CBR1 knockdown or overexpressionwere culturedwith or without T0901317, under low- or high-glucose conditions.Results showed that the levels of lipid peroxidation products, HNE andMDA, are significantly increased by CBR1 knockdown, but decreasedin CBR1-overexpressing cells under all conditions (Figs. 4A and B). Tofurther verify these observations, we measured the expression levelsof carbonyl-reducing enzymes, such as AR and ALDH, by Western blotanalysis. Results showed that AR and ALDH expression levels followpatterns similar to those of HNE and MDA (Figs. 4A and B). Takentogether, these results suggest that CBR1 plays a protective roleagainst oxidative damage and promotes pancreatic β-cell survival inglucotoxicity and glucolipotoxicity.

Carbonyl reductase 1 suppresses ROS accumulation and endoplasmicreticulum (ER) stress in pancreatic β-cells

It was shown that ROS increase lipid accumulation in HepG2 cells,though not in COS cells, via SREBP1c activation [21]. Also, hypoxia wasshown to upregulate FAS gene expression via activation of Akt andSREBP1c [32]. Therefore, we assumed that ROS increase lipidaccumulation in pancreatic β-cells during development of type 2diabetes. ROS levels were measured by flow cytometry and confocalmicroscopy in CBR1-knockdown or -overexpressing cells under high-glucose conditions, with or without T0901317. ROS generation wassignificantly increased by CBR1 knockdown (Figs. 5A and B). Incontrast, CBR1 overexpression decreased ROS generation (Figs. 5Aand B). Consistently, the levels of ER stress markers, such as Bip/Grp78, PDI, CHOP, and p-eIF2α, were also increased by CBR1knockdown, but decreased by its overexpression (Fig. 5C). Asexpected, insulin levels were downregulated by CBR1 knockdown,but upregulated by CBR1 overexpression (Fig. 5C).

image of Fig.�4

Fig. 5. CBR1 suppresses ROS generation and ER stress in pancreatic β-cells. (A) RINm5F cells transfected with scrambled siRNA, CBR1 siRNA, mock, and CBR1/WT were treated withor without T0901317 (10 μmol/L) for 2 days in medium containing high glucose (31.2 mmol/L). ROS levels were measured by fluorescence of DCF-DA (10 μmol/L). The data areexpressed as the means±SE of at least three independent experiments. *, #Pb0.05 compared with scrambled-siRNA- and mock-transfected controls. (B) ROS generation wasobserved via confocal microscopy in cells loaded with DCF-DA (10 μmol/L). (C) Cells were treated with or without T0901317 (10 μmol/L) for 36 h inmedium containing low glucose(11.2 mmol/L) or high glucose (31.2 mmol/L). The levels of ER stress markers (Bip/Grp78, PDI, CHOP, and p-eIF2α) and insulin were determined byWestern blot analysis. The dataare expressed as the means±SE of at least three independent experiments. *, #Pb0.05 compared with scrambled-siRNA- and mock-transfected controls. SC, scrambled; M, mock.

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Antioxidants attenuate lipogenesis and ER stress caused by knockdownof carbonyl reductase 1

Next, to confirm whether CBR1 regulates the expression oflipogenic enzymes, lipid accumulation, ER stress, and apoptotic celldeath by decreasing ROS generation, we pretreated CBR1-knockdowncells with antioxidants, NAC and Tiron, followed by growing them inmedium containing high glucose concentrations and T0901317. Then,we observed how these antioxidants affect the cells. Also, we testedthe effect of a FAS inhibitor, cerulenin, under the same conditions,because it has been reported that cerulenin protects cells from high-

glucose plus T0901317 conditions [25]. As expected, pretreatmentwith the antioxidants and cerulenin significantly reduced lipogenicenzymes, lipid accumulation, ER stress, apoptotic cell death, and ROSgeneration compared with control groups (Figs. 6 A–G).

Carbonyl reductase 1 suppresses the nuclear active form of SREBP1c andincreases glucose-stimulated insulin secretion in pancreatic β-cells

The GSIS is defective in RINm5F insulinoma cells [33]. In contrast,hamster insulinoma HIT-T15 cells produce and secrete insulin inresponse to glucose and stimulation by other secretagogues [34,35].

image of Fig.�5

Fig. 6. Attenuation of lipogenesis and ER stress by antioxidants in CBR1-knockdown cells. RINm5F cells transfected with scrambled siRNA and CBR1 siRNA were treated with orwithout T0901317 (10 μmol/L) inmedium containing high glucose (31.2 mmol/L). After pretreatment with orwithout cerulenin (0.5 mg/L), NAC (2 mmol/L), and Tiron (1 μmol/L),cells were incubated for 36 h, and then (A) lipogenic enzyme, (E) ER stress marker, (F) apoptosis-related protein, and (G) MTT assays were performed. (B and C) Cells were culturedfor 3 days for oil red O staining and (D) for 2 days for ROS generation. The data are expressed as the means±SE of at least three independent experiments. *Pb0.01 compared withuntreated CBR1-knockdown cells; #P b 0.05 compared with T0901317-treated CBR1-knockdown cells. Arrowheads indicate lipid accumulation within cells.

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Therefore, we used the HIT-T15 cell line for determining SREBP1cactivation and GSIS. The SREBP1c activation was measured byassessing the expression levels of the precursor and nuclear active

forms of SREBP1c. In our experiments, the expression levels ofprecursor and cleaved nuclear forms of SREBP1c were significantlyincreased in CBR1-knockdown cells, but decreased in CBR1-

image of Fig.�6

Fig. 7. CBR1 attenuates the nuclear active formof SREBP1c and increases GSIS in pancreaticβ-cells. (A) Expression levels of precursor and nuclear active forms of SREBP1cweremonitoredby Western blot analysis in HIT-T15 cells transfected with scrambled siRNA, CBR1 siRNA, mock, and CBR1/WT. Cells were treated with or without T0901317 (10 μmol/L) for 36 h inmedium containing low glucose (11.2 mmol/L) or high glucose (31.2 mmol/L). The data are expressed as themeans±SE of at least three independent experiments. *, #Pb0.05 comparedwith scrambled-siRNA- and mock-transfected controls. (B) Effect of CBR1 on GSIS in HIT-T15 cells was measured using an enzyme-linked immunoassay. The data are expressed as themeans±SEof at least three independent experiments. *, #Pb0.05 comparedwith scrambled-siRNA- andmock-transfected controls. SC, scrambled;M,mock; p-SREBP1c, precursor formofSREBP1c; n-SREBP1c, nuclear form of SREBP1c.

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overexpressing cells (Fig. 7A). The magnitude of GSIS, as determinedby enzyme-linked immunoassays, paralleled the pattern observedwith SREBP1c activation (Fig. 7B). Thus, CBR1 knockdown led to areduction in GSIS, whereas CBR1 overexpression was associated withan increase in GSIS.

Expression level of carbonyl reductase 1 is distinctly decreased inpancreatic islets of db/db mice

To explore the involvement of CBR1 in the pathogenesis of pancreaticβ-cell failure in an experimental model of type 2 diabetes, we examinedthemRNA expression level of CBR1 in pancreatic islets isolated from db/db mice. Compared with preproinsulin, the expression level of CBR1mRNA was relatively low in pancreatic β-cells (Fig. 8A). Moreimportantly, CBR1 mRNA expression was significantly lower in db/dbmice compared with nondiabetic wild-typemice (Fig. 8B). Preproinsulinand pancreatic and duodenal homeobox factor (PDX-1) gene expressionlevels were also decreased, as previously reported [36,37]. Finally, weexamined CBR1 protein expression and activity in pancreatic isletsisolated from db/db mice. Both CBR1 expression and enzyme activitylevels were significantly decreased in db/db mice compared withnondiabetic wild-type mice (Figs. 8 C and D). Our results suggest thatdecreased expression and enzyme activity of CBR1 might be one of themajor factors causing pancreatic β-cell failure in type 2 diabetic patients.

Discussion

In this study, we demonstrate for the first time that CBR1attenuates apoptosis and increases cell survival and insulin secretion

in pancreatic β-cell lines under glucotoxic and glucolipotoxicconditions. These effects were mediated by reducing oxidative stressand associated with decreases in ROS generation, ER stress, and lipidaccumulation. In addition, CBR1 expression level and enzymeactivity were decreased in pancreatic islets of diabetic db/db mice,indicating that CBR1 is regulated under pathophysiological condi-tions. These data support the possibility that CBR1 plays a role in thedysfunction of pancreatic β-cells in db/dbmice and/or human type 2diabetes.

ROS production in pancreatic β-cells is increased with hyperglycemiaas this increases glucose metabolism and thus ROS production inmitochondria. In addition, hyperglycemia is known to increase ROSgeneration by NADPH oxidase. Increased ROS production increasesoxidative stress, which causes β-cell dysfunction and/or apoptosis[3,13,14,38–40].Hyperlipidemia is alsoknownto increaseROSproductionand inflammatory stress in various cells [8], including pancreatic β-cells[41], causing lipotoxicity. Lipotoxicity in β-cells has been shown to beaugmented by hyperglycemia, leading to the glucolipotoxicity hypothesis[42]. In our experiments, we observed that CBR1 overexpressionsuppressed, whereas CBR1 knockdown promoted, apoptosis in RIN5mFcells under both glucotoxic and glucolipotoxic conditions. CBR1 suppres-sion (or promotion) of apoptosis was associated with decreased (orincreased) levels of ROS and oxidative or ER stress markers. In addition,two antioxidants, NAC and Tiron, prevented the detrimental effects ofCBR1 knockdown under glucolipotoxic conditions. Taken together,these data suggest that CBR1 regulates apoptosis and cell survival ininsulin-secreting cells by reducing oxidative stress. This is consistentwiththe knowneffect of CBR1 todetoxify endogenous andxenobiotic carbonylcompounds [17].

image of Fig.�7

Fig. 8. Decreased CBR1 expression in pancreatic islets of db/dbmice. (A) Gene expressionlevels of preproinsulin and CBR1 in RINm5F cells. The mRNA levels of the indicated geneswere analyzed by real-time quantitative RT-PCR and normalized to cyclophilin levels.*Pb0.001 compared with preproinsulin gene. (B) Gene expression profiles of pancreaticislets in db/db mice. The mRNA levels of the indicated genes in pancreatic islets isolatedfrom 12-week-old wild-type and db/db mice (n=4) were measured by real-timequantitative RT-PCR. *Pb0.05 compared with wild-type control. (C) Expression of CBR1was measured by Western blot analysis in pancreatic islets isolated from 12-week-oldwild-type and db/db mice (n=4). *Pb0.05 compared with wild-type control. (D) CBR1activity wasmeasured in pancreatic islets isolated from 12-week-oldwild-type and db/dbmice (n=4) using the specific NQO1 inhibitor dicoumarol in the presence of the substratemenadione and the NADPH cofactor. *Pb0.05 compared with wild-type control.

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Interestingly, we found that CBR1 regulated lipogenesis by alteringthe expression of SREBP1c and several lipogenic enzymes, such asACC, FAS, and ABCA1; CBR1 overexpression decreased, whereas CBR1knockdown increased, lipogenic enzyme expression and lipogenesis.These effects seem to be mediated by CBR1's effect of decreasing ROS(and/or oxidative stress). There is evidence that ROS are lipogenicfactors. First, Sekiya et al. reported that ROS, especially H2O2,increased SREBP1c transcriptional activity and induced lipogenicenzymes to increase lipid accumulation in HepG2 cells [21]. Second,hypoxia, which increased ROS production in human breast cancercells, was shown to upregulate FAS gene expression via activation ofAkt and SREBP1c [32]. Finally, ROS, especially H2O2, stimulateddifferentiation of preadipocytes to adipocytes [43,44]. In our ownexperiments, the antioxidants NAC and Tiron prevented the increasesin ROS production, ER stress, and lipid accumulation induced by CBR1knockdown, further supporting the notion that increased ROS and/oroxidative stress levels might be responsible for the increases in lipidaccumulation and lipogenic enzyme expression in CBR1-knockdowncells. On the other hand, there is evidence that elevated free fattyacids induce oxidative stress [39], and we also observed that the

FAS inhibitor cerulenin decreased ROS production and ER stress.Taken together, it is conceivable that increased lipid availability(hyperlipidemia) or accumulation in pancreatic β-cells stimulatesROS production, which may in turn increase lipid accumulation,resulting in a vicious cycle of increased ROS and lipid accumulation,leading to β-cell dysfunction.

Pancreatic β-cells are highly active cells, which are most susceptibleto ER stress. It iswell established that ROS induce ER stress,which in turninterferes with insulin secretion and contributes to β-cell apoptosis intype 2 diabetes [45–48]. Consistent with this concept, we observed thatCBR1, which controlled ROS production, regulated insulin secretion inHIT-T15 insulinomacells,whichwas strongly associatedwithexpressionchanges in ER stressmarkers suchasBip/Grp78, PDI, CHOP, andp-eIF2α.These data suggest that the effect of CBR1 on insulin secretion may bemediated by its effect of regulating ER stress (via ROS), and CBR1may improve insulin secretion under glucotoxic and glucolipotoxicconditions by suppressing ROS production and reducing ER stress.

ROS are also known to cause lipid peroxidation and reactivelipid aldehyde formation, which contribute to progressive deteriorationofβ-cell function in type 2diabetes [17,49]. Lipid peroxidation is amajormechanism of oxygen free radical toxicity to cellular organelles andmembrane-bound enzymes, and aldehydes are cytotoxic products oflipid peroxidation [50–52]. CBR1 catalyzes the reduction of the reactivelipid peroxidation product ONE to HNE, HNO, and ONA. It also catalyzesthe reduction of the glutathione adduct of ONE (GS–ONE) to GS–HNE.Because the CBR1products, HNE andGS–HNE, aremetabolized byALDHand AR [53], we had expected that the levels of HNE, AR, and ALDHwould be increased by CBR1 overexpression and decreased by CBR1knockdown. However, our results were opposite to this expectation,indicating that in addition to the metabolic pathways for the lipidperoxidation products mentioned above, other unknown pathways arealso involved in the reduction of oxidative stress by CBR1. A protectiverole for CBR1 in the pathogenesis of diabetes has been suggested; CBR1may participate in detoxification of reactive carbonyl products duringoxidative stress [54]. Because CBR1 also controls the redox state byreducing oxidized ubiquinones (coenzyme Q) and tocopherolquinone(vitaminE) [16],webelieve thatCBR1mayact as anantioxidant enzymein a broader sense, rather than merely reducing reactive lipidperoxidation products, in promoting pancreatic β-cell survival underglucotoxic or glucolipotoxic conditions.

CBR1 was upregulated by the transcription factor Nrf2 underoxidative stress conditions [55]. Therefore, we had expected that CBR1gene expressionwould be increased in the pancreatic islets of diabeticdb/dbmice, which are under oxidative stress. However, we found thatCBR1 mRNA levels were decreased, together with preproinsulin andPDX1 expression, in pancreatic islets isolated from db/db mice. Theprotein level and enzyme activity of CBR1 were also decreased. Thesedata indicate that CBR1 expression/activity is regulated underpathophysiological conditions. In addition, because CBR1 attenuatesapoptosis and promotes cell survival of insulin-secreting cells underoxidative stress or glucolipotoxic conditions, our data suggest thatdecreased CBR1 expression/activitymay play a role in the dysfunctionof pancreatic β-cells during the development of diabetes in db/dbmice and/or type 2 diabetes. It would be important to test whetherCBR1 expression is indeed downregulated in pancreatic β-cells of type2 diabetic patients.

In summary, this studydemonstrates that CBR1 attenuates apoptosisand promotes cell survival in pancreatic β-cell lines under glucotoxicand glucolipotoxic conditions via reducing ROS generation. In addition,our data demonstrate that CBR1 expression level and enzyme activityare decreased in pancreatic islets isolated from db/db mice, an animalmodel of type2diabetes. These results suggest thatCBR1mayplaya rolein protectingpancreaticβ-cells against oxidative stress under glucotoxicor glucolipotoxic conditions, and its reduced expression or activity maycontribute toβ-cell dysfunction indb/dbmiceorhuman type2diabetes.Further studies arewarranted todirectly testwhether CBR1expression/

image of Fig.�8

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activity is causally related to β-cell dysfunction or is reduced inpancreatic β-cells of type 2 diabetic patients.

Acknowledgments

We are grateful to Dr. Byung-Hyun Park at Chonbuk NationalUniversity for critically reading our manuscript. This work wassupported by Korea Science and Engineering Foundation Grant20090091346 to S.S. Kim from the Korea government.

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Carbonyl reductase 1 protects pancreatic β-cells against oxidative stress-induced apoptosis in glucotoxicity and glucolipot...Materials and methodsMaterialsCell culture and stable transfectionSmall interfering RNAMTT assayHoechst 33342 stainingMeasurements of cellular triglycerides, free fatty acids, and cholesterolOil red O stainingAnalysis of cellular ROS levelsConfocal microscopyFlow cytometry

Preparation of nuclear extractsMeasurement of insulin secretionPancreatic islet isolationReal-time quantitative RT-PCRMeasurement of CBR1 activityWestern blot analysisDensitometry and statistical analysis

ResultsCarbonyl reductase 1 regulates pancreatic β-cell survival in glucotoxicity and glucolipotoxicityCarbonyl reductase 1 regulates lipogenesis in pancreatic β-cellsCarbonyl reductase 1 attenuates lipid peroxidation in pancreatic β-cellsCarbonyl reductase 1 suppresses ROS accumulation and endoplasmic reticulum (ER) stress in pancreatic β-cellsAntioxidants attenuate lipogenesis and ER stress caused by knockdown of carbonyl reductase 1Carbonyl reductase 1 suppresses the nuclear active form of SREBP1c and increases glucose-stimulated insulin secretion in pa...Expression level of carbonyl reductase 1 is distinctly decreased in pancreatic islets of db/db mice

DiscussionAcknowledgmentsReferences