Insulin is the main regulator of carbohydrate and fat metabolism.

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1 DIABETES-INSULIN-GLUCAGON-GASTROINTESTINAL The Lipid Peroxidtion By-Product 4-Hydroxy-2- Nonenl (4-HNE) Induces Insulin Resistnce in Skeletl Muscle through Both Crbonyl nd Oxidtive Stress Nicols J. Pillon, Mrine L. Croze, Roxne E. Vell, Lurent Soulère, Michel Lgrde, nd Christophe O. Soulge Université de Lyon (N.J.P., M.L.C., R.E.V., L.S., M.L., C.O.S.), F Lyon, Frnce; Institut Ntionl de l Snté et de l Recherche Médicle Unité Mixte de Recherche 16 (N.J.P., M.L.C., R.E.V., M.L., C.O.S.), CrMeN, nd Institut Ntionl des Sciences Appliquées-Lyon (N.J.P., M.L.C., R.E.V., M.L., C.O.S.), Institut Multidisciplinire de Biochimie des Lipides, F Villeurbnne, Frnce; nd Lbortoire de Chimie Orgnique et Bioorgnique (L.S.), Institut Ntionl des Sciences Appliquées-Lyon, Centre Ntionl de l Recherche Scientifique Unité Mixte de Recherche 5246 Institut de Chimie et Biochimie Moléculires et Suprmoléculires, F Villeurbnne, Frnce Numerous oxidnts re produced s by-products of erobic cell metbolism, nd there is growing evidence tht they ply key roles in the pthogenesis of insulin resistnce. Under conditions of oxidtive stress, lipid peroxidtion of 6-polyunsturted ftty cids leds to the production of 4-hydroxy-2-nonenl (4-HNE). Severl lines of evidence suggest tht 4-HNE could be involved in the pthophysiology of metbolic diseses; therefore, in this study we ssessed the direct effects of 4-HNE on skeletl muscle insulin sensitivity. Gstrocnemius muscle nd L6 muscle cells were treted with 4-HNE. Insulin signling ws mesured by Western blotting nd glucose uptke using 2-deoxy- D-[3H]glucose. Crbonyl stress, glutthione content, nd oxidtive stress were ssessed s potentil mechnisms leding to insulin resistnce. Protection of cells ws induced by pretretment with 3H-1,2-dithiole-3-thione, N-cetyl-cysteine, minogunidine, or S-denosyl-methionine. 4-HNE induced time- nd dose-dependent decrese in insulin signling nd insulin-induced glucose uptke in muscle. It induced stte of crbonyl stress through dduction of proteins s well s depletion in reduced glutthione nd production of rdicl oxygen species. A phrmcologicl increse in glutthione pools ws chieved by 3H-1,2-dithiole-3-thione nd protected the cells ginst ll deleterious effects of 4-HNE; furthermore, N-cetylcysteine, minogunidine, nd S- denosylmethionine prevented 4-HNE noxious effects. 4-HNE cn impir insulin ction in muscle cells through oxidtive stress nd oxidtive dmge to proteins, eventully leding to insulin resistnce. These deleterious effects cn be prevented by pretretment with ntioxidnts, scvengers, or n increse in intrcellulr glutthione pools. Use of such molecules could represent novel strtegy to combt insulin resistnce nd other oxidtive stress-ssocited pthologies. (Endocrinology 153: , 212) ISSN Print ISSN Online Printed in U.S.A. Copyright 212 by The Endocrine Society doi: 1.121/en Received November 7, 211. Accepted Jnury 3, 212. First Published Online Mrch 6, 212 Insulin is the min regultor of crbohydrte nd ft metbolism. Impirment of its function on trget tissues leds to insulin resistnce, common pthologicl stte ssocited with dibetes mellitus, obesity, nd therosclerosis. There is growing body of evidence suggesting tht oxidtive stress is involved in the pthophysiology of insulin resistnce (1 4). Indeed, excessive production of rective oxygen species (ROS) is deleterious in itself, in ddition to triggering the production of severl secondry by-products. Possibly more deleterious thn ROS themselves, these secondry oxidtion by-products cn diffuse within the cells nd tissues to propgte the noxious effects Abbrevitions: AGD, Aminogunidine; CM-H2DCFDA, chloromethyl-2,7-dichlorodihydrofluorescein dicette, cetyl ester; DMSO, dimethylsulfoxide; DNPH, 2,4-dinitrophenylhydrzine; D3T, 3H-1,2-dithiole-3-thione; GSH, glutthione; 4-HNE, 4-hydroxy-2-nonenl; IRS-1, insulin receptor substrte-1; JNK, Jun kinse; NAC, N-cetylcysteine; PKB, protein kinse B; ROS, rective oxygen species; SAM, S-denosylmethionine. Endocrinology, My 212, 153(5): endo.endojournls.org 299

2 21 Pillon et l. 4-HNE-Induced Muscle Insulin Resistnce Endocrinology, My 212, 153(5): of oxidtive stress. Severl by-products re the result of lipid peroxidtion, nonenzymtic process initited by ROS ttck on the double bonds of membrne polyunsturted ftty cids. Lipid peroxidtion of cell membrne phospholipids leds to the production of severl ldehyde by-products, such s mlondildehyde, crolein, nd 4-hydroxy-2-nonenl. 4-Hydroxy-2-nonenl (4-HNE) is nine-crbon mphiphilic lipid ldehyde relesed from the peroxidtion of n-6 polyunsturted ftty cids. 4-HNE hs importnt electrophilic properties nd rects with mny clsses of biomolecules such s phospholipids, proteins, nd nucleotides, forming covlent dducts (5 7). Severl lines of evidence suggest tht 4-HNE could be involved in the pthophysiology of metbolic diseses (8). For instnce, levels of 4-HNE re incresed in the blood nd muscles of obese subjects (2, 9, 1), nd ccumultion of 4-HNEmodified proteins occurs in the pncretic -cells of Goto Kkizki rts s result of hyperglycemi (11). Moreover, 4-HNE nd other lipid peroxidtion by-products impir glucose-stimulted insulin secretion in isolted -cells (12) nd could contribute to the deth of pncretic -cells in dibetes (13). 4-HNE is incresed in dipocytes during obesity in which it my impir the function of key proteins involved in lipid metbolism (14). Indeed, dipocytes exposed to 1 M 4-HNE exhibit impired insulin ction (15), nd we recently demonstrted tht dduction of insulin by 4-HNE disrupts its biologicl ctivity (16), indicting puttive role of lipid ldehydes in the development of insulin resistnce. Improved insulin sensitivity resulting from exercise or cloric restriction is ssocited with reduced levels of 4-HNE (9, 17, 18). Additionlly, mice lcking the 4-HNE-conjugting enzyme glutthione S-trnsferse (lso known s mgsta4 4 null mice) exhibit n ccumultion of 4-HNE in multiple tissues nd spontneously develop obesity nd insulin resistnce (19), suggesting tht 4-HNE could per se promote obesity nd metbolic syndrome. Skeletl muscle ccounts for more thn 8% of whole-body postprndil glucose uptke nd is mjor site of insulin resistnce in type 2 dibetes (2); nevertheless, very few dt re vilble in the literture regrding the direct effect of lipid ldehydes on skeletl muscle insulin sensitivity. The purpose of the present study ws to determine whether nd how 4-HNE could impir insulin responsiveness in skeletl muscle nd contribute to insulin resistnce. Becuse protection ginst lipid ldehyde toxicity could represent promising strtegy to fight oxidtive stressssocited pthologies such s insulin resistnce nd dibetes, we further tested whether glutthione, ldehyde scvengers, or ntioxidnts could counterblnce deleterious effects of 4-HNE on insulin sensitivity. Mterils nd Methods Regents nd ntibodies 4-HNE ws synthesized s previously described (21). Insulin (Actrpid, 1 IU/ml) ws from Novo Nordisk (Copenhgen, Denmrk), nd 2-deoxy-D-[2,6-3 H]glucose ws from GE Helthcre (Orsy, Frnce). 3H-1,2-dithiole-3-thione (D3T) ws from LKT Lbortories (St. Pul, MN). Other regents were from Sigm-Aldrich (Sint Quentin Fllvier, Frnce). The primry ntibody ginst 4-HNE Michel Adducts ws from Clbiochem (reference 39327; Clbiochem, Sn Diego, CA). The nti-phospho-akt 1/2/3 rbbit IgG (7985R) nd nti-akt 1/2/3 rbbit IgG (8312) ntibodies were purchsed from Snt Cruz Biotechnology (Heidelberg, Germny), the ntitubulin mouse IgG ntibody ws from Sigm-Aldrich, nd the ntimouse IgG nd ntirbbit IgG ntibodies were from Bio-Rd (Mrnes-l- Coquette, Frnce). Super Signl West Pico Chemiluminescent substrte nd Restore Western blot stripping buffer were obtined from Thermo Scientific (Perbio, Brebières, Frnce). Other chemicls were obtined from Sigm-Aldrich when no origin is specified. Animls Mle CD-1 Swiss mice were purchsed from Jnvier SA (Le Genest-Sint-Isle, Frnce) nd housed in n ir-conditioned room with controlled environment of 21.5 C nd 6 7% humidity, under 12 h light, 12-h drk cycle (light on from 7 to 19 h) with free ccess to food [13.4 kj/g, 65% crbohydrtes, 11% ft, 24% proteins (wt/wt), AO3; SAFE, Augy, Frnce] nd wter. Animl experiments were performed under the uthoriztion no (CrMeN Lbortory, Direction Déprtementle des Services Vétérinires du Rhône). Authors N.J.P. (no ) nd C.O.S. (no ) hold license to experiment on living vertebrtes issued by the French Ministry of Agriculture nd Veterinry Service Deprtment. All experiments were crried out ccording to the guidelines lid out by the French Ministère de l Agriculture (no ) nd the Europen Union Council Directive for the Cre nd Use of Lbortory Animls of November 24, 1986 (86/69/EEC). Glucose uptke nd insulin signling in skeletl muscle: ex vivo insulin stimultion Mice were killed by cervicl disloction. Gstrocnemius muscles were dissected out, cut into strips of 1 15 mg, nd incubted for 15 min t 37 C in 3 ml of Krebs-Ringer bicrbonte contining 1 M 4-HNE or dimethylsulfoxide (DMSO) s control. Muscles were blotted dry nd incubted for 15 min with 1 nm recombinnt humn insulin. For glucose uptke, muscles were further incubted with 1 Ci of [ 3 H]-2-deoxy-glucose nd.5 Ci of [ 14 C]-mnnitol for 15 min nd then blotted dry nd snp frozen t 8 C. Muscles were digested overnight in sodium hydroxide [3% (wt/vol)] t room temperture nd centrifuged for 1 min t 14, g, nd liquots of the superntnt were quntified for [ 3 H] nd [ 14 C] using liquid scintilltion counting. Finlly, the 2-deoxy-D-glucose uptke ws corrected for residul interstitil mounts using [ 14 C]-mnnitol. For Western blotting, muscle pieces were blotted dry nd snp frozen in liquid nitrogen. Muscle smples were stored t 8 C for protein kinse B (PKB)/Akt phosphoryltion s described below.

3 Endocrinology, My 212, 153(5): endo.endojournls.org 211 Cell culture Rt L6 muscle cells were obtined from the Americn Type Culture Collection (reference CRL-1458, LGC stndrds; Molsheim, Frnce). Cells were grown in DMEM (Sigm-Aldrich) contining 1 U/ml penicillin, 1 g/ml streptomycin, 1% (vol/vol) fetl clf serum (Life Technologies, Inc., Krlsruhe, Germny), nd 2 mm glutmine. Cultures were mintined t 37 C in humidified tmosphere contining 5% (vol/vol) CO 2. Cells were serum strved for t lest 3 h before tretments. 4-HNE ws diluted in serum-free DMEM nd pplied on cells for 3 min unless otherwise specified. Cells were then thoroughly wshed to remove the remining 4-HNE nd stimulted with 1 nm insulin for nother 2 min. Cell vibility Cells seeded in 96-well pltes were treted with 4-HNE t indicted concentrtions nd times. Vibility ws determined vi 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrzolium bromide (Cell Prolifertion Kit I; Roche, Hvidovre, Denmrk). Plsm membrne integrity ws estimted by lctte dehydrogense ssy (in vitro toxicology ssy kit; Sigm-Aldrich) nd poptosis by the mesurement of cspse-3 ctivity (cspse-3 ssy kit; Sigm-Aldrich). Immunohistofluorescence L6 cells grown on coverslips were incubted with 4-HNE in PBS for 3 min. Cells were fixed for 3 min with 3% prformldehyde nd quenched with 5 mm NH 4 Cl for 5 min. After wshing, cells were permebilized with 5 g/ml digitonin for 1 min nd blocked with.1% BSA in PBS for 3 min. After tretment with the primry ntibody to 4-HNE Michel dducts, cells were wshed nd lbeled with fluorescent secondry ntibodies. The stined cells were mounted in Mowiol nd exmined under Zeiss LSM 51 confocl microscope (Jen, Germny) equipped with 63W oil objective nd fluorescence ws quntified using Imge J softwre (Ntionl Institutes of Helth, Bethesd, MD). Spectrophotometric 2,4-dinitrophenylhydrzine (DNPH) ssy for crbonyl content determintion Crbonyl groups on proteins were determined using DNPH s previously described (22). Crbonyl content ws determined from the bsorbnce t 37 nm using molr bsorption coefficient of 22, M 1 /cm 1 nd normlized to the protein concentrtion mesured t 28 nm. Intrcellulr ROS production We nlyzed the intrcellulr ROS level in L6 myoblsts by the 5- (nd-6)-chloromethyl-2,7 -dichlorodihydrofluorescein dicette, cetyl ester (CM-H2DCFDA; Invitrogen, Courtboeuf, Frnce) method s described previously (23). Briefly, cells grown in 12-well pltes were pretreted with 1 M CM- H2DCFDA t 37 C for 3 min in Krebs buffer, rinsed, nd then incubted for 3 min with 5 M 4-HNE or 1 M H 2 O 2 s positive control. Cells were solubilized in Triton-X1 1% (vol/ vol) in distillted wter, nd the chnge in fluorescence ws mesured by fluorometer t excittion nd emission wvelengths of 495 nd 52 nm, respectively. Results were normlized to the protein content of cell lystes (Brdford ssy). Protein extrction nd immunoblotting L6 cells were incubted with 4-HNE in PBS for 3 min. Cells were scrpped in stndrd lysis buffer (2 mm Tris-HCl, 138 mm NCl, 2.7 mm KCl, 1 mm MgCl 2, 5% glycerol, nd 1% Nonidet- P4) supplemented with protese nd phosphtse inhibitors (5 mm EDTA, 1 mm N 3 VO 4,2mM NF, 1 mm dithiothreitol, protein inhibitor cocktil; Sigm-Aldrich). Insoluble mterils were eliminted by centrifugtion (13, g, 15 min, 4 C), nd protein concentrtion in the superntnt ws determined by Brdford ssy (Bio-Rd Lbortories). For Western blotting, proteins were boiled in Lemmli buffer, seprted by SDS- PAGE, nd trnsferred onto nitrocellulose membrnes. For dot blotting, 2 g of protein from whole cell lyste ws loded directly onto nitrocellulose membrnes using the Bio-Dot pprtus (Bio-Rd Lbortories). After sturtion with 5% BSA, membrnes were probed with primry ntibodies. After incubtion with horserdish peroxidse-coupled secondry ntibody, membrnes were processed for chemiluminescence nd quntified by densitometry using ImgeJ softwre (Ntionl Institutes of Helth). 2-Deoxy-D-[ 3 H]-glucose uptke mesurement L6 cells grown in 12-well pltes were treted with 4-HNE for indicted concentrtions nd times. Cells were then incubted for 2 min with 1 nm insulin or 2 M cytochlsin B. Glucose uptke ws initited by the ddition of 2-deoxy-D-[ 3 H]-glucose (747 GBq/mmol) to finl concentrtion of.1 mm ( Bq/ml) for 5 min t 37 C. Uptke ws terminted by removl of the ssy buffer, followed by three wshes in ice-cold PBS. Cells were solubilized with.1% sodium dodecyl sulfte, nd tritium ws detected by liquid scintilltion counting. Results were normlized to protein concentrtion, nd nonspecific uptke mesured in presence of cytochlsin B ws subtrcted from ech determintion. Glutthione (GSH) ssy Reduced GSH intrcellulr pools were ssyed using commercilly vilble kit from BioVision (Clinisciences, Montrouge, Frnce) nd normlized to the protein concentrtion in smples. Sttistics All dt were nlyzed using Prism (GrphPd Softwre Inc., Sn Diego, CA) nd unless otherwise mentioned, mens re presented SEM. Results were compred by two-wy ANOVA followed when pproprite by post hoc Fisher protected lest significnt difference tests. One-wy ANOVA ws used to test differences between severl groups with equl vrinces. Simple comprisons were performed using Student s t test with Welch s correction for vrince inhomogeneity whenever needed. Differences were considered significnt t the P.5 level. Results 4-HNE impirs insulin signling in mouse skeletl muscle Glucose uptke nd phosphoryltion of PKB/Akt were explored in mouse skeletl muscle fter in vitro incubtion

4 212 Pillon et l. 4-HNE-Induced Muscle Insulin Resistnce Endocrinology, My 212, 153(5): with 4-HNE (1 M for 15 min) nd insulin stimultion (1 nm, 15 min). Insulin ws responsible for 2-fold increse in glucose uptke, which ws significntly blunted by the pretretment of muscle with 4-HNE (Fig. 1A). Results from immunoblotting showed 5-fold increse in phosphoryltion level of PKB/Akt in the mouse gstrocnemius muscle in response to insulin (Fig. 1B). In contrst, 4-HNE tretment did not ffect the bsl PKB/ Akt phosphoryltion, wheres it completely inhibited insulin-induced phosphoryltion. Aldehydes re known to form covlent dducts within tissues, nd indeed, incubtion of gstrocnemius muscle with 4-HNE resulted in incresed crbonyl content (Fig. 1C) nd decrese in totl GSH content (Fig. 1D). The increse in crbonyl content ws negtively correlted with muscle GSH content (Fig. 1E, n 9, P.5). We then ttempted to reverse the 4-HNE noxious effects using N-cetylcysteine (NAC). Mice were given NAC (1 mm) for 1 wk in drinking wter, nd gstrocnemius muscle were then dissected nd nlyzed s bove. In NAC-treted mice, insulin-induced Akt phosphoryltion ws preserved in isolted muscles treted with 4-HNE (Fig. 1F). 4-HNE tretment does not impir vibility of L6 muscle cell To gin insight into the mechnism of 4-HNE-induced insulin resistnce, we used the prototypic muscle cell line L6C5. We previously reported tht ldehydes cn be toxic to cultured cells through the induction of both poptosis nd necrosis (24); however, most studies describing cytotoxic effects were focused on high concentrtions (1 M) nd long-lsting exposure ( 12 h). To test whether 4-HNE ws cytotoxic to L6 cells, three prmeters of cytotoxicity were estimted in L6 muscle cells (Supplementl Tble 1, published on The Endocrine Society s Journls Online web site t A 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrzolium bromide (MTT) demonstrted tht there ws no differ- A 2-DG uptke nmol.min -1.1mg -1 Crbonyls, nmol/mg protein DMSO Insulin HNE b C D E ** 4-HNE GSH, µmol/mg protein DMSO ** 4-HNE B Ser 473 p-pkb/akt Insulin HNE Ser 473 p-akt Totl Akt Tubulin Crbonyls, nmol/mg protein b r 2 =.599 p=.14 DMSO 4-HNE GSH, µmol/mg protein Ser 473 p-pkb/akt Insulin HNE Ser 473 p-akt Totl Akt Tubulin FIG HNE impired insulin signling in mouse skeletl muscle. Mouse gstrocnemius muscle ws incubted for 15 min with 1 M 4-HNE or DMSO (control) nd then stimulted with insulin (1 nm, 15 min). A, Glucose uptke ws mesured using [ 3 H]-2-deoxyglucose s described in Mterils nd Methods (n 5). B, Immunoblotting study of in vitro insulin-induced phosphoryltion of PKB/Akt in gtrocnemius muscle. Note tht 4-HNE impired insulin-induced serine phosphoryltion of PKB/Akt. C, 4-HNE-induced crbonyltion of proteins ws ssyed using DNPH ssy. D, Tretment of muscle with 4-HNE depletes intrcellulr GSH. E, Correltion between GSH depletion nd crbonyl content in gstrocnemius muscle incubted in vitro with 4-HNE or DMSO. Note the negtive correltion between GSH nd crbonyl contents. F, In vivo pretretment with NAC prevented 4-HNE-induced disruption of insulin signling pthwys in skeletl muscle. Mice were given NAC (1 mm) for 1 wk in drinking wter. Gstrocnemius muscle ws dissected out, incubted with 4-HNE (1 M, 15 min) before insulin stimultion (1 nm, 15 min) nd phosphoryltion of PKB/Akt ws nlyzed by Western blotting. Results re mens SEM (n 4 for ech set of experiments). Different letters indicte significnt difference t P.5 level. **, P.1 vs. untreted control. F b b

5 Endocrinology, My 212, 153(5): endo.endojournls.org 213 ence in vibility between treted nd untreted cells. The percentge of necrotic cells, estimted by the mesurement of lctte dehydrogense ctivity, remined t the bseline level nd did not increse significntly, s did poptosis (mesured by cspse-3 ctivity). Under our experimentl conditions (1 1 M, 3 min exposure), 4-HNE did not exhibit significnt deleterious effects on vibility in L6 muscle cells; therefore, differences in glucose metbolism nd insulin signling reported herefter cnnot be regrded s cytotoxic effect on cell vibility (i.e. number of living cells). Insulin-stimulted glucose uptke is impired in 4-HNE exposed cells To determine whether 4-HNE could impir glucose metbolism in muscle cells, we studied the [ 3 H]-2-deoxy- D-glucose trnsport. In control cells, stimultion with 1 nm insulin for 2 min induced 7% increse in [ 3 H]-2- deoxy-d-glucose uptke. A 3-min pretretment with 4-HNE thoroughly diminished the insulin-stimulted glucose uptke without ffecting the bsl glucose uptke (Fig. 2A). A mximl inhibition of glucose uptke ws observed t 5 M 4-HNE. To study the time course of the inhibition of glucose trnsport, cells were preincubted for.5 4 h with 5 M 4-HNE. Mximl inhibition of insulin-induced glucose uptke ws observed s soon s 3 min fter induction of tretment (Fig. 2B). 4-HNE impirs insulin signling in L6 muscle cells To gin further understnding of the mechnisms underlying the defect in insulin-stimulted glucose uptke, Akt nd insulin receptor substrte-1 (IRS-1) phosphoryltion were nlyzed. Stimultion with 1 nm insulin for 2 min induced 4-fold increse in Akt Ser 473 phosphoryltion. This ws dose dependently inhibited by 3-min pretretment of the cells with 4-HNE, with mximl effect obtined t 25 M (Fig. 3A). Pretretment of the cells with 5 M 4-HNE for 3 min ws responsible for 4% decrese in Akt phosphoryltion (Fig. 3C), 4% decrese in IRS-1 phosphoryltion (Fig. 3D), nd 45% decrese in p85 coimmunoprecipittion (Fig. 3E). Representtive blots re shown in Fig. 3B. A recent report demonstrted tht tretment of 3T3-L1 dipocytes with 4-HNE (1 M) induced shrp decrese in IRS-1 nd PKB/Akt protein levels due to covlent binding of 4-HNE, which in turn promoted their degrdtion (15). We, however, did not find ny chnge in the totl mount of both PKB/Akt nd IRS-1 proteins (see Supplementl Fig. 1) nd concluded tht the decrese in the phosphoryltion of PKB/Akt nd A Glucose Uptke (% of unstimulted control) B Glucose Uptke (% of unstimulted control) Concentrtion (µm) IRS-1 did not result from decrese in the totl content of these proteins. We then ttempted to detect 4-HNE Michel dducts on IRS-1. To this end, cells were treted with 4-HNE, IRS-1 ws immunoprecipitted using specific ntibodies, nd the formtion of 4-HNE Michel dduct ws nlyzed by Western blotting. In n dditionl set of experiments, immunoprecipitted IRS-1 ws incubted in vitro with 1 M 4-HNE. Under ll these conditions, we filed to detect the formtion of 4-HNE Michel dduct on IRS-1 protein (dt not shown); thus, the defult in IRS-1 tyrosine phosphoryltion unlikely resulted from the direct dduction of the IRS-1 protein. Oxidtive stress ctivtes stress signling pthwys such s ERK nd Jun kinse (JNK), which in turn my induce serine phosphoryltion nd inhibit insulin-induced tyrosine phosphoryltion of IRS, thus contributing to the disruption of insulin signling. We therefore tested the * ** Time (min) FIG HNE impired insulin-induced glucose uptke in L6 muscle cells. L6 muscle cells were treted for 3 min with 75 M 4-HNE (A); in time course of 4-HNE, the effect ws studied t 5 M 4-HNE (B). Cells were then stimulted by 1 nm insulin for 2 min nd glucose uptke ws mesured using tritited 2-deoxyglucose (mens SEM, n 5 for ech set of experiments). *, P.5; **, P.1. White circles, Bsl; Blck squres, insulin-stimulted. *

6 214 Pillon et l. 4-HNE-Induced Muscle Insulin Resistnce Endocrinology, My 212, 153(5): FIG HNE disrupted insulin signling pthwy in L6 muscle cells. L6 muscle cells were treted for 3 min with 4-HNE. A, Akt phosphoryltion ws detected fter tretment of cells with incresing concentrtion of 4-HNE. *, P.5. White circle, bsl; blck squres, insulin-stimulted. B, Representtive blots of Akt nd IRS-1 phosphoryltion, p85 subunit of phosphtidylinositol 3-kinse (PI3K) coimmunoprecipittion nd their respective loding controls tubulin nd IgG. C, D, nd E, Quntifiction of phosphoryltion of Akt, IRS, nd p85 coimmunoprecipittion with IRS-1, respectively. Results re men SEM (n 4 for ech set of experiments), nd different letters indicte significnt difference t P.5 level. involvement of ERK nd JNK in hydroxylkenl-impired insulin signling. In the bsence of insulin, tretment with 5 M 4-HNE induced significnt 25% increse in ERK, JNK, nd P38 phosphoryltion (see Supplementl Fig 2). As previously reported, insulin triggered similr increse in MAPK phosphoryltion. Pretretment of cells with 4-HNE did not show further effects compred with insulin lone or 4-HNE lone. Tken together, these results suggest tht MAPK were ctivted by 4-HNE, but their ctivtion did not likely contributed to the 4-HNE-dependent inhibition of insulin signling. 4-HNE genertes covlent dducts, crbonyl stress, nd GSH depletion in L6 muscle cells 4-HNE rects with cell proteins to form covlent dducts resulting in protein dysfunction nd impirment of cellulr responses. We used specific ntibodies directed ginst 4-HNE Michel dducts nd confocl microscopy to estimte the formtion of dducts in situ. 4-HNE induced dose-dependent increse in Michel dduct formtion minly restricted to the cytoplsm, wheres the nucleus ws spred (Fig. 4, A nd B). Dose-dependent dduction of proteins ws evidenced using dot blotting for 4-HNE Michel dducts (Fig. 4C), nd SDS-PAGE nlysis reveled the formtion of dducts on mny proteins of different sizes rnging from 25 to 2 kd (Fig. 4D). The detection of dducted proteins ws further evidenced through the rection of DNPH with protein crbonyl groups. Incubtion of L6 muscle cells with 4-HNE resulted in dose-dependent increse in crbonyl content (Fig. 4E). Conjugtion with GSH is known to be mjor pthwy of detoxifiction of lipid ldehydes, nd we noticed tht in isolted muscle, tretment with 4-HNE induced n increse in crbonyl content, which ws correlted with depletion in GSH (Fig. 1). We therefore performed the mesurement of intrcellulr GSH pool in L6 muscle cells fter tretment with 4-HNE (Fig. 4F). A 3-min tretment with incresing concentrtions of 4-HNE resulted in shrp decrese in GSH content in L6 muscle cells. Indeed, GSH concentrtion ws reduced by 15 nd 3% for 5 nd 1 M, respectively. Similr to wht ws observed in gstrocnemius muscle, crbonyl formtion ws significntly correlted with GSH concentrtion (Fig. 4G). 4-HNE induces intrcellulr ROS production Intrcellulr ROS genertion ws determined through the fluorescence intensity of the intrcellulr fluoroprobe (CM-H2DCFDA) (Fig. 5). ROS were incresed by 2-fold in cells treted with 5 M 4-HNE for 3 min compred with unstimulted cells. Increse in GSH pools reverses the noxious effects of 4-HNE Becuse GSH nd glutthione-s-trnsferses re known to be involved in the detoxifiction of 4-HNE, we tested whether incresing the intrcellulr pools of glutthione could protect cells from hydroxy-lkenls. We used N-cetyl-cysteine nd D3T, nturl compound found in

7 Endocrinology, My 212, 153(5): endo.endojournls.org 215 A µm 1µM B D F 4-HNE Michel dducts (rbitrry units) MW mrkers 25 kd 15 kd 1 kd 75 kd GSH content (nmol /mg protein) 1 5 kd 37 kd 25 kd *** Concentrtion (µm) 4-HNE *** HNE (µm) 5µM C 4-HNE Michel dducts (% of control) E Crbonyls content (nmol/mg de proteins) 4 *** µM 1µm 1µm 1µm 1µm G Crbonyls, nmol/mg protein Concentrtion (µm) *** Concentrtion (µm) 15 r² =.933 p <.1 1 µm µm 1 µm µm GSH, nmol/mg protein FIG HNE induces crbonyl stress nd GSH depletion in L6 muscle cells. L6 muscle cells were treted for 3 min with 4-HNE ( 1 M). A, Adduct formtion within the cells ws determined s described in Mterils nd Methods using ntibodies ginst 4-HNE Michel dducts. B, Quntifiction of immunofluorescence ws performed with confocl microscopy in t lest five different fields. C, Michel dducts with proteins were detected fter dot blot using specific ntibodies to 4-HNE. D, Michel dducts with proteins were detected fter protein extrction nd Western blot. E, Crbonyltion of proteins ws ssyed using DNPH ssy. F, Tretment of L6 cells with incresing concentrtions of 4-HNE depletes cellulr GSH. GSH content ws mesured using fluorescent ssy s described in Mterils nd Methods. G, Correltion between GSH depletion nd crbonyl content in L6 cells incubted with 4-HNE. All results re mens SD from t lest three independent experiments (n 3). ***, P.1. cruciferous vegetbles, which ws reported to increse the intrcellulr pools of glutthione s well s glutthione-s-trnsferses ctivity (25). In L6 cells, pretretment with D3T (1 M for 24 h) nerly doubled the intrcellulr mount of reduced glutthione nd reversed the decrese in GSH induced by 4-HNE (Fig. 6A). In contrst, NAC did not ffect GSH content, nd the reduction induced by 4-HNE ws similr to tht without pretretment. The use of either D3T or NAC reversed the increse in crbonyl content observed fter 3 min tretment with 4-HNE (Fig. 6C) s well s Michel dduct formtion detected by dot blot (Fig. 6B). Similrly, pretretment of cells with D3T nd NAC prevented the 4-HNE induced increse in intrcellulr ROS production (Fig. 6D). Moreover, pretretment of L6 muscle cells with NAC or D3T completely restored the insulin-stimulted glucose uptke (Fig. 6E) s well s the insulininduced Akt phosphoryltion (Fig. 6F). Of note, the common ntioxidnt NAC did not increse GSH pools but led to the sme results s D3T on ROS, crbonyls, signling, nd glucose uptke, suggesting protective ntioxidnt effect out of GSH restortion. Tken together, these results suggested pivotl roles for both crbonyl stress nd oxidtive stress in 4-HNE-induced insulin resistnce in muscle. Independently of these short-term, nontoxic effects, we previously reported tht long-term exposure to 4-HNE induced dose-dependent cytotoxic effects on L6 muscle cells (24). Indeed, the lethl concentrtion 5 clculted fter 16 h tretment with 1 M 4-HNE ws 4 1. M (see Supplementl Fig. 3). Phrmcologicl increse in GSH through pretretment for 24 h with D3T mrkedly reduced the cytotoxicity induced by 4-HNE, leding to 2-fold increse in the lethl concentrtion 5 ( M, P.1).

8 216 Pillon et l. 4-HNE-Induced Muscle Insulin Resistnce Endocrinology, My 212, 153(5): Intrcellulr ROS (UFR/ mg protein) Aminogunidine (AGD) nd S-denosylmethionine (SAM) reversed the deleterious effects of 4-HNE AGD nd SAM re nucleophilic gents known to ct s scvengers of dvnced lipid peroxidtion by-products. In L6 muscle cells, pretretment with AGD or SAM prevented the crbonyltion of proteins (Fig. 7A). On insulininduced glucose uptke, both AGD nd SAM induced significnt increse in insulin response, but only AGD completely reversed the inhibition of insulin-induced glucose uptke (Fig. 7B); however, the insulin-induced glucose uptke in the presence of SAM nd 4-HNE ws still higher tht in the control sitution. In summry, pretretment of L6 cells with these molecules confers protection ginst the deleterious effects of 4-HNE on glucose metbolism nd insulin signling. Discussion Bsl 4-HNE H2O2 FIG HNE stimulted intrcellulr production of ROS in L6 muscle cells. After incubtion in Krebs buffer during 3 min, 1 M of CM- H2DCFDA ws dded to the medium for nother 3 min. Then cells were wshed nd treted with 5 M 4-HNE for 3 min. Cells were incubted with H 2 O 2 s positive control. After lysis in wter, fluorescence ws red using fluorometer, nd results were normlized to the protein concentrtion. Results re mens SEM (n 4). *, P.5; **, P.1. A number of oxidnts re produced s by-products of norml erobic cell metbolism, nd there is growing evidence tht they ply key roles in the pthogenesis of metbolic disorders. Oxidtive stress is ssocited with obesity nd insulin resistnce s evidenced by the systemic increse of 8-epi-prostglndin F2 in obese rts (26), the elevted plsm F2 isoprostnes in type 2 dibetic ptients (27) nd the incresed level of plsm mlondildehyde in obese humns (2). More evidences cn be found within tissues, such s n increse in protein nitrosyltion in dibetic mouse muscle (28), n increse in mlondildehyde in liver of obese rts (29), nd decresed ntioxidnt enzymes in dipose tissue from obese KKAy mice (2). Oxidtive stress cn led to lipid peroxidtion nd to the formtion of secondry by-products such s toxic lipid * ** ldehydes. Among them, 4-HNE is produced from the peroxidtion of n-6 unsturted ftty cids nd its levels in tissues of norml nimls rnge from.5 to 1 M. Indeed, 4-HNE concentrtion in the ventriculr fluid of ptients with Alzheimer s disese ws reported s high s 12 M (3), wheres its concentrtion is bout 3 M in rt isolted pncretic islets (12). 4-HNE is highly rective nd hs minly been detected in the form of covlent dducts, wheres fewer dt re vilble regrding the free 4-HNE concentrtion. It hs been suggested tht 4-HNE cn rech locl concentrtion of up to the millimolr rnge close to membrnes undergoing peroxidtion (31). 4-HNE is highly rective species prone to forming covlent dducts on mny clsses of biomolecules present in culture medium (e.g. mino cids). To determine the ctul concentrtion of 4-HNE tht the cells or tissues were exposed to in our experiments, we performed the gs chromtogrphy/mss spectrometry mesurement of free 4-HNE in culture medium (without cells) 3 min fter ddition of 1 1 M 4-HNE (Supplementl Fig. 4). The free concentrtion of 4-HNE in culture medium ccounted for 11, 4, 27, 27, nd 19% of initil mount of 4-HNE for 1, 1, 25, 5, nd 1 M, respectively; therefore, most of the 4-HNE rected with constituents of cell culture medium, nd tretment of L6 muscle cells with 5 M likely leds to n vilble concentrtion of 15 M free 4-HNE. The concentrtion rnge used in this study ws therefore physiologiclly relevnt, nd we were ble to show tht 4-HNE forms mny covlent dducts in muscle cells without ffecting cell vibility under these conditions. The mechnism of oxidtive-induced insulin resistnce is complex nd still uncler, even though severl mechnisms hve been proposed: degrdtion or serine phosphoryltion of IRS-1, ctivtion of MAPK signling pthwys, nd direct nitrosyltion of elements of the insulin pthwy (reviewed in Ref. 3). Recently the role of lipid ldehydes, especilly 4-HNE, in insulin resistnce hs been outlined (8). 4-HNE nd other lipid peroxidtion products hve been shown to impir insulin secretion induced by glucose in pncretic -cells (12). 4-HNE is lso ble to form covlent dducts on IRS-1 nd Akt (15, 32), nd it hs been proposed tht it cts s second messenger to ctivte signl trnsduction pthwys, especilly MAPK-signling pthwys (33), which re known to impir IRS ctivtion. In 3T3-L1 dipocytes, 4-HNE exhibited incresed IRS degrdtion due to covlent modifiction nd elevted phosphoryltion on Ser 37, leding to impired insulin signling nd glucose uptke (15). Skeletl muscle ccounts for more thn 8% of wholebody postprndil glucose uptke, nd it is only when the

9 Endocrinology, My 212, 153(5): endo.endojournls.org 217 FIG. 6. D3T nd NAC reversed 4-HNE noxious effects on glutthione levels, dduct formtion, glucose uptke, nd insulin signling. L6 cells were preincubted with 1 M of D3T for 24 h nd then treted by 4-HNE for 3 min A, Pretretment with D3T but not NAC increses intrcellulr GSH. GSH content ws mesured using fluorometric ssy s described in Mterils nd Methods. B nd C, Pretretment with D3T or NAC decreses crbonyl formtion in L6 cells. Crbonyls were mesured using DNPH nd dducts were detected fter dot blot using specific ntibody ginst 4-HNE Michel dducts. D, D3T nd NAC prevent ROS production mesured using the CM-H2DCFDA fluorescent probe. E, Pretretment with D3T or NAC reverses deleterious effects of 4-HNE on insulin-stimulted glucose uptke. Glucose uptke ws mesured using tritited 2- deoxyglucose s described previously. F, D3T nd NAC reverse the deleterious effects of 4-HNE on insulin-induced Akt phosphoryltion s mesured by Western blotting. All results re men SD for n 3. *, P.5; **, P.1. Different letters indicte significnt difference t P.5 level. skeletl muscle becomes insulin resistnt tht systemic insulin resistnce occurs (2, 34); however, to the best of our knowledge, very few dt re vilble in the literture regrding the direct effect of lipid ldehydes on skeletl muscle insulin sensitivity. This report is the first to demonstrte tht 4-HNE cn blunt insulin ction in gstroc-

10 218 Pillon et l. 4-HNE-Induced Muscle Insulin Resistnce Endocrinology, My 212, 153(5): FIG. 7. Aldehyde scvengers AGD nd SAM reversed the deleterious effects of 4-HNE. L6 muscle cells were pretreted with AGD or SAM before tretment with 4-HNE. A, Pretretment with AGD or SAM reverses crbonyl formtion in L6 cells. Michel dducts with proteins were detected in dot blot using specific ntibodies to 4-HNE dducts. B, Pretretment with AGD or SAM reverses deleterious effects of 4- HNE on insulin-stimulted glucose uptke. Glucose uptke ws mesured s described in Mterils nd Methods using tritited 2- deoxyglucose. All results re men SEM (n 4). Different letters indicte significnt difference t P.5 level. *, P.5. nemius muscle nd L6 muscle cells. 4-HNE induced crbonyl stress nd triggered intrcellulr ROS production, leding to impirment of glucose uptke s well s Akt nd IRS signling, hllmrks of insulin resistnce. This cn be defined s stte of cellulr insulin resistnce, which cn led to the development of type 2 dibetes in the orgnism. We recently reported tht direct dduction of the insulin protein by 4-HNE disrupts its biologicl ctivity (16). To exclude such direct effect on insulin, the medium contining 4-HNE ws removed nd cells thoroughly wshed before insulin stimultion. Using this protocol, insulin stimultion ws not performed in medium contining 4-HNE, excluding the possibility tht the observed effects were due to defect in insulin itself. We lso demonstrted previously tht the cytotoxic effect of hydroxy-lkenls is due to their bility to form covlent dducts on proteins (24). In contrst to wht hs been shown in 3T3-L1 dipocytes, we filed to detect ny 4-HNE dduct on IRS-1, even if covlent dduction remins possible mechnism for 4-HNE toxicity. Numerous studies hve shown tht both endogenous nd dietry ntioxidnts cn protect tissue from oxidtive dmge, nd mny recent studies re focused on the identifiction of nturl substnces with protective potentil tht could scvenge free rdicls or their secondry byproducts. Tretment of dibetes with therpies involving ntioxidnts nd/or scvenging drugs hs been evluted in severl studies, nd plethor of scientific reports demonstrte protection of -cells ginst oxidtive stress by ntioxidnt drugs (35, 36). Moreover, -lipoic cid, biologicl ntioxidnt nd nturl cofctor of the mitochondril dehydrogense complex, hs been found to increse insulin sensitivity in niml models of type 2 dibetes nd insulin resistnce (4, 37). Consequently, we chose to use phrmcologicl pproch using severl compounds which could potentilly protect the cells from the deleterious effects of 4-HNE. We used the nturl compound D3T, found in cruciferous vegetbles, to increse the intrcellulr pools of glutthione s well s glutthione-s-trnsferses ctivity. Indeed, detoxifiction of 4-HNE in vivo is minly chieved through GSH nd glutthione-s-trnsferses, nd severl groups suggested tht n increse in intrcellulr glutthione pools my protect from ldehyde toxicity (38, 39). In prllel, we used, SAM, cosubstrte of severl enzymes leding to cysteine synthesis, precursor for glutthione production, which is the proposed mechnism for the ntioxidnt effects of SAM (4, 41). We lso tested NAC, well-known ntioxidnt (42, 43), nd AGD, nucleophilic scvenger ble to trp crbohydrte-derived crbonyls (44). In clinicl studies, there is growing evidence tht -lipoic cid hs beneficil effects on the tretment of type 2 dibetes nd some of its complictions (45). D3T, SAM, NAC, nd -lipoic cid re ll sulfur-contining components nd could consequently shre some chrcteristics, such s increse in GSH contents nd ntioxidnt properties (42). In our muscle cell model, D3T ws ble to significntly increse the GSH pools, which prevented the deleterious effects of 4-HNE on dduct formtion nd glucose uptke. The sme beneficil effects were observed fter pretretment of the cells with SAM. This importnt role of glutthione in protecting ginst oxidtive stress hs been described in different models, but we showed here for the first time tht GSH is importnt to mintin the insulin response in muscle during n ldehyde chllenge. In greement with previous reports (46), 4-HNE tretment induced n increse in intrcellulr ROS production in L6 muscle cells. ROS is clerly possible mechnism for insulin signling inhibition, but bsed on our results, we re proposing model in which redox imblnce cretes

11 Endocrinology, My 212, 153(5): endo.endojournls.org 219 Redox imblnce components. Increse in glutthione pools, scvenging of ldehydes nd ntioxidnt tretment re ll vible strtegies to reverse the deleterious effects of 4-HNE on dduct formtion nd glucose uptke. D3T, NAC, SAM, nd AGD could therefore be potentil cndidtes for the tretment of oxidtive stress-ssocited diseses such s type 2 dibetes. 4-HNE production «Vicious cycle» ROS formtion Acknowledgments Crbonyl stress We grtefully cknowledge Dr. Asmi Mkino for technicl help in immunohistofluorescence nd confocl microscopy nlysis. We lso wrmly thnk Dr. Sheil Costford for proofreding nd corrections. Insulin resistnce FIG. 8. Proposed mechnism for 4-HNE-induced insulin resistnce in muscle. Under conditions of oxidtive stress, ccumultion of ROS leds to the production of toxic ldehydes, which in turn induce ROS production. This vicious circle eventully leds to crbonyl stress through dduct formtion on cell proteins. vicious circle linking ROS nd ldehyde production (Fig. 8). ROS re inducing lipid peroxidtion which genertes ldehydes, wheres these ldehydes re generting more ROS, leding to n mplifiction of the toxicity of oxidtive stress. Indeed, under our conditions, NAC did not increse glutthione pools, even if exhibiting significnt protection ginst 4-HNE noxious effects. The beneficil effects of NAC were therefore likely due to its direct ntioxidnt properties: by decresing ROS production, NAC could reduce secondry ldehyde production nd therefore stop the vicious circulr mechnism described bove. Unmngeble crbonyl stress only occurs when ntioxidnt defenses nd/or glutthione pools of the cell re overtken. Accumultion of dducted proteins is lso to relte to the endoplsmic stress, which hs been shown to induce stte of insulin resistnce (47). Moreover, overexpression of ldehyde dehydrogense-2, involved in ldehyde detoxifiction, meliortes both endoplsmic reticulum stress nd insulin resistnce (48), suggesting role for 4-HNE nd ldehydes in this phenomenon. In conclusion, we were ble to demonstrte tht the lipid peroxidtion by-product 4-HNE cn impir insulin signling nd glucose uptke in muscle cells s well s in isolted skeletl muscle. Moreover, we highlighted the importnt role of both oxidtive stress nd glutthione in this mechnism nd showed tht deleterious effects of 4-HNE cn be prevented by tretment with sulfur-contining Address ll correspondence nd requests for reprints to: Dr. Nicols J. Pillon, CrMeN (Crdiovsculire, Métbolisme, Dibétologie, et Nutrition), Bâtiment IMBL, Institut Ntionl des Sciences Appliquées-Lyon, 2 Avenue Albert Einstein, F Villeurbnne cedex, Frnce. E-mil: n.pillon@gmil.com. This work ws supported by the Institut Ntionl de l Snté et de l Recherche Médicle nd the Institut Ntionl des Sciences Appliquées de Lyon. N.J.P., M.L.C., nd R.E.V. were supported by grnts from the French Ministère de l Eduction Ntionle, de l Recherche et de l Technologie. Disclosure Summry: The uthors hve nothing to declre. References 1. Evns JL, Goldfine ID, Mddux BA, Grodsky GM 22 Oxidtive stress nd stress-ctivted signling pthwys: unifying hypothesis of type 2 dibetes. Endocr Rev 23: Furukw S, Fujit T, Shimbukuro M, Iwki M, Ymd Y, Nkjim Y, Nkym O, Mkishim M, Mtsud M, Shimomur I 24 Incresed oxidtive stress in obesity nd its impct on metbolic syndrome. J Clin Invest 114: Bshn N, Kovsn J, Kchko I, Ovdi H, Rudich A 29 Positive nd negtive regultion of insulin signling by rective oxygen nd nitrogen species. Physiol Rev 89: Henriksen EJ, Jcob S, Streeper RS, Fogt DL, Hokm JY, Tritschler HJ 1997 Stimultion by -lipoic cid of glucose trnsport ctivity in skeletl muscle of len nd obese Zucker rts. Life Sci 61: Negre-Slvyre A, Cotrieux C, Ingueneu C, Slvyre R 28 Advnced lipid peroxidtion end products in oxidtive dmge to proteins. Potentil role in diseses nd therpeutic prospects for the inhibitors. Br J Phrmcol 153: Bcot S, Bernoud-Hubc N, Chntegrel B, Deshyes C, Doutheu A, Ponsin G, Lgrde M, Guichrdnt M 27 Evidence for in situ ethnolmine phospholipid dducts with hydroxy-lkenls. J Lipid Res 48: Hu W, Feng Z, Eveleigh J, Iyer G, Pn J, Amin S, Chung FL, Tng MS 22 The mjor lipid peroxidtion product, trns-4-hydroxy- 2-nonenl, preferentilly forms DNA dducts t codon 249 of humn p53 gene, unique muttionl hotspot in heptocellulr crcinom. Crcinogenesis 23: Mttson MP 29 Roles of the lipid peroxidtion product 4-hydroxynonenl in obesity, the metbolic syndrome, nd ssocited

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13 Endocrinology, My 212, 153(5): endo.endojournls.org 2111 dmge by lipid peroxidtion products. Toxicology : Poh ZX, Goh KP 29 A current updte on the use of lipoic cid in the mngement of type 2 dibetes mellitus. Endocr Metb Immune Disord Drug Trgets 9: Ustyuk PV, Prinndi NL, Ntrjn V 26 Redox regultion of 4-hydroxy-2-nonenl-medited endothelil brrier dysfunction by focl dhesion, dherens, nd tight junction proteins. J Biol Chem 281: Ozcn U, Co Q, Yilmz E, Lee AH, Iwkoshi NN, Ozdelen E, Tuncmn G, Görgün C, Glimcher LH, Hotmisligil GS 24 Endoplsmic reticulum stress links obesity, insulin ction, nd type 2 dibetes. Science 36: Li SY, Gilbert SA, Li Q, Ren J 29 Aldehyde dehydrogense-2 (ALDH2) meliortes chronic lcohol ingestion-induced myocrdil insulin resistnce nd endoplsmic reticulum stress. J Mol Cell Crdiol 47: Michlski MC, Clzd C, Mkino A, Michud S, Guichrdnt M 28 Oxidtion products of polyunsturted ftty cids in infnt formuls compred to humn milk preliminry study. Mol Nutr Food Res 52: Members hve FREE online ccess to the journl Hormones & Cncer.