Diabetic Downregulation of Nrf2 Activity via ERK Contributes to Oxidative Stress-Induced Insulin Resistance in Cardiac Cells In Vitro and In Vivo.

1.5 MB·English

by  Tan, Yi

ORIGINALARTICLE
Diabetic Downregulation of Nrf2 Activity via ERK
–
Contributes to Oxidative Stress Induced Insulin
Resistance in Cardiac Cells In Vitro and In Vivo
Yi Tan,1,2 Tomonaga Ichikawa,3 Jinqing Li,3 Qiusheng Si,4 Huaitao Yang,4 Xiangbai Chen,4
Curtis S. Goldblatt,4 Colin J. Meyer,5 Xiaokun Li,1 Lu Cai,1,2 and Taixing Cui1,3
OBJECTIVE—Oxidative stress is implicated in cardiac insulin
resistance,acriticalriskfactorforcardiacfailure,butthedirect I
evidence remains missing. This study explored a causal link nsulin resistance usually refers to a defect in the
between oxidative stress and insulin resistance with a focus on ability of insulin to stimulate glucose uptake. Im-
a regulatory role of redox sensitive transcription factor NF-E2– portantly, insulin resistance is not only a character-
related factor2 (Nrf2)in thecardiaccellsinvitro andinvivo.
isticfeatureoftype2diabetesbutalsoimplicatedin
RESEARCHDESIGNANDMETHODS—Chronictreatmentof the heart of type 1 diabetes (1,2). The severity of insulin
HL-1 adultcardiomyocyte withhydrogenperoxide led toinsulin resistance independently predicts mortality in patients
resistance, reflected by a significant suppression of the insulin- with heart failure (3,4). At a molecular level, insulin re-
inducedglucoseuptake.Thiswasassociatedwithanexaggerated sistance is characterized by impairment of the insulin-
phosphorylation of extracellular signal–related kinase (ERK).
induced activation of insulin receptor substrate (IRS)/
Although U0126, an ERK inhibitor, enhanced insulin sensitivity
phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)
and attenuated oxidative stress–induced insulin resistance,
pathway, the major player of the metabolic action of in-
LY294002,aninhibitorofphosphoinositide3-kinase(PI3K),wors-
ened the insulin resistance. Moreover, insulin increased Nrf2 sulin,leadingtosuppressionoftheinsulin-inducedglucose
transcriptional activity, which was blocked by LY294002 but en- uptake in the insulin-sensitive organs, including the heart
hanced byU0126. Forcedactivation of Nrf2 byadenoviral over- (3,4). Indeed, reduced IRS-1 associated PI3K activity in
expression of Nrf2 inhibited the increased ERK activity and skeletal muscle has been demonstrated to contribute to
recovered the blunted insulin sensitivity on glucose uptake in wholebodyinsulinresistanceindiabetes(5).Theseresults
cardiomyocytes that were chronically treated with H O . In the
2 2 reveal a unique feature of cardiac insulin resistance that
hearts of streptozotocin-induced diabetic mice and diabetic
patientsNrf2expressionsignificantlydecreasedalongwithsignif- might play a crucial role in the pathogenesis of diabetic
cardiomyopathy.
icantincreasesin3-nitrotyrosineaccumulationandERKphosphor-
ylation, whereas these pathogenic changes were not observed in Although a pivotal role of insulin resistance in cardiac
theheartofdiabeticmicewithcardiac-specificoverexpressionof dysfunction appears to be recognized, the mechanisms
a potent antioxidant metallothionein. Upregulation of Nrf2 by its contributing to insulin resistance are poorly understood.
activator, Dh404, in cardiomyocytes in vitro and in vivo pre- Notably, increasing evidence has suggested that oxidative
ventedhydrogenperoxide–anddiabetes-inducedERKactivation stress plays a causal role in the cardiac complications of
andinsulin-signaling downregulation.
insulin resistance, and the overgenerated reactive oxygen
CONCLUSIONS—ERK-mediated suppression of Nrf2 activity species (ROS) or reactive nitrogen species (RNS) and in-
leads to the oxidative stress–induced insulin resistance in adult sulin resistance may be coconspirators in cardiac dys-
cardiomyocytes and downregulated glucose utilization in the di- function,eachcapableoftriggeringorworseningtheother
abeticheart.Diabetes60:625–633,2011 (6–8). However, a direct, clear, and causal relationship
between oxidative stress and insulin resistance in the
heart is not yet established.
Cells have evolved endogenous defense mechanisms
against sustained oxidative stress such as the redox sen-
sitive transcription factor NF-E2–related factor 2 (Nrf2),
which regulates antioxidant response element (ARE/
EpRE)-mediatedexpressionofdetoxifyingandantioxidant
From the 1Chinese-American Research Institute for Diabetic Complications,
WenzhouMedical College,Wenzhou,Zhejiang,China;the 2Departmentof enzymesandthecystine/glutamatetransporterinvolvedin
Pediatrics,UniversityofLouisville,Louisville,Kentucky;the3Departmentof glutathionebiosynthesis(9).DiminishedNrf2/AREactivity
CellBiologyandAnatomy,UniversityofSouthCarolinaSchoolofMedicine,
contributes to increased oxidative stress and mitochon-
Columbia,SouthCarolina;the4DepartmentofPathology,MemorialMedical
Center,Johnstown,Pennsylvania;andthe5ReataPharmaceuticals,Irving, drialdysfunctioninthevasculature,leadingtoendothelial
Texas. dysfunction,insulinresistance,andabnormalangiogenesis
Correspondingauthors:TaixingCui,[email protected],andLuCai,
observedindiabetes(10).Recentlywehavedemonstrated
[email protected].
Received17August2010andaccepted20November2010. the critical role of Nrf2 expression in protecting the car-
DOI:10.2337/db10-1164 diaccellsfromoxidativedamageanddeathcausedbyhigh
This article contains Supplementary Data online at http://diabetes.
levels of glucose (11).
diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1164/-/DC1.
Y.T.andT.I.contributedequallytothiswork. Herein, we demonstrate that oxidative stress directly in-
(cid:1)2011bytheAmericanDiabetesAssociation.Readersmayusethisarticleas ducesinsulinresistanceincardiomyocytesviaexaggerating
longastheworkisproperlycited,theuseiseducationalandnotforprofit, extracellularsignal–relatedkinase(ERK)activityinvitro.In
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/fordetails. the heart of streptozotocin (STZ)-induced type 1 diabetic
diabetes.diabetesjournals.org DIABETES,VOL.60,FEBRUARY2011 625
Nrf2ANDINSULINRESISTANCEINCARDIOMYOCYTES
mice,cardiacexpressionofNrf2wassignificantlydepressed a Beckman GS-6R centrifuge for 15 min. The protein concentration was
at the late stage of diabetes, the time period at which the measuredbyBradfordassay.Thesample,dilutedinloadingbufferandheated
heart showed a significant decrease in glucose metabolism at95°Cfor5min,wasthensubjectedtoelectrophoresison10%SDS-PAGEgel
along with the development of significant diabetic cardio- at120V.Afterelectrophoresisofthegelandtransfer oftheproteinstoni-
trocellulose membrane, the membranes were rinsed briefly in Tris-buffered
myopathy (12–14). Depressed expression of cardiac Nrf2
saline,blockedinblockingbuffer(5%milkand0.5%BSA)for1h,andwashed
was associated with significant increases in nitrosative three times withTris-buffered salinecontaining 0.05%Tween 20. Themem-
damage and phosphorylation of ERK, all of which were branes were incubated with different primary antibodies at a dilution of
prevented in the hearts of diabetic mice with cardiac over- 1:1,000 for 2 h and then washed and reacted with secondary horseradish
expression of a potent antioxidant metallothionein (MT). In peroxidase–conjugated antibody for 1 h. Antigen-antibody complexes were
thenvisualizedusinganECLkit(Amersham,Piscataway,NJ).
addition,upregulationofcardiacNrf2byitsactivatordihydro-
Humanheartspecimens.Tissuesectionsofleftventricleswerepreparedfrom
CDDO-trifluoroethyl amide (Dh404) significantly prevented
autopsyheartspecimensofhumanswithorwithoutadiseasehistoryordiabetes.
diabetes-induced nitrosative damage, ERK activation, and All diabetic males had histories of hypertension and cardiac dysfunction
insulinsignalingdownregulation.Thesefindingssuggestthat (SupplementaryTable1).ThisstudywasapprovedbytheInstitutionalReview
oxidative stress–depressed expression of cardiac Nrf2 is BoardforhumansubjectresearchattheUniversityofSouthCarolinaSchoolof
associatedwithcardiacactivationERKanddownregulation MedicineandtheMemorialMedicalCenterofJohnstown.
Statisticalanalysis. Values are expressed as means 6 SD. The data were
of glucose metabolism. Therefore, Nrf2, a master transcrip-
analyzedusingANOVAwiththeNewman-Keulstest.ValuesofP,0.05were
tional factor of antioxidative defense system (15), may be consideredtobestatisticallysignificant.
a novel negative regulator of oxidative stress–mediated in-
sulin resistance in cardiomyocytes and the heart.
RESULTS
Oxidative stress induces insulin resistance via
RESEARCHDESIGNANDMETHODS
overactivation of ERK in adult cardiomyocytes.
Cellcultureandadenoviralinfection.HL-1cellswereobtainedfromDr. Chronic treatment with H O has been demonstrated to
Claycomb (Louisiana State University Health Science Center, New Orleans, 2 2
induce oxidative stress leading to insulin resistance in
LA),whofirstestablishedandcharacterizedthecelllinederivedfromadult
various cell types (18); therefore, we tested whether such
murineatrialcardiomyocytetumorlineage,andculturedinClaycombMedium
(Sigma-Aldrich)(16).Cellswereinfectedwithadenovirusofb-galactosidase a treatment induces insulin resistance in HL-1 adult car-
(Ad-bGal)ormurineNrf2(Ad-Nrf2)aspreviouslydescribed(15). diomyocytes. Consistent with a previous report (19), in-
Glucoseuptakeassay.CellswereincubatedinKrebs-Ringer-HEPESbuffer(15 sulin stimulated 20–30% increases in glucose uptake in
mmol/LHEPES[pH7.4],105mmol/LNaCl,5mmol/LKCl,1.4mmol/LCaCl2, HL-1 cells (Fig. 1A), and treatment with H O at a nontoxic
1mmol/LKHPO,1.4mmol/LMgSO,and10mmol/LNaHCO)for30min.Cells 2 2
2 4 4 3
nextwereincubatedwithinsulin(100nmol/L)inKrebs-Ringer-HEPESbufferfor
30minandthenaddedto2-deoxy-D-glucose(0.2mmol/L)and2-deoxy-D-[3H]
glucose(2-DG)(1mCi/mL,MPBiomedicals)foranadditional10min.Thecells
werewashedthreetimeswithice-coldPBS,solubilizedin0.5mol/LNaOH,and
neutralizedwith0.5mol/LHCl,andtheradioactivityinaliquotsfromcellextracts
wasmeasuredusingaliquidscintillationcounter.Proteinconcentrationofthe
cellextractswasmeasuredusingaproteinassaykit(Bio-Rad).Deoxyglucose
uptakewasexpressedascountsperminutepermicrogramofprotein.
Nrf2 transcriptional reporter assay. Cells were transfected with Nrf2
transcription reporter gene ARE-luc (firefly luciferase) and internal control
pRL-TK-luc(renillaluciferase)plasmids,andNrf2transcriptionalactivitywas
quantifiedbymeasuringluciferaseactivitiesaspreviouslydescribed(17).
Diabetes models and drug administration. MT-overexpressing transgenic
(MT-TG)micewereproducedfromtheFVBmicethathavebeenwellcharac-
terized (12–14). Both8- to10-week-old MT-TG positive mice (heterozygotes)
andnegativelittermates(wildtype[WT])werekeptinthesamecageswithfree
accesstorodentchowandtapwater.Allanimalprocedureswereapprovedby
theInstitutionalAnimalCareandUseCommitteeoftheUniversityofLouisville,
whichiscertifiedbytheAmericanAssociationforAccreditationofLaboratory
AnimalCare.STZ(Sigma,St.Louis,MO)wasdissolvedinsodiumcitratebuffer
(pH4.5).MalemiceweregivenanintraperitonealinjectionofSTZat40mg/kg
bodywtdailyforfivedays.Wholebloodglucoseobtainedfromthemurinetail
veinwasdetectedusingaSureStepcompletebloodglucosemonitor(LifeScan,
Milpitas, CA) five days after the last STZ injection. STZ-injected mice with
glucose levels .12 mmol/L were considered diabetic, and mice serving as
controlsweregiventhesamevolumeofsodiumcitrate(12–14).
Twoanimalstudieswereperformed.Thefirststudywastoinvestigatethe
diabetic effect on cardiac Nrf2 expression along with nitrosative damage,
measuredby3-nitrotyrosine(3-NT)andERKexpression,usingMT-TGandWT
diabeticmiceat2and5monthsafterdiabetesonset.Thesecondstudywasto
investigate the preventive effect of Nrf2 activator Dh404 (provided by Reata
Pharmaceuticals)oncardiacNrf2expressionand3-NTaswellasinsulinsig-
nalingtargetsincludingphosphorylationofAktandglycogensynthasekinase-3b
(GSK-3b)at2weeksafterdiabetesonset.ForDh404treatment,diabeticand
age-matchednondiabeticmicewereadministratedwithDh404(10mg/kgbody FIG.1. H2O2-induced insulin resistance in HL-1 cells. Cellswerepre-
wt)orvehicle(sesameoil;Sigma)everyotherdaybygavage.Twoweekslater, treated with H2O2 (100 mmol/L) with or without NAC (1 mmol/L) in
6hafterthelastdoseadministration,micewerekilledandhearttissueswere 0.1%FBSmediumintheabsenceofnorepinephrinefor16handthen
subjectedtobasalorinsulin(100nmol/Lfor10min)-stimulated2-DG
collectedforthestudy.
uptakeassay(A),followedbyWesternblottingofphosphorylatedand
Immunochemicalstainingandimmunoblotanalysis.StainingandWestern totalERK(B).Resultsarerepresentativesof3separatedexperiments
blotanalysisforNrf2,totalandphosphor-ERK1/2,totalandphosphor-Akt,total (n=4).*P<0.05vs.control(H O 2andinsulin2),#P<0.05vs.H O
and phosphor-GSK-3b, and 3-NT were performed as previously described (+) without NAC; otherwise st2ati2stical difference was indicated.2(A2
(9,12–14). Briefly, heart tissues were homogenized in lysis buffer using ho- high-qualitycolorrepresentationofthisfigureisavailableintheonline
mogenizer.Tissueproteinswerecollectedbycentrifugingat12,000gat4°Cin issue.)
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FIG.3.AnegativeregulationofNrf2byERKworsensinsulinsensitivity
inHL-1cells.A:InsulinactivatesNrf2viaanegativecross-talkbetween
PI3KandERK.CellsweretransfectedwithARE-lucandpRL-TK-lucin
Opti-MEM (Invitrogen) for 6 h and then changed with 0.1% FBS me-
dium in the absence of norepinephrine for 48 h. The cells were next
stimulatedwith100nmol/Lofinsulin,10mmol/LofU0126,and10mmol/
LofLY294002asindicatedfor12h.Nrf2transcriptionalactivitywas
measuredbyadualluciferaseassaykit(Promega).*P<0.05vs.con-
trol(2),#P<0.05vs.insulin(+);n=4.B:Effectofoverexpressionof
FIG.2.Effectofinhibitorsofmitogen-activatedproteinkinasekinase Nrf2oninsulinsensitivityinHL-1cells.CellsinfectedwithAd-bGal(20
(MEK) and PI3K on basal or insulin-induced glucose uptake in HL-1 multiplicityofinfection[MOI])andAd-Nrf2(20MOI)werepretreated
cells with or without oxidative stress. Cells were pretreated with or withH2O2(100mmol/L)asinFig.1andsubjectedto2-DGuptakeassay.
withoutH O (100mmol/L)asinFig.1,withabsence(A)andpresence *P<0.05vs.control(2),n=4.C:Infectedcellswerepretreatedasin
2 2
(B)of100nmol/Linsulinpretreatmentfor10min.Cellsweretreated Fig.1andstimulatedwithorwithoutinsulin(100nmol/L)for10min.
with or without U0126 (10 mmol/L) or LY294002 (10 mmol/L) as in- *P<0.05vs.control(H2O22andinsulin2);otherwisestatisticaldif-
dicatedfor30minandthensubjectedto2-DGuptakeassay.*P<0.05 ferencewasindicated.
vs.control(2),#P<0.05vs.insulin(+)plusH O (+);n=4.
2 2
dose of 100 mmol/L for 16 h suppressed not only the basal possible role of ERK in oxidative stress–induced insulin
but also the insulin-induced glucose uptake (Fig. 1A). Pre- resistance in cardiomyocytes.
treatment of N-acetyl-cysteine (NAC), a ROS scavenger, ERK negatively regulates insulin-induced glucose
partially reversed H O -induced inhibition of the basal and uptake in adult cardiomyocytes. We next examined
2 2
insulin-induced glucose uptake (Fig. 1A), suggesting that the role of ERK and PI3K in regulating insulin-mediated
oxidative stress directly causes insulin resistance in adult glucose uptake in HL-1 cells. In control cells, addition of
cardiomyocytes. U0126,anERKinhibitor,enhancedthebasal(Fig.2A)and
Potential link of ERK1/2 with oxidative stress and in- insulin-induced glucose uptake (Fig. 2B), respectively;
sulin resistance in cardiomyocytes has been documented surprisingly, LY294002, a PI3K inhibitor, had no effect
(19). Accordingly, we examined the effect of chronic (Fig. 2A and B). In the cells with chronic H O treatment,
2 2
treatmentofH O onERK1/2phosphorylationinHL-1with U0126 was able to partially reverse, but LY294002 wors-
2 2
andwithoutinsulinstimulation.AsshowninFig.1B,acute ened,theH O -mediatedinhibitionofglucoseuptake(Fig.
2 2
stimulation of insulin (30 min) or chronic treatment of 2A)andinsulinresistance(Fig.2B).Theseresultssupport
H O (16h)increasedERKphosphorylation;however,the the above hypothesis that ERK as a negative regulator of
2 2
insulin-inducedERKactivitywassignificantlyenhancedby insulin-mediated glucose uptake plays a mediator role in
H O . NAC did not affect the insulin-induced ERK activity oxidative stress–induced insulin resistance in adult car-
2 2
but suppressed ERK activity induced by H O per se or diomyocytes.
2 2
H O with insulin (Fig. 1B). The reservation of H O - Positive regulation of insulin by Nrf2 to stimulate
2 2 2 2
induced insulin resistance (Fig. 1A) and suppression of glucose uptake in adult cardiomyocytes. Because we
H O -mediated ERK activity (Fig. 1B) by NAC reflects a havedemonstrated thatNrf2negativelyregulates oxidative
2 2
diabetes.diabetesjournals.org DIABETES,VOL.60,FEBRUARY2011 627
Nrf2ANDINSULINRESISTANCEINCARDIOMYOCYTES
FIG.4.CardiacNrf2expressioninthemicewithandwithoutcardiac-specificMToverexpression.Diabeteswasinducedbymultiplelowdosesof
STZinmicewithcardiac-specificMToverexpression(MT-TG)andlittermateWTmice.A:CardiacNrf2expressionat2monthsaftertheonsetof
diabeteswasdetectedbyWesternblot(WT,n=6forcontrol[Ctrl],n=8fordiabetesmellitus[DM];MT-TG,n=7forcontrol,n=8forDM).At
5monthsaftertheonsetofdiabetes,cardiacNrf2expression(B);nitrosativedamage,measuredby3-NT(C);andERKphosphorylation(D)were
detectedbyWesternblot,respectively(WT,n=7forcontrol,n=8forDM;MT-TG,n=6forcontrol,n=7forDM).b-ActinortotalERKwasused
asloadingcontrol.*P<0.05vs.control.
stressintheheart(15),wethendeterminedwhetherNrf2 shown in Fig. 4, cardiac Nrf2 expression was slightly in-
plays a role in regulating the oxidative stress–induced in- creased at 2 months (Fig. 4A) but significantly decreased
sulin resistance in HL-1 cells. Nrf2 transcription reporter at 5 months (Fig. 4B) after diabetes onset. The decreased
assay showed that insulin activated Nrf2, which was en- cardiacNrf2expressionatthelatestagewasaccompanied
hancedbyU0126,whereasblockedbyLY294002(Fig.3A), with oxidative and nitrosative damage, shown by signifi-
suggesting that the PI3K-mediated Nrf2 activation is neg- cantly increased cardiac 3-NT accumulation (Fig. 4C) and
atively regulated by ERK in insulin-stimulated HL-1 cells. overactivation of ERK function (Fig. 4D), as observed in
To further test whether Nrf2 plays a critical role in regu- theinvitrostudy(Fig.3).Importantly,allthesepathogenic
lating insulin-mediated glucose uptake, especially under changes were not observed in the hearts of the MT-TG
an oxidative status, we examined effects of forced acti- diabetic mice (Fig. 4B–D). These findings suggest that di-
vation of Nrf2 on the oxidative stress–induced activation abetic oxidative and nitrosative stress may induce ERK
of ERK as well as the subsequent insulin resistance in activation that downregulates Nrf2 expression.
adult cardiomyocytes. As shown in Fig. 3B, a forced activa- Consistent with the finding from diabetic mice, immu-
tion of Nrf2 by adenoviral overexpression of Nrf2 signifi- nochemical staining of human normal and diabetic hearts
cantlysuppressed H O -inducedERKactivityinHL-1cells alsoshowedthatNrf2proteinexpressionwasdramatically
2 2
with the presence of insulin. Most importantly, the forced suppressed in the diabetic failing hearts of both males
activation of Nrf2 enhanced the insulin-mediated glucose (Fig. 5A) and females (Fig. 5B), as compared with non-
uptakeinnon–H O -treatedcells,relativetothecontrolof diabetic hearts.
2 2
Ad-bGal overexpression and completely recovered insulin PharmacologicalactivationofNrf2attenuatesoxidative
sensitivity to stimulate glucose uptake in H O -treated stress–inducedinsulinresistanceinadultcardiomyocytes
2 2
cells (Fig. 3C). in vitro and in the diabetic heart in vivo. Werecently
Cardiac Nrf2 expression was downregulated in demonstrated that a novel synthetic triterpenoid deriv-
diabetic animals and patients. To explore a pathologi- ative, Dh404, is a potent Nrf2 activator and suppresses
cal relevance of Nrf2 in diabetic hearts, we examined oxidative stress in cardiomyocytes (20). We next deter-
cardiac Nrf2 expression in STZ-induced diabetic mice at 2 mined whether Dh404 could mimic the inhibitory effect
and 5 months after diabetes onset. The glucose levels of of Nrf2 overexpression on H O -induced insulin resis-
2 2
thesediabeticmicewere25.7662.14mmol/Lat2months tance in HL-1 cells. As expected, Dh404 strongly acti-
and 26.61 6 1.70 mmol/L at 5 months, respectively. As vated Nrf2 transcriptional activity and additively increased
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FIG.5.DownregulationofNrf2expressioninhumandiabetichearts.A:RepresentativesofNrf2stainingonleftventriculartissuesectionsof
normal(males,n=5;females,n=5)anddiabetic(males,n=4;females,n=2)humanhearts.RedisNrf2,andgreenisa-myosinheavychain.Blue
isnuclei.ThesemiquantificationofNrf2proteinlevelsbymeasuringmeanintegratedopticaldensity(IOD)ofeightrandomlychosenfieldsofeach
tissuesectionformales(B)andfemales(C)arepresented,respectively.Twosectionsofeachheartspecimenhavebeenanalyzed.*P<0.05vs.
normalgroup.(Ahigh-qualitydigitalrepresentationofthisfigureisavailableintheonlineissue.)
the insulin-induced Nrf2 transcriptional activity (Fig. 6A). decreased phosphorylation of Akt (Fig. 7D) and GSK-3b
Dh404 treatment did not change insulin-induced ERK (Fig. 7E). Activation of Nrf2 by Dh404 preserved the nor-
phosphorylation (Fig. 6B) and glucose uptake (Fig. 6C) mallevelsofAktandGSK-3bphosphorylationintheheart
but inhibited H O -induced ERK phosphorylation and of diabetic mice (Fig. 7D and E).
2 2
partially reversed H O -inhibited basal glucose uptake or
2 2
insulin-stimulated glucose uptake (Fig. 6B and C). These
results further support the notion that Nrf2 is a critical DISCUSSION
regulator of insulin-mediated glucose uptake in car- Oxidative stress is causative of insulin resistance in vari-
diomyocytes in vitro. ous cell types (2,6,7,18), which was confirmed by our
Tovalidatetheinvitrofinding,diabeticmice,inducedby present study using HL-1 adult cardiomyocytes treated
multiple-low doses of STZ, were treated with Dh404 at 10 with H O . Reportedly the stimulating effect of acute
2 2
mg/kg every other day for 2 weeks. Cardiac tissues were treatment with H O on glucose uptake has been recog-
2 2
collectedfromthesediabeticmicewithandwithoutDh404 nized for years in adipocytes (21). In fact, the cellular
treatmentandsubjecttoWesternblottinganalysisforNrf2 generation of ROS such as H O by insulin has been con-
2 2
and 3-NT. Dh404 treatment did not change glucose levels sidered as an integral part of the insulin signaling (21).
of these mice (control 8.40 6 1.20; diabetes: 25.40 6 2.89; TheseresultsemphasizethebiologicalsignificanceofROS
Dh404:7.9960.56;diabetes/Dh404:27.1261.22mmol/L). as signaling molecules, e.g., for the insulin-mediated glu-
Cardiac Nrf2 expression was increased in the hearts of cose metabolism in cardiomyocytes; however, chronic
either diabetic or Dh404-treated nondiabetic mice and accumulation of ROS causes oxidative stress, thereby
synergistically increased in the hearts of Dh404-treated contributing to the pathogenesis of various diseases such
diabetic mice (Fig. 7A). Diabetes significantly induces as cardiac dysfunction (9). Cardiac oxidative stress is of-
3-NT accumulation (Fig. 7B) and ERK1/2 phosphorylation ten observed coincident with insulin resistance, and there
(Fig. 7C), which were prevented by Dh404 treatment, is accumulating evidence that ROS and/or RNS mediates
suggesting that activation of Nrf2 expression by Dh404 in deleterious effects in the insulin-resistant heart although
the heart plays an important role in the prevention of di- the mechanisms by which oxidative stress induces insulin
abetes-induced cardiac oxidative and/or nitrosative dam- resistance remain not well understood.
age and ERK1/2 activation. As expected, diabetes-induced IncreasingevidenceindicatesthatERKactivationseems
oxidative damage and ERK1/2 activation is accompa- a causal factor for insulin resistance caused by oxidative
nied with downregulation of insulin signaling, shown by stress in cardiomyocytes. For instance, through activation
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FIG.6.AtherapeuticeffectofDh404oncardiomyocyteinsulinresistance.A:EffectofDh404oninsulin-inducedNrf2transcriptionalactivityin
HL-1cells.CellsweretransfectedasinFig.3.Thecellswerestimulatedwith100nmol/Lofinsulinand200nmol/LofDh404asindicatedfor12h.
Nrf2transcriptionalactivitywasmeasuredasinFig.3.EffectofDh404onoxidativestress–inducedERKphosphorylation(B)andglucoseuptake
inHL-1cells(C)isshown.CellswerepretreatedwithH O (100mmol/L)withorwithoutDh404(200nmol/L)in0.1%FBSmediumintheabsence
2 2
ofnorephineprinefor16handthenstimulatedwithorwithoutinsulin(100nmol/Lfor10min)asindicated.2-DGuptakeassayandWestern
blottingofphosphorylatedandtotalERKwereperformedasdescribedinRESEARCHDESIGNANDMETHODS.*P<0.05vs.normalgroup;#P<0.05vs.
insulinalone(A)orH O alone(C);†P<0.05vs.H O plusinsulin(C).
2 2 2 2
ofERK1/2,highlevelsofglucoseorangiotensinIIinduces production in both primary neonatal and adult car-
insulinresistanceinseveralconditions(19,22,23).Arecent diomyocytes from the heart of Nrf2 WT (C57BL/6 back-
study that showed that deficiency in the ERK1 protects ground) murine heart, whereas, in Nrf2 knockout cells,
leptin-deficient mice from insulin resistance without af- ROS was significantly higher under basal conditions and
fecting obesity further confirms the causative of ERK ac- further enhanced under high glucose exposure conditions
tivationintheinsulinresistance(24).Inthecurrentstudy, than that in WT cells (11). Concomitantly high glucose
we demonstrated for the first time that chronic treatment induced significantly higher levels of apoptosis at lower
of HL-1 cells with H O increased ERK phosphorylation concentrations and in shorter time in Nrf2 knockout cells
2 2
alongwithasignificantinhibition ofglucose uptake.More than in Nrf2 WT cells (11). To explore a pathological rel-
importantly we also demonstrated in the diabetic hearts evance of Nrf2 in diabetic hearts, we examined cardiac
thatupregulationofERK1/2alongwithnitrosativedamage Nrf2 expression in STZ-induced diabetic mice. As shown
is associated with downregulation of phosphorylation of in Figs. 4 and 7, cardiac Nrf2 expression was slightly in-
both Akt and GSK-3b, two key components of insulin creased in the heart of diabetic mice at 2 weeks (Fig. 7A)
signaling cascade. In the cells with chronic H O treat- and 2 months (Fig. 4A) but significantly decreased in the
2 2
ment,inhibitionofERK1/2activationbyU0126wasableto heartsofdiabeticmiceat5months(Fig.4B)afterdiabetes
partially reverse glucose uptake and insulin resistance onset.TheslightincreaseincardiacNrf2expressionatthe
(Fig. 2B). Findings that NAC prevents H O -mediated early stages of diabetes was consistent with our previous
2 2
activation of ERK1/2 in vitro (Fig. 1B) and MT prevents study (11) in which Nrf2 was increased in the heart of
diabetes-induced activation of ERK1/2 in vivo (Fig. 4D) diabetic mice with C57BL/6 background at 2 weeks after
also suggest that the activation of ERK1/2 is associated STZ-induced hyperglycemia. These results suggest that
with oxidative stress induced either by H O in vitro or Nrf2isadaptivelytryingtoremainfunctionaltoovercome
2 2
diabetes in vivo. diabetic damage at the early stage of diabetes. At the late
Nrf2 has been shown to negatively regulate oxidative stage of diabetes, however, cardiac antioxidant function
stress in the heart (15). To support this notion, our recent is further impaired, leading to a decrease in cardiac
study showed that high levels of glucose induced ROS Nrf2 expression. Importantly, the decreased cardiac Nrf2
630 DIABETES,VOL.60,FEBRUARY2011 diabetes.diabetesjournals.org
Y.TANANDASSOCIATES
FIG.7.Dh404cardiacpreventionofdiabeticoxidativestressandinhibitionofglucosemetabolism.Diabeteswasinducedbymultiplelowdosesof
STZasusedinFig.4.Diabeticandage-matchednondiabeticmiceweretreatedwithDh404at10mg/kgbodywteveryotherdayfromtheonsetof
diabetesfor2weeks.CardiacNrf2expression(A);nitrosativedamage,measuredby3-NT(B);ERKphosphorylation(C);Aktphosphorylation
(D);andGSK-3bphosphorylation(E)weredetectedbyWesternblot,respectively(n=5forcontrol[Ctrl],n=7forDM).b-Actin,totalERK,total
Akt,ortotalGSK-3bwasusedasloadingcontrol.*P<0.05vs.control;#P<0.05vs.DM;$P<0.05vs.Dh404.F:AworkinghypothesisofNrf2-
mediatedregulationofinsulinsensitivityinthecardiomyocytes.(Ahigh-qualitycolorrepresentationofthisfigureisavailableintheonlineissue.)
expression in the diabetic hearts was accompanied with cardiomyocytes from H O downregulation of Nrf2 in the
2 2
oxidative and nitrosative damage, shown by significantly invitrostudy(Fig.3),MTasapotentantioxidantprevents
increased 3-NT accumulation (Fig. 4C) and overactivation diabetic oxidative and nitrosative stress, leading to a sup-
of ERK function (Fig. 4D) as observed in the in vitro pression of ERK activation and a preservation of cardiac
study (Fig. 3). Peroxynitrite interacts with lipids, DNA, Nrf2 function, as illustrated in Fig. 7F, to protect the heart
and proteins via direct nitration or via indirect, radical- fromdiabetes-andLPS-induceddamage.Werecentlyalso
mediated mechanisms. These reactions trigger cellular showed that inactivationof GSK-3bbyoverexpressionof
responses ranging from subtle modulations of cell signal- cardiac MT gene results in an improvement of cardiac
ing to overwhelming oxidative and nitrosative injury. In glucose metabolism in the diabetic hearts along with
vivo, peroxynitrite generation represents a crucial patho- a significant suppression of cardiac nitrosative damage,
genic mechanism in conditions such as diabetic cardio- inflammation, and remodeling (14).
myopathy (12,25). In the current study, we demonstrated for the first time
WehavedemonstratedthatMT-TGmiceareresistantto that Nrf2 plays a critical role in insulin-mediated glucose
diabetes-induced oxidative and/or nitrosative damage and uptake, especially under an oxidative status. By Nrf2
development of cardiomyopathy (13,26). Here we further transcription reporter assay we found that insulin acti-
showed that MT-TG mice are also significantly resistant vated Nrf2 (Fig. 3A), which could be enhanced by in-
to diabetes-induced decrease in cardiac Nrf2 expression hibition of ERK1/2 with U0126, whereas it is blocked by
(Fig.4B)andincreasesin3-NTaccumulation(Fig.4C)and PI3K inhibition with LY294002 (Fig. 3A), indicating that
ERK activation (Fig. 4D) at the late stage of diabetes insulin activates Nrf2 via PI3K-mediated signaling that is
(5 months after diabetes onset). Our finding is consistent negatively regulatedbyERKinHL-1cells,asillustratedin
with a recent study in which MT-TG mice are resistant to Fig. 7F. Furthermore, forced activation of Nrf2 by adeno-
lipopolysaccharide (LPS)-induced cardiac oxidative stress, viraloverexpressionofNrf2significantlysuppressedinsulin-
ERK1/2 activation, and cardiac dysfunction as compared inducedERK activity in HL-1 cells with and without H O
2 2.
with WT mice (27). Therefore, like NAC that protects This finding suggests that Nrf2 activation could prevent
diabetes.diabetesjournals.org DIABETES,VOL.60,FEBRUARY2011 631
Nrf2ANDINSULINRESISTANCEINCARDIOMYOCYTES
oxidativestress–inducedERKactivityinadultcardiomyocytes REFERENCES
(Fig. 7F). Most importantly, the forced activation of Nrf2 1. Davidoff AJ, Davidson MB, Carmody MW, Davis ME, Ren J. Diabetic
further enhances insulin-mediated glucose uptake in non– cardiomyocyte dysfunction and myocyte insulin resistance: role of glu-
H O -treated cells and also completely recovered insulin- cose-inducedPKCactivity.MolCellBiochem2004;262:155–163
2 2
mediated glucose uptake in H O -treated cells (Fig. 3C). 2. Mellor KM, Ritchie RH, Delbridge LM. Reactive oxygen species and
The in vitro finding was furt2he2r supported by in vivo insulin-resistantcardiomyopathy.ClinExpPharmacolPhysiol2010;37:
222–228
results showing that diabetes inhibits cardiac Akt and
3. DoehnerW,RauchhausM,PonikowskiP,etal.Impairedinsulinsensitivity
GSK-3b phosphorylation, effects that were prevented by asanindependentriskfactorformortalityinpatientswithstablechronic
enhanced expression of Nrf2 with Dh404 (Fig. 7D and E). heartfailure.JAmCollCardiol2005;46:1019–1026
Therefore, these results not only establish a key role of 4. IngelssonE,SundströmJ,ArnlövJ,ZetheliusB,LindL.Insulinresistance
Nrf2 in the maintaining of insulin sensitivity but also raise andriskofcongestiveheartfailure.JAMA2005;294:334–341
5. Björnholm M, Kawano Y, Lehtihet M, Zierath JR. Insulin receptor sub-
a possibility that Nrf2 is a crucial player in the regulation
strate-1 phosphorylation and phosphatidylinositol 3-kinase activity in
cardiac insulin signaling in the cardiomyocytes and the
skeletal muscle from NIDDM subjects after in vivo insulin stimulation.
heart, as illustrated in Fig. 7F. These findings provide Diabetes1997;46:524–527
further support for other recent findings implying the link 6. RitchieRH.Evidenceforacausalroleofoxidativestressinthemyocardial
ofNrf2deficiencytooxidativestressandinsulinresistance complicationsofinsulinresistance.HeartLungCirc2009;18:11–18
in other cells and tissues (28,29). Therefore, diabetes-in- 7. Dong F, Fang CX, Yang X, Zhang X, Lopez FL, Ren J. Cardiac over-
ducedoxidativestresssuppressesNrf2expression(Fig.5) expressionofcatalaserescuescardiaccontractiledysfunctioninducedby
insulinresistance:roleofoxidativestress,proteincarbonylformationand
(30),andlackofNrf2expressioninthediabeticheartsand
insulinsensitivity.Diabetologia2006;49:1421–1433
kidney contributes to an insulin resistance and eventually
8. KocaSS,KaracaI,YavuzkirMF,etal.Insulinresistanceisrelatedwith
cardiac dysfunction (11) or renal dysfunction (31,32). oxidativestressinsystemiclupuserythematosus.AnadoluKardiyolDerg
In summary, our data provide several novel findings of 2009;9:23–28
redoxsignalingintheregulationofinsulinsensitivityinthe 9. Li J, Ichikawa T, Janicki JS, Cui T. Targeting the Nrf2 pathway against
cardiac cells in vitro and in vivo, as illustrated by Fig. 7F. cardiovasculardisease.ExpertOpinTherTargets2009;13:785–794
10. ChengX,Siow RC,Mann GE. Impairedredox signalingandantioxidant
First, oxidative stress plays a causative role in the de-
geneexpressioninendothelialcellsindiabetes:aroleformitochondria
velopment of insulin resistance. Second, ERK is a nega-
andthenuclearfactor-E2-relatedfactor2-kelch-likeECH-associatedpro-
tive regulator of glucose uptake and mediates oxidative tein 1 defense pathway. Antioxid Redox Signal. 20 August 2010 [Epub
stress–induced insulin resistance. Third, ERK-mediated aheadofprint]
suppression of Nrf2 activity is linked to the oxidative 11. HeX,KanH,CaiL,MaQ.Nrf2iscriticalindefenseagainsthighglucose-
stress–induced insulin resistance. Fourth, Nrf2 expression inducedoxidativedamageincardiomyocytes.JMolCellCardiol2009;46:
is upregulated at early stage of diabetic heart but signifi- 47–58
12. CaiL,WangJ,LiY,etal.Inhibitionofsuperoxidegenerationandasso-
cantly downregulated at the late stage in diabetic hearts.
ciatednitrosativedamageisinvolvedinmetallothioneinpreventionofdi-
Fifth, Nrf2 activation suppresses the oxidative stress– abeticcardiomyopathy.Diabetes2005;54:1829–1837
induced ERK activity and reverses the oxidative stress– 13. Cai L, Wang Y, Zhou G, et al. Attenuation by metallothionein of early
induced insulin resistance. Finally, antioxidants such as cardiaccelldeathviasuppressionofmitochondrialoxidativestressresults
NAC and MT can prevent oxidative stress–induced ERK in a prevention of diabetic cardiomyopathy. J Am Coll Cardiol 2006;48:
activation and Nrf2 downregulation. These results sug- 1688–1697
14. Wang Y, Feng W, Xue W, et al. Inactivation of GSK-3beta by metal-
gest that Nrf2 plays a critical role in regulating insulin
lothionein prevents diabetes-related changes in cardiac energy metabo-
sensitivity in the heart, and targeting Nrf2 might provide lism,inflammation,nitrosativedamage,andremodeling.Diabetes2009;58:
a novel therapeutic approach for the treatment of cardiac 1391–1402
insulinresistanceanddiabeticcardiomyopathy.Importantly 15. Li J, Ichikawa T, Villacorta L, et al. Nrf2 protects against maladaptive
a close structural analog of Dh404 that also potently in- cardiacresponsestohemodynamicstress.ArteriosclerThrombVascBiol
duces Nrf2, bardoxolone methyl (CDDO-Me; RTA 402), is 2009;29:1843–1850
in a pivotal clinical study in patients with type 2 diabetes 16. Claycomb WC,LansonNAJr,Stallworth BS,etal.HL-1cells:acardiac
muscle cell line that contracts and retains phenotypic characteristics of
mellitus with advanced kidney disease to assess changes
theadultcardiomyocyte.ProcNatlAcadSciUSA1998;95:2979–2984
in renal function and glucose control (33,34).
17. Ichikawa T, Li J, Nagarkatti P, et al. American ginseng preferentially
suppresses STAT/iNOS signaling in activated macrophages. J Ethno-
ACKNOWLEDGMENTS pharmacol2009;125:145–150
This study was supported in part by the American Heart 18. HoustisN,RosenED,LanderES.Reactiveoxygenspecieshaveacausal
AssociationasaBeginningGrantinAid(BGIA)(0865101E roleinmultipleformsofinsulinresistance.Nature2006;440:944–948
19. AlfaranoC,SartianiL,NedianiC,etal.Functionalcouplingofangiotensin
to T.C.), the American Diabetes Association as a Career
IItype1receptorwithinsulinresistanceofenergysubstrateuptakesin
Development Award (05-07-CD-02 to L.C.), a Starting-Up immortalizedcardiomyocytes(HL-1cells).BrJPharmacol2008;153:907–
FundforChinese-AmericanResearchInstituteforDiabetic 914
ComplicationsfromWenzhouMedicalCollege(toX.L.and 20. IchikawaT,LiJ,MeyerCJ,JanickiJS,HanninkM,CuiT.Dihydro-CDDO-
L.C.), and a Zhejiang Provincial Extremely Key Subject trifluoroethylamide(dh404),anovelNrf2activator,suppressesoxidative
BuildingProject“PharmacologyandBiochemicalPharma- stressincardiomyocytes.PLoSONE2009;4:e8391
ceutics 2009” (to X.L. and L.C.). 21. GoldsteinBJ,MahadevK,WuX,ZhuL,MotoshimaH.Roleofinsulin-in-
ducedreactiveoxygenspeciesintheinsulinsignalingpathway.Antioxid
C.J.M. is employed by Reata Pharmaceuticals. No other RedoxSignal2005;7:1021–1031
potential conflicts of interest relevant to this article were 22. IzawaY,YoshizumiM,FujitaY,etal.ERK1/2activationbyangiotensinII
reported. inhibitsinsulin-inducedglucoseuptakeinvascularsmoothmusclecells.
Y.T. and T.I. researched data and reviewed and edited ExpCellRes2005;308:291–299
the article. J.L., Q.S., H.Y., X.C., C.S.G., and C.J.M. 23. NazariH,TakahashiA,HaradaN,etal.AngiotensinIIinhibitsinsulin-induced
researched data. X.L. contributed initial discussion of the actinstressfiberformationandglucoseuptakeviaERK1/2.JMedInvest2007;
54:19–27
project and reviewed the article. L.C. and T.C. wrote the
24. Jager J, Corcelle V, Gremeaux T, et al. Deficiency in the extracellular
article, contributed to discussion, and reviewed and edited signal-regulated kinase 1 (ERK1) protects leptin-deficient mice from in-
the article. sulinresistancewithoutaffectingobesity.Diabetologia2011;54:180–189
632 DIABETES,VOL.60,FEBRUARY2011 diabetes.diabetesjournals.org
Y.TANANDASSOCIATES
25. PacherP,BeckmanJS,LiaudetL.Nitricoxideandperoxynitriteinhealth 30. MorrisonCD,PistellPJ,IngramDK,etal.Highfatdietincreaseshippo-
anddisease.PhysiolRev2007;87:315–424 campal oxidative stress and cognitive impairment in aged mice: im-
26. Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ. Hyperglycemia-induced plicationsfordecreasedNrf2signaling.JNeurochem2010;114:1581–1589
apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated 31. YohK,HirayamaA,IshizakiK,etal.Hyperglycemiainducesoxidativeand
caspase-3activationpathway.Diabetes2002;51:1938–1948 nitrosativestressandincreasesrenalfunctionalimpairmentinNrf2-deficient
27. Ceylan-Isik AF, Zhao P, Zhang B, Xiao X, Su G, Ren J. Cardiac over- mice.GenesCells2008;13:1159–1170
expressionofmetallothioneinrescuescardiaccontractiledysfunctionand 32. JiangT,HuangZ,LinY,ZhangZ,FangD,ZhangDD.Theprotectiveroleof
endoplasmic reticulum stress but not autophagy in sepsis. J Mol Cell Nrf2instreptozotocin-induceddiabeticnephropathy.Diabetes2010;59:850–860
Cardiol2010;48:367–378 33. Thimmulappa RK, FuchsRJ, Malhotra D,etal. Preclinical evaluation of
28. Beyer TA, Xu W, Teupser D, et al. Impaired liver regeneration in Nrf2 targetingtheNrf2pathwaybytriterpenoids(CDDO-ImandCDDO-Me)for
knockout mice: role of ROS-mediated insulin/IGF-1 resistance. EMBO J protectionfromLPS-inducedinflammatoryresponseandreactiveoxygen
2008;27:212–223 species in human peripheral blood mononuclear cells and neutrophils.
29. Aleksunes LM, Reisman SA, Yeager RL, Goedken MJ, Klaassen CD. Nu- AntioxidRedoxSignal2007;9:1963–1970
clearfactorerythroid2-relatedfactor2deletionimpairsglucosetolerance 34. YatesMS,TauchiM,KatsuokaF,etal.Pharmacodynamiccharacterization
andexacerbateshyperglycemiaintype1diabeticmice.JPharmacolExp ofchemopreventivetriterpenoidsasexceptionallypotentinducersofNrf2-
Ther2010;333:140–151 regulatedgenes.MolCancerTher2007;6:154–162
diabetes.diabetesjournals.org DIABETES,VOL.60,FEBRUARY2011 633