Biochimica et Biophysica Acta 1852 (2015) 343–352

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bbad is

Antioxidant catalase rescues against high fat diet-induced cardiacdysfunction via an IKKβ-AMPK-dependent regulation of autophagy☆

Lei Liang a,b,1, Xi-Ling Shou a,b,1, Hai-Kang Zhao c,1, Gu-qun Ren d, Jian-Bang Wang d, Xi-Hui Wang d,Wen-Ting Ai b, Jackie R. Maris e, Lindsay K. Hueckstaedt e, Ai-qun Ma b,⁎, Yingmei Zhang e,⁎⁎a Department of Cardiology, The People's Hospital of Shaanxi Province, Xi'an, Chinab Department of Cardiology, The First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Chinac Department of Neurosurgery, The Second Affiliated Hospital, Xi'an Medical University, Xi'an, Chinad Department of Cardiology, The Second Affiliated Hospital, Xi'an Medical University, Xi'an, Chinae Center for Cardiovascular Research and Alternative Medicine, University of Wyoming College of Health Sciences, Laramie, WY 82071, USA

☆ This article is part of a Special Issue entitled: Autophagcardiometabolic diseases.⁎ Correspondence to: A. Ma, Department of Cardiolog

School of Medicine, Xi'an Jiaotong University, 277 Yanta WTel.: +86 29 85323338.⁎⁎ Correspondence to: Y. Zhang, Center for CardiovascMedicine, University of Wyoming College of Health ScieTel.: +1 307 766 6133; fax: +1 307 766 2953.

E-mail addresses: maaiqun@medmail.com.cn (A. Ma),(Y. Zhang).

1 These authors contributed equally.

http://dx.doi.org/10.1016/j.bbadis.2014.06.0270925-4439/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 May 2014Received in revised form 10 June 2014Accepted 22 June 2014Available online 30 June 2014

Keywords:Fat dietCatalaseAutophagyCardiomyocyteIKKβAMPK

Autophagy, a conservative degradation process for long-lived and damaged proteins, participates in a variety ofbiological processes including obesity. However, the precisemechanism of action behind obesity-induced chang-es in autophagy still remains elusive. This study was designed to examine the role of the antioxidant catalase inhigh fat diet-induced changes in cardiac geometry and function as well as the underlying mechanism of actioninvolvedwith a focus on autophagy.Wild-type (WT) and transgenicmicewith cardiac overexpression of catalasewere fed low or high fat diet for 20 weeks prior to assessment ofmyocardial geometry and function. High fat dietintake triggered obesity, hyperinsulinemia, and hypertriglyceridemia, the effects of which were unaffected bycatalase transgene. Myocardial geometry and function were compromised with fat diet intake as manifestedby cardiac hypertrophy, enlarged left ventricular end systolic and diastolic diameters, fractional shortening, car-diomyocyte contractile capacity and intracellular Ca2+ mishandling, the effects of which were ameliorated bycatalase. High fat diet intake promoted reactive oxygen species production and suppressed autophagy in theheart, the effects of which were attenuated by catalase. High fat diet intake dampened phosphorylation of inhib-itor kappa B kinase β(IKKβ), AMP-activated protein kinase (AMPK) and tuberous sclerosis 2 (TSC2) whilepromoting phosphorylation of mTOR, the effects of which were ablated by catalase. In vitro study revealed thatpalmitic acid compromised cardiomyocyte autophagy and contractile function in a manner reminiscent of fatdiet intake, the effect ofwhichwas significantly alleviated by inhibition of IKKβ, activation of AMPK and inductionof autophagy. Taken together, our data revealed that the antioxidant catalase counteracts against high fat diet-induced cardiac geometric and functional anomalies possibly via an IKKβ-AMPK-dependent restoration of myo-cardial autophagy. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardio-metabolic diseases.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Autophagy is a conservative bulk degradation cellular process toclear long-lived proteins and organelles [1]. It is well conceived that

y and protein quality control in

y, The First Affiliated Hospital,est Road, 710061 Xi'an, China.

ular Research and Alternativences, Laramie, WY 82071, USA.

zhangym197951@126.com

autophagy plays a pivotal role in both physiological and pathologicalconditions such as development, immunity, cancer, nutrient deficiency,aging and neurodegenerative diseases [2–4]. A number of cell signalingmolecules have been identified participating in the regulation of au-tophagy. The serine/threonine kinase, mammalian target of rapamycin(mTOR) serves as perhaps the most predominant signaling moleculeto negatively regulate autophagy through mTOR complex 1 (mTORC1)[1,5,6]. It is known that mTORC1maymediate the regulation of autoph-agy in response to rapamycin, starvation, growth factors, amino acids,and energy excess, through a cascade of autophagy signaling includingmammalian Atg13, ULK1 and FIP200 [1,2,6]. Evidence further depicteda role for IκB kinase (IKK) in the regulation of autophagy through amechanism independently of nuclear factor κ B (NFκB) [3]. Althoughautophagy serves as a housekeeper under physiological condition tomaintain cardiac homeostasis [7], dysregulated especially excessive

344 L. Liang et al. / Biochimica et Biophysica Acta 1852 (2015) 343–352

autophagy may prove to be devastating under pathological conditionssuch as ischemia/reperfusion injury, dilated cardiomyopathy, alcoholiccardiomyopathy, myocardial hypertrophy and heart failure [8–10].Up-to-date, the precise role of autophagy and the underlying autophagyregulatory mechanisms remain essentially unclear in obesity-inducedmyocardial anomalies. Epidemiological survey has revealed that ap-proximately 35% of the US population are considered obese or over-weight [11–14]. Not surprisingly, uncorrected obesity is deemed anindependent risk factor for the pathogenesis of cardiovascular compli-cations leading to high morbidity and mortality in obese populations.Severalmechanisms have been postulated for the etiology of obesity co-morbidities including c-Jun N terminal kinase (JNK) activation [15–17],protein kinase C induction [18,19], oxidative stress [20,21], alteredsubstrate metabolism [22] and dysregulation of autophagy [23–25].To this end, the present work was designed to examine the possibleimpact of antioxidant catalase in the regulation of cardiac autophagyand homeostasis in the face of high fat diet-induced obesity. To eval-uate a possible role of the antioxidant catalase in the regulation ofmyocardial autophagy and function, wild-type (WT) and transgenicmice with cardiac-specific overexpression of catalase were fed lowor high fat diet for 20 weeks. Plasma profile, cardiac geometry,contractile function, cardiomyocyte contractile properties and intra-cellular Ca2+ homeostasis, production of reactive oxygen species(ROS) and autophagy were examined. The mTOR-dependent(IKKβ-AMPK-TSC-Rheb-mTOR) autophagy regulatory machinerywas examined in an effort to discern high fat diet- and catalase-induced changes in myocardial autophagy and function in WT andcatalase transgenic mice.

2. Methods and materials

2.1. Experimental animals and high fat diet feeding

The animal procedures described herewere approved by the Institu-tional Animal Care and Use Committee at the University of Wyoming(Laramie, WY, USA). Cardiac-specific catalase overexpression micewere used as described [26,27]. FVB littermates were used as wild-type. A primer pair derived from the MHC promoter and rat catalasecDNAwas used for identification of catalase transgene with the reversesequence of 5′-aat atc gtg ggt gac ctc aa-3′ and the forward sequence of5′-cag atg aag cag tgg aag ga-3′. All micewere housed in a temperature-controlled room under a 12 h/12 h-light/dark and allowed access tofood and tap water ad libitum. Five-week-old male WT and catalasetransgenic mice were subjected to a normal (10% calories from fat,

Table 1Biometric and myocardial contractile parameters of WT and catalase (CAT) transgenic mice fed

Parameter WT-low fat

Body weight (g) 26.0 ± 0.5Heart weight (mg) 143 ± 4Heart/body weight (mg/g) 5.50 ± 0.10Heart/tibial length (mg/mm) 8.21 ± 0.23Liver weight (g) 1.33 ± 0.02Liver/body weight (mg/g) 51.4 ± 1.0Kidney weight (g) 0.380 ± 0.010Kidney/body weight (mg/g) 15.2 ± 0.6Fasting blood glucose (mg/dl) 102.3 ± 3.9Plasma insulin (ng/ml) 0.254 ± 0.030Plasma triglycerides (mg/dl) 43.1 ± 4.0Cardiac catalase activity (μmol/min/mg protein) 36.8 ± 3.3Heart rate (bpm) 445 ± 26LV wall thickness (mm) 1.14 ± 0.15LVESD (mm) 1.09 ± 0.07LVEDD (mm) 2.03 ± 0.08Fractional shortening (%) 50.3 ± 1.4

HW = heart weight; Mean ± SEM, n = 11–12 mice per group, *p b 0.05 vs. WT-Low fat grou

3.91 kcal/g) or high fat (45% calories from fat, 4.83 kcal/g; ResearchDiets, New Brunswick, NJ, USA) diet feeding for 20 weeks [28]. Plasmalevels of glucose, insulin and triglycerides were measured using com-mercial kits.

2.2. Catalase activity

Myocardial tissues were homogenized in 1% Triton X-100 in anassay buffer described below using a variable-speed tissue tearer(Biospec Products, Racine, WI) at 20,000 rpm for 30 s on ice. Thehomogenates were centrifuged at 6000 ×g at 4 °C for 20 min. Thesupernatant was diluted with 1.5 volumes of the assay buffer(50 mM KH2PO4/50 mM Na2HPO4, pH 7.0). The enzyme activitywas determined by the method described previously [29].Briefly, in a cuvette 2 ml of sample was added and the reactionwas initiated by adding 1 ml 30 mM H2O2 and the change in absor-bance at 240 nm was monitored at 25 °C for 1 min. A portion of theremaining sample was used for protein determination. Specific ac-tivity is expressed as μmole H2O2/min/mg protein.

2.3. Echocardiographic assessment

Cardiac geometry and contractile function were evaluated in anes-thetized (ketamine 80 mg/kg and xylazine 12 mg/kg, i.p.) mice usinga 2-D guided M-mode echocardiography (Sonos 5500) equipped witha 15–6 MHz linear transducer. Diastolic and systolic left ventricular(LV) dimensions were recorded from M-mode images using methodadopted by the American Society of Echocardiography [30]. Fractionalshortening was calculated from end-diastolic diameter (EDD) andend-systolic diameter (ESD) using the equation of (EDD− ESD) / EDD[29].

2.4. Isolation of mouse cardiomyocytes and in vitro drug treatment

Hearts were mounted onto a temperature-controlled (37 °C)Langendorff system. After perfusion with a modified Tyrode's solution,the heart was digested with a Ca2+-free KHB buffer containing liberaseblendzyme 4 (Hoffmann-La Roche Inc., Indianapolis, IN) for 20min. Themodified Tyrode solution (pH 7.4) contained the following (in mM):NaCl 135, KCl 4.0, MgCl2 1.0, HEPES 10, NaH2PO4 0.33, glucose 10,butanedione monoxime 10, and the solution was gassed with 5% CO2/95% O2. The digested heart was then removed from the cannula andthe left ventricle was cut into small pieces in the modified Tyrode's so-lution. Tissue pieces were gently agitated and pellet of cells was

a low or high fat diet for 20 weeks.

WT-high fat CAT-low fat CAT-high fat

32.4 ± 0.8⁎ 25.8 ± 0.5 31.4 ± 0.6⁎

178 ± 4⁎ 143 ± 4 161 ± 3⁎,⁎⁎

5.49 ± 0.09 5.58 ± 0.22 5.16 ± 0.139.94 ± 0.33⁎ 8.09 ± 0.21 8.88 ± 0.15⁎,#

1.63 ± 0.03⁎ 1.30 ± 0.05 1.57 ± 0.03⁎

50.7 ± 1.6 50.5 ± 2.2 50.2 ± 1.50.480 ± 0.010⁎ 0.389 ± 0.012 0.488 ± 0.014⁎

14.9 ± 0.6 15.1 ± 0.5 15.6 ± 0.5100.1 ± 4.1 99.1 ± 4.1 97.0 ± 4.10.698 ± 0.073⁎ 0.351 ± 0.053 0.717 ± 0.076⁎

104.8 ± 5.0⁎ 46.9 ± 6.1 106.1 ± 7.7⁎

24.4 ± 3.3⁎ 184.9 ± 17.6⁎ 184.0 ± 17.2⁎

452 ± 18 453 ± 27 459 ± 221.14 ± 0.05 1.03 ± 0.06 1.02 ± 0.041.58 ± 0.19⁎ 1.10 ± 0.06 1.15 ± 0.06#

2.49 ± 0.18⁎ 2.09 ± 0.10 2.26 ± 0.10⁎,#

37.6 ± 3.9⁎ 47.2 ± 2.5 49.2 ± 0.6#

p, #p b 0.05 vs. WT-High fat group.

345L. Liang et al. / Biochimica et Biophysica Acta 1852 (2015) 343–352

resuspended. A yield of 60–70% viable rod-shaped cardiomyocyteswithclear sacromere striations was achieved. Only rod-shaped myocyteswith clear edges were selected for contractile studies [31]. To eluci-date the role for IKKβ, AMPK and autophagy in fat diet-induced re-sponse on cardiomyocyte autophagy and contractile function,cardiomyocytes from WT mice were treated with palmitic acid(75 μM) [32] for 6 h in the presence or absence of the IKKβ inhibitor(phosphorylation stimulator) sodium salicylate (5 mg/ml) [33], theAMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside(AICAR, 500 μM) [34,35] or the autophagy inducer rapamycin (20 μM)[34] prior to assessment of cardiomyocyte autophagy (using LC3II-to-LC3I ratio) and function.

2.5. Cell shortening/relengthening

Mechanical properties of cardiomyocytes were assessed using aSoftEdge MyoCam® system (IonOptix Corporation, Milton, MA, USA)[29]. Cells were placed in a Warner chamber mounted on the stage ofan inverted microscope (Olympus, IX-70) and superfused (~1 ml/min

Fig. 1. Cardiomyocyte mechanical function inWT and catalase transgenic mice fed low or highlength); C:maximal velocity of shortening (+dL/dt); D:maximal velocity of relengthening (−dcells from 4 mice per group, *p b 0.05 vs.WT-low fat group, #p b 0.05 vs.WT-high fat group.

at 25 °C) with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2,1 MgCl2, 10 glucose, and 10 HEPES, at pH 7.4. The cells were field stim-ulatedwith supra-threshold voltage at a frequency of 0.5 Hz and a dura-tion of 3 msec. The cardiomyocyte being studied was displayed on acomputer monitor using an IonOptix MyoCam camera. An IonOptixSoftEdge software was used to capture changes in cell length duringshortening and relengthening. Cell shortening and relengthening wereassessed using the following indices: peak shortening (PS), time-to-PS(TPS), time-to-90% relengthening (TR90), maximal velocities of shorten-ing (+dL/dt) and relengthening (−dL/dt).

2.6. Intracellular Ca2+ transients

Isolated cardiomyocytes were loaded with fura-2/AM (0.5 μM)for 10 min, and fluorescence intensity was recorded with a dual-excitation fluorescence photomultiplier system (IonOptix). Myocyteswere placed onto an Olympus IX-70 inverted microscope and imagedthrough a Fluor 40x oil objective. Cells were exposed to light emittedby a 75-W lamp and passed through either a 360- or a 380-nm filter

fat diet for 20 weeks. A: Resting cell length; B: peak shortening (normalized to resting cellL/dt); E: time-to-PS (TPS); and F: time-to-90% relengthening (TR90). Mean± SEM, n=94

346 L. Liang et al. / Biochimica et Biophysica Acta 1852 (2015) 343–352

while being stimulated to contract at 0.5 Hz. Fluorescence emissionswere detected between 480 and 520 nm, and qualitative change infura-2 fluorescence intensity was inferred from the fura-2 fluorescenceintensity ratio at the 2wavelengths (360/380). Fluorescence decay timewas calculated as an indicator of intracellular Ca2+ clearing [36].

2.7. Histological examination

Following anesthesia, hearts were excised and immediately placedin 10% neutral-buffered formalin at room temperature for 24 h after abrief rinse with PBS. The specimen was embedded in paraffin, cut in5-μm sections and stained with hematoxylin and eosin (H&E). Cardio-myocyte cross-sectional areas were calculated on a digital microscope(×400) using the Image J (version 1.34S) software [37].

2.8. Generation of intracellular reactive oxygen species (ROS)

To evaluate the generation of myocardial ROS, freshly frozen leftventricular myocardium (10-μm slices) was incubated for 1 h at 37 °Cwith 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate(DCF, 4 μM,Molecular Probes, Eugene, OR, USA) [38]. Tissueswere sam-pled using an Olympus BX-51 microscope equipped with an OlympusMagnaFire™ SP digital camera and ImagePro image analysis software(Media Cybernetics, Silver Spring, MD, USA). Fluorescence was calibrat-ed with InSpeck microspheres (Molecular Probes).

2.9. Western blot analysis

Murine heart tissues or dispersed cardiomyocytes were homogenizedand sonicated in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mMNaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 0.1% sodium dodecyl sulfate(SDS), and a protease inhibitor cocktail. Samples were incubated withanti-Atg5, anti-Beclin-1, anti-LC3, anti-p62, anti-Rheb, anti-IKKβ, anti-

Fig. 2. Intracellular Ca2+ property in cardiomyocytes from WT and catalase transgenic micB: electrically-stimulated rise in FFI (ΔFFI); C: single exponential intracellular Ca2+ decay; anper group, *p b 0.05 vs. WT-low fat group, #p b 0.05 vs. WT-high fat group.

phosphorylated IKKβ (pIKKβ, Ser180), anti-AMPK, anti-phosphorylatedAMPK (pAMPK, Thr172), anti-mTOR, anti-phosphorylated mTOR (pmTOR,Ser2448), anti-TSC2, anti-phosphorylated TSC2 (pTSC2, Ser1387) and anti-GAPDH (as loading control) antibodies. The membranes were incubatedwith horseradish peroxidase (HRP)-coupled secondary antibodies. Afterimmunoblotting, the film was scanned and detected with a Bio-Rad Cali-bratedDensitometer and the intensity of immunoblot bandswas normal-ized to that of GAPDH [38].

2.10. Data analysis

Data were Mean ± SEM. Statistical significance (p b 0.05) was esti-mated by one-way analysis of variation (ANOVA) followed by a Tukey'stest for post hoc analysis.

3. Results

3.1. Effect of catalase overexpression on biometric properties andechocardiographic function following high fat diet feeding

Chronic high fat diet intake overtly increased body and organweights (heart, liver and kidney) aswell as heartweight-to-tibial lengthratio (although not organ-to-body weight ratio), the effects of whichwere unaffected by catalase overexpression. Catalase transgene didnot affect body or organ weights under low fat diet intake. Plasma glu-cose levels were comparable in all mouse groups. High fat diet feedingovertly elevated plasma levels of insulin and triglycerides. Catalasetransgene did not affect levels of insulin and triglycerides in either WTor catalasemice. High fat diet intake significantly suppressedmyocardi-al catalase activity, the effect of which was masked by cardiac catalaseoverexpression. Echocardiographic analysis revealed that high fat dietfeeding significantly decreased fractional shortening as well as in-creased LV end-diastolic diameter (LVEDD), LV end-systolic diameter

e fed low or high fat diet for 20 weeks. A: Resting fura-2 fluorescence intensity (FFI);d D: bi-exponential intracellular Ca2+ decay. Mean ± SEM, n = 56–57 cells from 4 mice

347L. Liang et al. / Biochimica et Biophysica Acta 1852 (2015) 343–352

(LVESD) but not LVwall thickness. Although catalase overexpression it-self failed to elicit any notable effect on these echocardiographic param-eters, it significantly attenuated high fat diet-inducedechocardiographic changes (Table 1).

3.2. Effect of catalase overexpression on cardiomyocyte mechanics followinghigh fat diet feeding

To evaluate the effect of catalase transgene on high fat diet-inducedcardiac responses, cardiomyocyte mechanical function was assessed inWT or catalase transgenic mice with low or high fat diet intake. Ourdata revealed comparable resting cell length in cardiomyocytes fromWT and catalase transgenic mice. High fat feeding for 20 weeks signifi-cantly suppressed peak shortening (PS), maximum velocity of shorten-ing/relengthening (±dL/dt) and prolonged time-to-90% relengthening(TR90) without affecting time-to-PS (TPS). Although catalase transgeneitself did not alter the mechanical parameters tested, it ablated high fatdiet-induced cardiomyocyte mechanical changes (Fig. 1). To examinethe role of intracellular Ca2+ homeostasis in catalase overexpressionand/or high fat diet-induced cardiac mechanical responses, intracellularCa2+ handling was evaluated using fura-2 fluorescence technique.Our data revealed that high fat diet feeding significantly decreasedelectrically-stimulated rise in intracellular Ca2+ levels (ΔFFI) andprolonged intracellular Ca2+ decay without affecting the resting intracel-lular Ca2+, the effects of which were ablated by catalase overexpression.

Fig. 3. Histological analysis and ROS levels in hearts fromWT and catalase transgenic mice fedtransverse myocardial section (×400); B: quantitative analysis of cardiomyocyte cross-seSEM, n = 120–125 cells (panel B) or 14–16 fields (panel C) from 4 mice per group, *p b

Catalase overexpression itself did not affect intracellular Ca2+ indicestested (Fig. 2).

3.3. Effect of catalase overexpression on cardiac hypertrophy and ROSproduction following high fat diet feeding

Consistent with the increased heart weight following high fat dietfeeding, cardiomyocyte cross-sectional area was significantly elevatedfollowing high fat diet feeding. Although catalase overexpression itselffailed to affect cardiomyocyte cross-sectional area, it attenuated highfat diet-induced rise in cardiomyocyte cross-sectional area (Fig. 3A–B).Our data further demonstrated elevated ROS production followinghigh fat diet feeding. Although catalase overexpression itself failed toaffect myocardial ROS production, it significantly attenuated high fatdiet-induced rise in ROS production (Fig. 3C).

3.4. Effect of catalase overexpression on autophagy and autophagy signalingfollowing high fat diet feeding

To elucidate possible mechanisms of action behind catalase trans-gene and/or high fat diet-induced cardiac derangements, autophagyand autophagy regulatory signaling molecules were examined. Ourdata depicted that fat diet intake significantly suppressed myocardialautophagy (Atg5 and LC3II-to-LC3I ratio although not Beclin-1) and au-tophagy flux (elevated autophagy adaptor protein p62) along with the

low or high fat diet for 20 weeks. A: Representative H&E staining micrographs displayingctional (transverse) area; and C: myocardial ROS levels using DCF staining. Mean ±0.05 vs. WT-low fat group, #p b 0.05 vs. WT-high fat group.

348 L. Liang et al. / Biochimica et Biophysica Acta 1852 (2015) 343–352

upregulated critical negative regulator of autophagy Rheb. Catalasetransgene itself did not affect myocardial autophagy or autophagy fluxalthough it significantly attenuated high fat diet-induced changes in au-tophagy (Atg5 and LC3 ratio) and Rheb but not p62 accumulation(Fig. 4).

Our observation revealed that high fat diet intake overtly suppressedphosphorylation (absolute or normalized value) of IKKβ, AMPK andTSC2 while promoting the phosphorylation of mTOR. Although catalasetransgene failed to affect phosphorylation of these autophagy signalingmolecules in low fat-fed mice, it ablated high fat intake-induced chang-es in the phosphorylation of IKKβ, AMPK, TSC2 and mTOR (Fig. 5). Nei-ther high fat diet nor catalase overexpression affected pan proteinexpression of IKKβ, AMPK, TSC2 and mTOR (data not shown).

3.5. Effects of IKKβ inhibition as well as activation of AMPK and autophagyon palmitic acid-induced changes in cardiomyocyte autophagy andcontractile function

To elucidate a role for IKKβ and AMPK in fat diet intake-induced car-diac dysfunction, cardiomyocytes fromWTmice (fed low fat diet) weretreated with palmitic acid (75 μM) in the presence and absence of theautophagy inducer rapamycin, the IKKβ inhibitor salicylate and AMPK

Fig. 4. Changes in autophagymarkers in hearts fromWT and catalase transgenic mice fed low oratio, p62, Rheb and GAPDH (loading control) using specific antibodies; B: Beclin-1; C: Atg5; D:group, #p b 0.05 vs. WT-high fat group.

activator AICAR. Our data shown in Fig. 6 revealed that palmitic acidovertly suppressed cardiomyocyte autophagy and contractile functionas manifested by decreased LC3II-to-LC3I ratio, PS, ±dL/dt, andprolonged TR90 without affecting TPS. Interestingly, palmitic acid-induced changes in cardiomyocyte autophagy and contractile functionwere significantly alleviated or mitigated by rapamycin, AICAR and sa-licylate. Little changes in cardiomyocyte autophagy and function werenoted for these pharmacological activators. Neither palmitic acid northe pharmacological regulators tested affected resting cardiomyocytecell length (data not shown).

4. Discussion

Although a number ofmechanisms have been speculated for high fatdiet-induced cardiac geometric and contractile changes including oxi-dative stress, lipotoxicity, mitochondrial damage, and intracellularCa2+ mishandling [15–17], the precise mechanism(s) in particular theinterplay among these concurrent contributing factors behind fat diet-induced obesity cardiomyopathy remains elusive. Our present study re-vealed that the antioxidant catalase significantly attenuated fat diet-induced cardiac geometric and contractile anomalies along with sup-pressed autophagy. Involvement of autophagy in high fat diet-induced

r high fat diet for 20 weeks. A: Representative gel blots for levels of Atg5, Beclin-1, LC3II/ILC3I/II; E: p62; and F: Rheb.Mean± SEM, n=5–7mice per group, *p b 0.05 vs.WT-low fat

Fig. 5.Changes in autophagy regulatory signalingmolecules in hearts fromWT and catalase transgenicmice fed low or high fat diet for 20 weeks. A: phosphorylated IKKβ (pIKKβ)-to-IKKβratio; B: phosphorylated AMPK (pAMPK)-to-AMPK ratio; C: phosphorylated TSC2 (pTSC2)-to-TSC2 ratio; and D: phosphorylated mTOR (pmTOR)-to-mTOR ratio. Insets: Representativegel blots for levels of total or phosphorylated IKKβ, AMPK, TSC2 andmTOR (GAPDH used as loading control) using specific antibodies; Mean± SEM, n= 5–7mice per group, *p b 0.05 vs.WT-low fat group, #p b 0.05 vs. WT-high fat group.

349L. Liang et al. / Biochimica et Biophysica Acta 1852 (2015) 343–352

cardiac contractile dysfunction was further consolidated by the benefi-cial effects from autophagy induction and AMPK activation againstpalmitic acid-induced cardiomyocyte functional anomalies. Our datafurther depicted that IKKβ inhibition protects against palmitic acid-induced cardiomyocyte autophagy and contractile defect, suggesting arole for IKKβ in fat diet-induced cardiac dysfunction. Taken together,these results have collectively prompted for a likelihood action of anti-oxidant and autophagy induction in the regulation of myocardial geom-etry and function in fat diet-induced obesity and more importantly, thetherapeutic potential of autophagy in obesity cardiac complications.

High fat diet intake triggers hyperinsulinemia, dyslipidemia, insulinresistance and onset of type 2 diabetes [23,37,39], all of which aremajorrisk factors for myocardial remodeling and dysfunction via sympatheticoveractivation, renin–angiotensin stimulation or intrinsic myopathicchanges [40]. Our high fat feeding regimen elicited elevated plasmamarkers for insulin, and triglycerides in the absence of full-blown diabe-tes as evidenced by unchanged blood glucose. More importantly, ourdata revealed decreased myocardial contractile capacity in fat diet-induced obese hearts, in line with earlier reports of compromisedmyo-cardial function and geometry in diet-induced obesity [15–17]. Interest-ingly, fat diet-induced cardiac remodeling (heart mass, heart size, andcardiomyocyte cross-sectional area) and contractile dysfunction werealleviated by the antioxidant catalase, favoring a permissive role for

oxidative stress in fat diet-induced obesity cardiomyopathy. The cata-lase enzyme did not exert any discernable “systemic effect” on bodyweight, glucose metabolism, as well as plasma insulin and triglyceridelevels in the face of fat diet intake, suggesting that catalase-induced ben-eficial effect is not secondary to changes in systemic metabolic profile.

Our experimental data revealed that catalase ameliorated diet-induced obesity-associated suppression of autophagy as evidenced byAtg5, LC3II-to-LC3I ratio and rise in the negative regulator of autophagyRheb but not accumulation of the autophagy adaptor protein p62. Thisis accompanied by rescued phosphorylation of IKKβ, APMK and TSC2along with the suppressed mTOR phosphorylation. These findingswere in line with the previous observation of impaired myocardial au-tophagy and autophagy flux (accumulation of p62) following high fatdiet intake [41]. The unchanged myocardial p62 levels between highfat diet-fedWT and catalasemice suggest an unlikely role for autophagyflux in catalase enzyme-offered protection of cardiac autophagy andcontractile function. Furthermore, the possible role of AMPK and au-tophagy in fat diet intake-induced cardiac anomalies was substantiatedby the fact that rapamycin and AMPK activation effectively negatedpalmitic acid-induced changes in cardiomyocyte autophagy and func-tion. These findings received further support from the IKKβ inhibition-elicited protective effects against palmitic acid-induced loss in cardio-myocyte autophagy and contractile capacity. These data favor the

Fig. 6. Effect of palmitic acid (PA) on cardiomyocyte autophagy and mechanical properties in the absence or presence of IKKβ inhibitor, AMPK activator or autophagy inducer.Cardiomyocytes from WT mice were incubated with palmitic acid (75 μM) for 6 h in the presence or absence of the IKKβ inhibitor (phosphorylation stimulator) sodium salicylate(5 mg/ml), the AMPK activator AICAR (500 μM) or the autophagy inducer rapamycin (20 μM) prior to assessment of cardiomyocyte function. A: Cardiomyocyte autophagy evaluatedusing LC3II-to-LC3I ratio, insets: representative gel blots for levels of LC3 and GAPDH (loading control) using specific antibodies; B: peak shortening (normalized to resting cell length);C:maximal velocity of shortening (+dL/dt); D:maximal velocity of relengthening (−dL/dt); E: time-to-PS (TPS); and F: time-to-90% relengthening (TR90). Mean± SEM, n=6 isolations(panel A) or 62 cells per group, *p b 0.05 vs. control group, #p b 0.05 vs. PA group.

350 L. Liang et al. / Biochimica et Biophysica Acta 1852 (2015) 343–352

notion that antioxidant catalase rescues against autophagy through amechanism involving IKKβ and AMPK to preserve cardiac homeostasisin fat diet-induced obesity. The ability of the antioxidant catalase to res-cue myocardial autophagy is consistent with the notion that oxidativestress or ROSmay promote excessive autophagy induction leading to un-desired changes in cardiac geometry and contractile function [42,43].However, given that catalase acts as an enzyme to detoxify the oxidativestress end product hydrogen peroxide (H2O2), our current study was un-able to elucidate the potential redox signaling pathway(s) responsible forthe regulation of autophagy en route to changes in cardiac structure andfunction. Recent evidence suggested that activation of NADPH oxidase 4in the endoplasmic reticulum may promote cardiomyocyte autophagyand survival during energy stress [44]. Further study is warranted toexamine the possible involvement of NADPH oxidase in fat diet-inducedinduction of autophagy in the heart.

Our data suggest that fat diet-induced obesity inhibits autophagypossibly via an IKKβ-AMPK-dependent mechanism. Loss of myocardialAMPK activation was reported in obesity [28,45]. AMPK negative regu-lates mTOR via a TSC2-mediated inhibition of Rheb, as supported by

our data of suppressed AMPK phosphorylation in conjunction with up-regulated Rheb following high fat diet intake. AMPK activation is wellknown to facilitate autophagy through a TSC-Rheb-mediated inhibitionof mTOR en route to the maintenance of cardiac homeostasis [46,47].Our present finding revealed dampened myocardial phosphorylationof IKKβ in associationwith decreased phosphorylation of the IKK down-stream signals AMPK-TSC2 in diet-induced obesity, the effect of whichwas ameliorated or mitigated by catalase enzyme. This finding is inline with the previous finding of suppressed IKKβ activity (phosphory-lation) under oxidative stress (such as accumulation of free radicals orfat diet intake in our current experimental setting) [48,49]. Theseobservations collectively provide a possible explanation for fat dietintake- and catalase-mediated regulation of autophagy through anIKKβ-AMPK-TSC-mTOR-dependent mechanism. A scheme is providedin Fig. 7 to summarize the proposed mechanism of action involved infat diet- and catalase-induced regulation of cardiac autophagy, geome-try and function. It should be pointed out that the involvement ofIKKβ-AMPK-TSC2-Rheb-mTOR pathway in the regulation of obesitycardiomyopathy received convincing support from our in vitro finding

Fig. 7. Schematic diagram depicting possible mechanism(s) involved in high fat dietintake- and catalase overexpression-induced changes in cardiac geometry, intracellularCa2+ homeostasis and contractile function. High fat diet intake suppressed activation ofIKKβ. Suppressed IKKβ phosphorylation inhibits activation of AMPK and the TSC complex(through phosphorylation) to alleviate the inhibition on Rheb and mTOR, leading tointerrupted autophagy and autophagyflux. Catalase restores activation of IKKβ and subse-quently AMPK activation, mTOR inhibition to preserve autophagy regulation and cardiachomeostasis under fat diet intake. Arrows denote stimulation whereas the lines with a“T” ending represent inhibition. IKKβ: IκB kinase β subunit; AMPK: AMP-activation pro-tein kinase; TSC: tuber sclerosis complex; mTOR: mammalian target of rapamycin.

351L. Liang et al. / Biochimica et Biophysica Acta 1852 (2015) 343–352

that salicylate reversed palmitic acid-induced cardiomyocyte dysfunc-tion. Last but not the least, it is noteworthy that possible contributionfrom other autophagy regulatory machineries such as JNK-mediatedphosphorylation of Bcl-2 to disengage the association between Bcl-2and the autophagy protein Beclin-1 [50] cannot be excluded at thistime. Nonetheless, our data indicated unchanged levels of Beclin-1,not favoring involvement of mTOR-independent regulation of autopha-gy although future study is needed to consolidate possible involvement(or non-involvement) of other autophagy regulatory pathways.

In conclusion, our finding suggests that catalase enzymemay rescueagainst diet-induced cardiac geometric and contractile anomalies. Thesefindings support a pivotal role for oxidative stress in the regulation ofcardiac autophagy en route to cardiac homeostasis in fat diet-inducedobesity. Our data favor the notion that catalase rescues myocardialautophagy possibly via an IKKβ-AMPK-TSC-Rheb-mTOR mechanism infat diet-induced obesity (Fig. 7). Although loss of the highly conservedself-repair autophagy capacity is well known in obesity [23,41], ourdata suggest a beneficial role for antioxidant in the restoration of au-tophagy. These findings support the beneficial role for dietary polyphe-nol antioxidants such as green tea catechins, resveratrol and curcuminin the management of obesity and obesity-related complications [51].Despite the pronounced effect fromanimal obesity studies includingde-creases in body weight, fat mass and triglycerides through enhancingenergy expenditure and fat utilization, and modulating glucose hemo-stasis, limited human evidence is available for dietary polyphenol anti-oxidants to support the antiobesity impact possibly due to variationsin study designs and durations, and human subjects (age, gender, eth-nicity) [51]. To this end, a better understanding of antioxidant-offeredbeneficial effects in cardiac geometry and function through autophagyregulation should shed some lights towards a more effective therapeuticapplication of antioxidant in the management of obesity and associatedcardiac anomalies.

Competing interests

The authors have declared that no competing interests exist.

Acknowledgements

The catalase foundermicewere kindly provided by Professor Paul N.Epstein from University of Louisville (Louisville, KY, USA). This workwas supported in part by a project from NIH P20 GM103432, the Na-tional Natural Science Foundation of China (NSFC 81200102), and theNatural Science Funds of Shaan'xi Province (2014JM4166, 13-LC058and 2011KJXX68).

References

[1] B. Levine, G. Kroemer, Autophagy in the pathogenesis of disease, Cell 132 (2008)27–42.

[2] D.J. Klionsky, B.A. Schulman, Dynamic regulation of macroautophagy by distinctiveubiquitin-like proteins, Nat. Struct. Mol. Biol. 21 (2014) 336–345.

[3] S. Sarkar, V.I. Korolchuk, M. Renna, S. Imarisio, A. Fleming, A. Williams, M. Garcia-Arencibia, C. Rose, S. Luo, B.R. Underwood, G. Kroemer, C.J. O'Kane, D.C.Rubinsztein, Complex inhibitory effects of nitric oxide on autophagy, Mol. Cell 43(2011) 19–32.

[4] D.R. Green, B. Levine, To be or not to be? How selective autophagy and cell deathgovern cell fate, Cell 157 (2014) 65–75.

[5] A.M. Cuervo, E. Bergamini, U.T. Brunk, W. Droge, M. Ffrench, A. Terman, Autoph-agy and aging: the importance of maintaining “clean” cells, Autophagy 1 (2005)131–140.

[6] D.C. Rubinsztein, P. Codogno, B. Levine, Autophagy modulation as a potential thera-peutic target for diverse diseases, Nat. Rev. Drug Discov. 11 (2012) 709–730.

[7] M. Xie, C.R. Morales, S. Lavandero, J.A. Hill, Tuning flux: autophagy as a target ofheart disease therapy, Curr. Opin. Cardiol. 26 (2011) 216–222.

[8] Y. Matsui, S. Kyoi, H. Takagi, C.P. Hsu, N. Hariharan, T. Ago, S.F. Vatner, J. Sadoshima,Molecular mechanisms and physiological significance of autophagy duringmyocardialischemia and reperfusion, Autophagy 4 (2008) 409–415.

[9] G.R. De Meyer, G.W. De Keulenaer, W. Martinet, Role of autophagy in heart failureassociated with aging, Heart Fail. Rev. 15 (2010) 423–430.

[10] A. Sun, Y. Cheng, Y. Zhang, Q. Zhang, S. Wang, S. Tian, Y. Zou, K. Hu, J. Ren, J. Ge,Aldehyde dehydrogenase 2 ameliorates doxorubicin-induced myocardial dysfunc-tion through detoxification of 4-HNE and suppression of autophagy, J. Mol. Cell.Cardiol. 71 (2014) 92–104.

[11] S. Smyth, A. Heron, Diabetes and obesity: the twin epidemics, Nat. Med. 12 (2006)75–80.

[12] J.E. Fradkin, G.P. Rodgers, Diabetes research: a perspective from the National Insti-tute of Diabetes and Digestive and Kidney Diseases, Diabetes 62 (2013) 320–326.

[13] Increasing Prevalence of Diagnosed Diabetes—United States and Puerto Rico, 1995–2010, MMWR, Morbidity and Mortality Weekly Report, 61, 2012, pp. 918–921.

[14] Awareness of Prediabetes—United States, 2005–2010, MMWR, Morbidity and Mor-tality Weekly Report, 62, 2013, pp. 209–212.

[15] M.S. Han, D.Y. Jung, C. Morel, S.A. Lakhani, J.K. Kim, R.A. Flavell, R.J. Davis, JNKexpression by macrophages promotes obesity-induced insulin resistance andinflammation, Science 339 (2013) 218–222.

[16] D.C. Henstridge, C.R. Bruce, C.P. Pang, G.I. Lancaster, T.L. Allen, E. Estevez, T. Gardner,J.M. Weir, P.J. Meikle, K.S. Lam, A. Xu, N. Fujii, L.J. Goodyear, M.A. Febbraio, Skeletalmuscle-specific overproduction of constitutively activated c-Jun N-terminal kinase(JNK) induces insulin resistance in mice, Diabetologia 55 (2012) 2769–2778.

[17] V. Aguirre, T. Uchida, L. Yenush, R. Davis, M.F. White, The c-Jun NH(2)-terminalkinase promotes insulin resistance during association with insulin receptorsubstrate-1 and phosphorylation of Ser(307), J. Biol. Chem. 275 (2000) 9047–9054.

[18] L.O. Leiria, C. Sollon, M.C. Calixto, L. Lintomen, F.Z. Monica, G.F. Anhe, G. De Nucci, A.Zanesco, A.D. Grant, E. Antunes, Role of PKC and CaV1.2 in detrusor overactivity in amodel of obesity associated with insulin resistance in mice, PLoS ONE 7 (2012)e48507.

[19] X. Lu, J.S. Bean, G.S. Kassab, M.D. Rekhter, Protein kinase C inhibition amelioratesfunctional endothelial insulin resistance and vascular smooth muscle cell hypersen-sitivity to insulin in diabetic hypertensive rats, Cardiovasc. Diabetol. 10 (2011) 48.

[20] J.L. Evans, I.D. Goldfine, B.A. Maddux, G.M. Grodsky, Are oxidative stress-activatedsignaling pathways mediators of insulin resistance and beta-cell dysfunction? Dia-betes 52 (2003) 1–8.

[21] A. Bloch-Damti, N. Bashan, Proposed mechanisms for the induction of insulin resis-tance by oxidative stress, Antioxid. Redox Signal. 7 (2005) 1553–1567.

[22] P.M. Rindler, C.L. Crewe, J. Fernandes, M. Kinter, L.I. Szweda, Redox regulation of in-sulin sensitivity due to enhanced fatty acid utilization in the mitochondria, Am. J.Physiol. Heart Circ. Physiol. 305 (2013) H634–H643.

[23] S.Y. Ren, X. Xu, Role of autophagy in metabolic syndrome-associated heart disease,Biochim. Biophys. Acta 1852 (2015) 225–231.

[24] A.L. Levonen, B.G. Hill, E. Kansanen, J. Zhang, V.M. Darley-Usmar, Redox regulation ofantioxidants, autophagy, and the response to stress: implications for electrophiletherapeutics, Free Radic. Biol. Med. 71C (2014) 196–207.

[25] W. Quan, M.S. Lee, Role of autophagy in the control of body metabolism, Endocrinol.Metab. (Seoul) 28 (2013) 6–11.

352 L. Liang et al. / Biochimica et Biophysica Acta 1852 (2015) 343–352

[26] F. Dong, C.X. Fang, X. Yang, X. Zhang, F.L. Lopez, J. Ren, Cardiac overexpression of cat-alase rescues cardiac contractile dysfunction induced by insulin resistance: role ofoxidative stress, protein carbonyl formation and insulin sensitivity, Diabetologia49 (2006) 1421–1433.

[27] Y.J. Kang, Y. Chen, P.N. Epstein, Suppression of doxorubicin cardiotoxicity by overex-pression of catalase in the heart of transgenic mice, J. Biol. Chem. 271 (1996)12610–12616.

[28] S. Turdi, M.R. Kandadi, J. Zhao, A.F. Huff, M. Du, J. Ren, Deficiency in AMP-activatedprotein kinase exaggerates high fat diet-induced cardiac hypertrophy and contrac-tile dysfunction, J. Mol. Cell. Cardiol. 50 (2011) 712–722.

[29] W. Ge, Y. Zhang, X. Han, J. Ren, Cardiac-specific overexpression of catalase attenu-ates paraquat-induced myocardial geometric and contractile alteration: role of ERstress, Free Radic. Biol. Med. 49 (2010) 2068–2077.

[30] J. Ren, J.R. Privratsky, X. Yang, F. Dong, E.C. Carlson, Metallothionein alleviates gluta-thione depletion-induced oxidative cardiomyopathy in murine hearts, Crit. CareMed. 36 (2008) 2106–2116.

[31] T.A. Doser, S. Turdi, D.P. Thomas, P.N. Epstein, S.Y. Li, J. Ren, Transgenic overexpressionofaldehyde dehydrogenase-2 rescues chronic alcohol intake-induced myocardial hyper-trophy and contractile dysfunction, Circulation 119 (2009) 1941–1949.

[32] J.J. Luiken, F.A. van Nieuwenhoven, G. America, G.J. van der Vusse, J.F. Glatz, Uptakeandmetabolismof palmitate by isolated cardiacmyocytes fromadult rats: involvementof sarcolemmal proteins, J. Lipid Res. 38 (1997) 745–758.

[33] A. Khoshnan, J. Ko, S. Tescu, P. Brundin, P.H. Patterson, IKKalpha and IKKbeta regu-lation of DNA damage-induced cleavage of huntingtin, PLoS ONE 4 (2009) e5768.

[34] W. Ge, R. Guo, J. Ren, AMP-dependent kinase and autophagic flux are involved inaldehyde dehydrogenase-2-induced protection against cardiac toxicity of ethanol,Free Radic. Biol. Med. 51 (2011) 1736–1748.

[35] H.K. Nyblom, E. Sargsyan, P. Bergsten, AMP-activated protein kinase agonist dosedependently improves function and reduces apoptosis in glucotoxic beta-cellswithout changing triglyceride levels, J. Mol. Endocrinol. 41 (2008) 187–194.

[36] M.R. Kandadi, X. Yu, A.E. Frankel, J. Ren, Cardiac-specific catalase overexpression res-cues anthrax lethal toxin-induced cardiac contractile dysfunction: role of oxidativestress and autophagy, BMC Med. 10 (2012) 134.

[37] Y. Zhang, M. Yuan, K.M. Bradley, F. Dong, P. Anversa, J. Ren, Insulin-like growthfactor 1 alleviates high-fat diet-induced myocardial contractile dysfunction:role of insulin signaling and mitochondrial function, Hypertension 59 (2012)680–693.

[38] S. Turdi, X. Han, A.F. Huff, N.D. Roe, N. Hu, F. Gao, J. Ren, Cardiac-specific overexpres-sion of catalase attenuates lipopolysaccharide-induced myocardial contractiledysfunction: role of autophagy, Free Radic. Biol. Med. 53 (2012) 1327–1338.

[39] M.J. Munsters, W.H. Saris, Body weight regulation and obesity: dietary strategies toimprove the metabolic profile, Annu. Rev. Food Sci. Technol. 5 (2014) 39–51.

[40] R.H. Eckel, W.W. Barouch, A.G. Ershow, Report of the National Heart, Lung, andBlood Institute—National Institute of Diabetes and Digestive and Kidney DiseasesWorking Group on the pathophysiology of obesity-associated cardiovasculardisease, Circulation 105 (2002) 2923–2928.

[41] X. Xu, Y. Hua, S. Nair, Y. Zhang, J. Ren, Akt2 knockout preserves cardiac functionin high-fat diet-induced obesity by rescuing cardiac autophagosome maturation, J.Mol. Cell Biol. 5 (2013) 61–63.

[42] Y. Mei, M.D. Thompson, R.A. Cohen, X. Tong, Autophagy and oxidative stress in car-diovascular diseases, Biochim. Biophys. Acta 1852 (2015) 243–251.

[43] D.F. Dai, S.C. Johnson, J.J. Villarin, M.T. Chin, M. Nieves-Cintron, T. Chen, D.J.Marcinek, G.W. Dorn II, Y.J. Kang, T.A. Prolla, L.F. Santana, P.S. Rabinovitch, Mito-chondrial oxidative stress mediates angiotensin II-induced cardiac hypertrophyand Galphaq overexpression-induced heart failure, Circ. Res. 108 (2011) 837–846.

[44] S. Sciarretta, P. Zhai, D. Shao, D. Zablocki, N. Nagarajan, L.S. Terada, M. Volpe, J.Sadoshima, Activation of NADPH oxidase 4 in the endoplasmic reticulum promotescardiomyocyte autophagy and survival during energy stress through the protein ki-nase RNA-activated-like endoplasmic reticulum kinase/eukaryotic initiation factor2alpha/activating transcription factor 4 pathway, Circ. Res. 113 (2013) 1253–1264.

[45] C.R. Lindholm, R.L. Ertel, J.D. Bauwens, E.G. Schmuck, J.D. Mulligan, K.W. Saupe, Ahigh-fat diet decreases AMPK activity inmultiple tissues in the absence of hypergly-cemia or systemic inflammation in rats, J. Physiol. Biochem. 69 (2013) 165–175.

[46] H. Ma, R. Guo, L. Yu, Y. Zhang, J. Ren, Aldehyde dehydrogenase 2 (ALDH2) rescuesmyocardial ischaemia/reperfusion injury: role of autophagy paradox and toxic alde-hyde, Eur. Heart J. 32 (2011) 1025–1038.

[47] Y. Zhang, J. Ren, Autophagy in ALDH2-elicited cardioprotection against ischemicheart disease: slayer or savior? Autophagy 6 (2010) 1212–1213.

[48] C. Bodur, O. Kutuk, T. Tezil, H. Basaga, Inactivation of Bcl-2 through IkappaBkinase (IKK)-dependent phosphorylation mediates apoptosis upon exposure to4-hydroxynonenal (HNE), J. Cell. Physiol. 227 (2012) 3556–3565.

[49] P. Zhou, S. Gross, J.H. Liu, B.Y. Yu, L.L. Feng, J. Nolta, V. Sharma, D. Piwnica-Worms, S.X.Qiu, Flavokawain B, the hepatotoxic constituent from kava root, induces GSH-sensitive oxidative stress through modulation of IKK/NF-kappaB and MAPK signalingpathways, FASEB J. 24 (2010) 4722–4732.

[50] Z. Ni, B. Wang, X. Dai, W. Ding, T. Yang, X. Li, S. Lewin, L. Xu, J. Lian, F. He, HCC cellswith high levels of Bcl-2 are resistant to ABT-737 via activation of the ROS-JNK-autophagy pathway, Free Radic. Biol. Med. 70 (2014) 194–203.

[51] S. Wang, N. Moustaid-Moussa, L. Chen, H. Mo, A. Shastri, R. Su, P. Bapat, I. Kwun, C.L.Hen, Novel insights of dietary polyphenols and obesity, J. Nutr. Biochem. 25 (2014)1–18.