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Mol. Nutr. Food Res. 00, 0, 2017, 1600433 (1 of 12) 1600433DOI 10.1002/mnfr.201600433

RESEARCH ARTICLE

A mix of apple pomace polysaccharide improvesmitochondrial function and reduces oxidative stress inthe liver of high-fat diet-induced obese miceLei Chen1, Lei Liu2, Caixia Li1, ChengLi Hu2, Fan Su3, Run Liu1, Mengqi Zeng1, Daina Zhao1,Jiankang Liu1, Yurong Guo3∗ and Jiangang Long1

1 Center for Mitochondrial Biology and Medicine and Center for Translational Medicine, The Key Laboratory ofBiomedical Information Engineering of Ministry of Education, School of Life Science and Technology and FrontierInstitute of Science and Technology, Xi’an Jiaotong University, Xi’an, China

2 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, China3 College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, China

Received: May 23, 2016Revised: August 28, 2016

Accepted: August 31, 2016

Scope: Apple pomace polysaccharides (APP), a free radical scavenger, is one of the majorcompounds derived from apple pomace. However, whether APP has beneficial effects onmetabolic disorders is still unknown.Methods and results: In the present study, water-soluble APP was isolated from the pomaceof the locally abundant “Qinguan” apple and chemically characterized. Then, APP was orallyadministrated to high-fat diet (HFD)-induced obese mice. We found that APP significantlyreduced HFD-induced body weight gain and ameliorated HFD-induced hepatic metabolicdisorders and oxidative stress. In a palmitate-loaded HepG2 cell model, APP protected the cellsfrom palmitate-induced insulin resistance and loss of viability by suppressing mitochondrialreactive oxygen species and rescuing mitochondrial respiratory function.Conclusion: Our work suggests that APP, a promising bioactive food component, successfullyimproved obesity-associated hepatic metabolic disorder, most likely though the activation ofhepatic mitochondrial function and the suppression of mitochondria oxidative stress.

Keywords:Apple pomace polysaccharides / Hepatic metabolic disorder / High-fat diet / Mito-chondrial function / “Qinguan”

1 Introduction

China is the largest apple juice concentrate producer and sup-plier in the world, accounting for 60% of the global trade vol-ume [1]. During apple juice processing, one abundant wasteproduct is pomace [2], with approximately 1 million tons pro-duced every year in China. The pomace represents a rich

Correspondence: Jiangang LongE-mail: [email protected]

Abbreviations: ALT, alanine aminotransferase; APP, apple po-mace polysaccharide; AST, aspartate aminotransferase; CAT, fun-gal catalase; FAS, fatty acid synthase; HDL-C, high density lipopro-tein cholesterol; HFD, high-fat diet; HI, hepatosomatic index; HK,hexokinase; LDH, lactate dehydrogenase; LDL-C, LDL cholesterol;MDA, malondialdehyde; MMP, mitochondrial membrane poten-tial; MPO, myeloperoxidase; PK, pyruvate kinase; ROS, reactiveoxygen species; SOD, superoxide dismutase; TC, total choles-terol; TGs, triglycerides; T-AOC, total antioxidant capacity

source of potentially bioactive apple ingredients [3], includ-ing polyphenols and flavonoids [4]. However, a major classof phytochemicals commonly found in apple pomace is thepolysaccharides, which play an important role as free radicalscavengers in the prevention of oxidative damage in livingorganisms and can be exploited as novel antioxidants [5, 6].

Numerous epidemiological studies have indicated thatconsumption of apple and apple products is correlated witha lower prevalence of chronic diseases and diabetes [7–9],which is believed to be partially due to oxidative stressreduction, weight management, and improvement in insulinresistance [5, 10, 11]. Hepatic metabolic disorder is oneof the most common chronic liver diseases in the world[12–15]. It is closely related to the increased frequency ofobesity, insulin resistance, nonalcoholic fatty liver disease,

Colour Online: See the article online to view Figs. 1 and 5 in colour.∗Additional corresponding author: Yurong Guo E-mail: [email protected]

C© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com

1600433 (2 of 12) L. Chen et al. Mol. Nutr. Food Res. 00, 0, 2017, 1600433

and diabetes [16, 17]. The unprecedented urbanization ofChina has led to rapid changes in lifestyle and a consequentincrease in the prevalence of metabolic disorder. TheInvestigation Reports of Chinese Residents Nutrition andChronic Diseases (2015) revealed that 30.1% of adults and9.6% of children are overweight, 11.9% of adults and 6.4%of children are obese, 9.7% of adults have type 2 diabetesmellitus, and 25.2% of adults have high blood pressure(http://news.xinhuanet.com/health/2015-06/30/c_127968555.htm). The increasing prevalence of obesity, coupled withtype 2 diabetes mellitus and hypertension, has rendereda large proportion of the Chinese population at a risk fordeveloping metabolic disorder in the coming decades.

Polysaccharides are a major ingredient in apple pomace,but whether they affect hepatic metabolic disorder remains tobe explored. In the present study, we analyzed the monosac-charide profiles of apple pomace polysaccharides (APPs) iso-lated from “Qinguan” and demonstrated the protective ef-fects of APP against high-fat diet (HFD)-induced hepaticmetabolic disorder in mice. We further found that APP im-proved palmitate-induced mitochondrial dysfunction and in-sulin resistance in HepG2 cells.

2 Materials and methods

2.1 Materials and chemicals

The apple pomace used in this study was collected fromShannxi Normal University, Xi’an, Shaanxi province, China.Insulin, adiponectin, leptin, and fatty acid synthase (FAS)were measured using commercial ELISA kits accordingto the manufacturer’s protocols (Shanghai Guduo Biolog-ical Technology Co., Ltd., China). Detection kits for to-tal cholesterol (TC), triglycerides (TGs), LDL cholesterol(LDL-C), HDL cholesterol (HDL-C), total antioxidant capac-ity (T-AOC), fungal catalase (CAT), myeloperoxidase (MPO),hexokinase (HK), pyruvate kinase (PK), FAS, alanine amino-transferase (ALT), aspartate aminotransferase (AST), lactatedehydrogenase (LDH), superoxide dismutase (SOD), glu-tathione peroxidase, and malondialdehyde (MDA) were fromthe Nanjing Jiancheng Bioengineering Institute (Nanjing,China). Oligomycin, FCCP, and antimycin A were fromSigma (St. Louis, MO, USA). Anti-p-AKT and anti-AKT anti-bodies were purchased from Santa Cruz Biotechnology. Allother reg reagents were purchased from Invitrogen.

2.2 Extraction and purification of APP

Based on our previous report [18], APP was extracted and pu-rified, with some modifications. The apple pomace was driedat 55�C and crushed into a powder (60 mesh) by a disintegra-tor. The powder was extracted with 95% ethanol (1:10, w/v)and refluxed at 80�C for 8 h to remove impurities and smalllipophilic molecules. Subsequently, the powders were dried

and extracted with hot water (1:40, w/v) at 90�C for 2 h forthree cycles. The combined aqueous extract was concentratedto 25% of the original volume by a rotary vacuum evaporatorat 60�C and then centrifuged at 4500 × g for 20 min. Thesupernatant was collected and precipitated for three cycles byadding five times the volume of 95% ethanol at 4�C for 24 h.After centrifuging, the separated precipitate was completelydissolved in an appropriate volume of water and intensivelydialyzed for 3 days against ultrapure water (cutoff Mw 8000Da) to remove the small molecular compounds. The remain-ing protein was deproteinized using a freeze-thaw process,which was repeated ten times in a plastic bottle, followed byfiltration. The extracts were centrifuged at 3000 × g for 10min to remove insoluble material, and the supernatant waslyophilized in the freeze-dry apparatus (Sihuan Co., China)to obtain the refined APP, which was brown and fluffy.

2.3 Chemical characterization of the polysaccharides

The monosaccharide composition of APP was analyzed byHPLC as described in Lv et al. [18, 19]. Briefly, 20 mg of APPsample was hydrolyzed with 2 mL of 3 M trifluoroacetic acidat 100�C for 8 h in an ampoule (5 mL) sealed under a nitro-gen atmosphere to release constituent monosaccharides, andderivatization was then carried out with 5-Methyl-2-phenyl-1,2-dihydropyrazol-3-one (PMP). The analysis of PMP-labeledmonosaccharides was performed using a reversed-phaseHPLC column (4.6 �m id × 250 mm, 5 �m, Venusil, USA)on a Shimadzu LC-2010A HPLC system equipped with aUV–Vis detector and a Shimadzu Class-VP 6.1 chromatog-raphy workstation (Shimadzu, Japan). The mobile phase Awas ACN, and B was 3.3 mM TEA (pH 7.5), with a gradientelution of 95-95-90-90% B by a linear decrease from 0-5-8-30 min, respectively. The wavelength for UV detection was250 nm.

2.4 Animals and experimental design

Ninety Kunming mice (weight 18–22 g, half male and half fe-male, and approximately 45 days of age) were provided by theExperimental Animal Center, Lanzhou University (Lanzhou,China). The mice were housed in a temperature (22–28�C)-and humidity (60 ± 5%)-controlled animal room and main-tained on a 12-h light/12-h dark cycle (light from 06:00 a.m.to 06:00 p.m.) with food and water provided during the ex-periments. After 1 wk of acclimatization, the mice were ran-domly distributed into the following five groups: (i) controlmice fed a standard chow diet (12% kcal fat content; KEAO,Beijing, China); (ii) mice fed an HFD (45% kcal fat content;KEAO); (iii) mice fed an HFD and administered a daily oralgavage of low-dose APP (200 mg/kg/day); (iv) mice fed anHFD and administered a daily oral gavage of middle-doseAPP (400 mg/kg/day); and (v) mice fed an HFD and admin-istered a daily oral gavage of high-dose APP (800 mg/kg/day).After 30 days of feeding, the mice were fasted overnight and


http://news.xinhuanet.com/health/2015-06/30/c_127968555.htmhttp://news.xinhuanet.com/health/2015-06/30/c_127968555.htm

Mol. Nutr. Food Res. 00, 0, 2017, 1600433 (3 of 12) 1600433

sacrificed. All procedures were performed in accordance withthe Regulations of Experimental Animal Administration is-sued by the State Committee of Science and Technology ofthe People’s Republic of China.

2.5 Blood samples and biochemical measurements

At the end of the experimental period, all mice were fullyanesthetized by the inhalation of ether, weighed, and thensacrificed to obtain blood and liver samples. The blood sam-ples were centrifuged at 1200 × g for 20 min and stored at 4�C,while the livers were frozen at −80�C. On the basis of bodyweight and corresponding liver weight of every mouse, wecalculated the hepatosomatic index (HI) according to the fol-lowing formula: HI = liver weight/body weight × 100%. Thelevels of TG, TC, LDL-C, HDL-C, T-AOC, AST, ALT, MDA,LDH, GSH-PX, SOD, CAT, MPO, HK, and PK were analyzedusing an automated biochemical analyzer (Nanjing JianchengBioengineering Institute, China). Serum levels of insulin,adiponectin, leptin, and FAS were measured using commer-cial ELISA kits according to the manufacturer’s protocols(Shanghai Guduo Biological Technology Co., Ltd., China).

2.6 HepG2 cell culture

HepG2 cells were cultured in DMEM supplemented with10% fetal calf serum, 100 U/mL penicillin G sodium, and 100�g/mL streptomycin sulfate at 37�C in a humidified incubatorwith 5% CO2, and the experiments were initiated once thecells reached 70% confluence [20].

2.7 Assessment of cell viability

The effect of APP on the viability of HepG2 cells was analyzedin vitro using MTT assays [21]. The cells were seeded at adensity of 5 × 104 cells/mL in 96-well plates and incubatedwith APP at concentrations of 0, 1, 2, 5, 10, 25, 50, 100, and200 �g/mL for 24 h. After incubation, 100 �L of MTT-DMEMsolution (1:9) was added to each well. The plates were furtherincubated for 4 h followed by addition of 100 �L of DMSO toeach well. The absorbance was measured at 490 nm on theBio-Rad model 680 Microplate Reader (PA, USA).

2.8 JC-1 assay for mitochondrial membranepotential

HepG2 were cultured at a density of 5 × 104 cells/mLin 96-well plates. After treatment, mitochondrial mem-brane potential (MMP) was measured with the lipophiliccationic probe 5, 5′, 6, 6′-terachloro-1, 1′, 3, 3′-tetraethyl-imidacarbocyanineiodide (JC-1). Cells were washed with PBSonce after JC-1 staining and scanned with a microplate fluo-

rometer (Fluoroskan Ascent, Thermo Fisher Scientific, Inc.)at 488 nm excitation and 538 and 590 nm emission wave-lengths to measure green and red JC-1 fluorescence, respec-tively. The red/green fluorescence intensity ratio reflects theMMP [22].

2.9 Determination of reactive oxygen species

HepG2 were cultured at a density of 2 × 105 cells/mL in six-well plates. After treatment, the intracellular reactive oxygenspecies (ROS) generation was determined by measuring thefluorescence of 2′, 7′-dichlorofluorescein diacetate (DCFH2-DA). Briefly, DCFH2-DA at a final concentration of 10 �Mwas incubated with HepG2 cells in serum-free medium for30 min, and cells were washed twice and collected with PBS.After centrifugation at 1000 × g for 1 min at 4�C, cells weresuspended with PBS. Cells were analyzed by flow cytometry(BD Bioscience, Franklin Lakes, NJ, USA) [22].

2.10 Mitochondrial ROS measurement

HepG2 were cultured at a density of 5 × 104 cells/mL in 12-well plates. After treatment, the generation of mitochondrialROS was determined by the MitoSOX ۛ Red Mitochondrial Su-peroxide Indicator (Thermo Fisher, USA), and the mitochon-dria were assessed with MitoTracker ۚ Green FM (Invitrogen,USA). Briefly, MitoSOX ۛ and MitoTracker ۚ Green FM at a fi-nal concentration of 10 �M were incubated with HepG2 cellsin serum-free medium for 30 min. After washing with PBS,the cells were visualized by confocal microscopy (Zeiss, Jena,Germany).

2.11 Western blot analyses

Samples were lysed with Western and IP lysis buffer (Bey-otime). The lysates were homogenized, and the homogenateswere centrifuged at 13 000 × g for 15 min at 4�C. The super-natants were collected, and the protein concentrations weredetermined with pierceTM BCA protein assay kit (Thermo,USA). Equal aliquots (20 �g) of the protein were separatedby 10% SDS-PAGE, transferred to pure nitrocellulose mem-branes (PerkinElmer Life Sciences), and blocked with 5%nonfat milk in tris-buffered saline and tween 20 (TBST)buffer. The membranes were incubated with anti-AKT andanti-p-AKT (1:1000) antibodies at 4�C overnight. Then, themembranes were incubated with anti-rabbit secondary an-tibodies at room temperature for 1.5 h. Chemiluminescentdetection was performed using an ECL Western blotting de-tection kit (Thermo Fisher, Rockford, IL, USA). The resultswere analyzed by Quantity One software (Bio-Rad, Shanghai,China) to obtain the optical density ratio of the target pro-teins relative to glyceraldehyde-3-phosphate dehydrogenase(GAPDH).



Figure 1. The HPLC chromatograms of13 standard monosaccharides (A) andthe component monosaccharides re-leased from APP (B). Peaks: (1) fucose;(2) glucosamine; (3) rhamnose; (4) ara-binose; (5) galactosamine; (6) galac-tose; (7) glucose; (8) xylose; (9) man-nose; (10) fructose; (11) ribose; (12)galacturonic acid; and (13) glucuronicacid.

Table 1. The monosaccharides in APP

Monosaccharide Molar contents (%)

Arabinose 32.52Galacturonic acid 29.96Galactose 20.60Rhamnose 7.33Xylose 6.61Glucose 2.98

2.12 Cell oxygen consumption rate measurement

HepG2 cells were seeded at a density of 1.5 × 104 cells/mLin XF 24-well microplates (Seahorse Bioscience, Biller-ica, MA, USA). After treatment, oxygen consumption wasmeasured using extracellular flux analysis (Seahorse Bio-science). The final concentrations of the mitochondrial in-hibitors were 1 �M antimycin A, 0.5 �M Carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP), and 1 �Moligomycin. Basal respiration is the baseline oxygen con-sumption reading before the compounds are injected. Maxi-mal respiration represents the maximum oxygen consump-tion rate (OCR) measurement value after the FCCP injection.Spare respiratory capacity is calculated by recording the OCRresponse to FCCP and dividing that number by the basal res-piration to obtain a percentage. After detection, cell proteinwas calculated, and the OCR was adjusted accordingly.

2.13 Statistical analysis

Data are presented as the mean ± SD. Statistical analysis wasperformed using one-way ANOVA followed by an LSD posthoc analysis (Graphpad Prism 6, USA). In all comparisons,the level of significance was set at p < 0.05.

3 Results

3.1 Chemical characterization of APP

APP was extracted from apple pomace using a multistep pu-rification procedure, including hot-water extraction and re-peated ethanol precipitation, and the yield of APP was ap-proximately 5.13% w/w of the dried apple pomace. The totalcarbohydrate content of APP was determined to be 65.5% bythe phenol-H2SO4 assay. In addition, the APP did not reactwith the Folin–Ciocalteu reagent, suggesting that the smallmolecular phenolic compounds in APP had been removed bydialysis processing against distilled water in the purificationof the macromolecular polysaccharides.

Chromatographic analysis was employed to identify andquantify the major monosaccharides present in APP. Asshown in Fig. 1A, 13 PMP-labeled standard monosaccharideswere rapidly separated in 32 min. The peaks were identifiedin the order of fucose, glucosamine, rhamnose, arabinose,



Table 2. Effects of APP on body weight and HI of mice subjected to HFD treatment

HFD

Control 0 200 400 800 (mg/kg/day)

Initial body weight (g) 19.51 ± 0.30 19.62 ± 0.40 20.14 ± 0.20 20.06 ± 0.30 17.69 ± 0.40Final body weight (g) 32.23 ± 0.25 36.70 ± 0.34a) 33.81 ± 0.43b) 32.21 ± 0.32b) 31.88 ± 0.45b)Body weight gain (g) 12.72 ± 0.13 17.08 ± 0.24a) 13.57 ± 0.23b) 12.15 ± 0.32b) 14.19 ± 0.40b)Food intake (g) 1621.28 1541.57 1567.89 1583.86 1605.85HI (%) 4.61 ± 0.056 5.26 ± 0.078a) 4.87 ± 0.172b) 4.71 ± 0.183b) 4.49 ± 0.014c)

Date are shown as means ± SD (n = 18).a) p < 0.05, as compared with the normal group.b) p < 0.05, as compared with the high-fat group.c) p < 0.01, as compared with the high-fat group.

galactosamine, galactose, glucose, xylose, mannose, fructose,ribose, galacturonic acid, and glucuronic acid by matchingtheir retention times with those of monosaccharide standardsunder the same analytical conditions. The monosaccharidecompositions of APP are shown in Fig. 1B, and the quanti-fied constituents are shown in Table 1. APP was composedof arabinose, galacturonic acid, galactose, rhamnose, xylose,and glucose with molar percentages of 32.52, 29.96, 20.60,7.33, 6.61, and 2.98%, respectively.

3.2 The effects of APP administration on bodyweight and HI in HFD-induced obese mice

An HFD mice model was used to investigate the poten-tial effects of APP on obesity-associated hepatic metabolicdisorder. Obesity was induced by the administration of anHFD over a 30 day period. APP was administered by oralgavage at dosages of 200, 400, and 800 mg/kg/day dur-ing the HFD treatment. As shown in Table 2, the HFD

Figure 2. Effects of APP onserum parameters in HFD-induced obese mice. Data areshown as the mean ± SD(n = 18). **p < 0.01 comparedwith the control group; #p <0.05, ##p < 0.01 compared withthe high-fat group.



Figure 3. Effects of HFD and APPon the enzymatic activities ofserum ALT (A), AST (B), and LDH(C); and the level of hepatic FAS(D) in mice. Data are shown asthe mean ± SD (n = 18). **p< 0.01 compared with the con-trol group. #p < 0.05, ##p <0.01 compared with the high-fatgroup.

significantly (p < 0.05) increased body weight. The APP treat-ment effectively reduced body weight (Table 2). Comparedwith the normal control group, the HFD-treated mice showedan increased HI (p < 0.05), which could be significantly(p < 0.05) decreased by pretreatment with APP at the testeddoses of 200, 400, and 800 mg/kg/day in a dose-dependentmanner.

3.3 The effects of APP on serum parameters inHFD-induced obese mice

The HFD-induced obesity model is usually accompanied byhyperlipidemia and impaired sensitivity. Figure 2 shows theserum levels of TC, TG, and LDL-C, which were increasedby HFD and effectively blocked by all low-, middle-, andhigh-dose APP treatments; only high-dose APP did not sig-nificantly inhibit the LDL-C induced by the HFD. The HFD-induced decrease in adiponectin and HDL-C levels was signif-icantly restored by all the three APP treatments. In addition,a significantly higher level of fasting insulin was induced byHFD and was effectively blocked by all three doses of APP.

3.4 The effects of APP on hepatic injury inHFD-induced obese mice

It is well known that liver damage with the leakage of cellularenzymes into plasma is a sign of hepatic injury [23]. The enzy-matic activities of serum ALT, AST, and LDH are consideredsensitive indicators of hepatic function [24, 25]. As shown inFig. 3A–C, the HFD treatment induced acute hepatotoxicityin mice, as indicated by the increases in serum ALT, AST,

and LDH levels relative to the control group (p < 0.01). As ex-pected, APP supplementation successfully restored all theseenzymatic activities to normal levels. The lipid biosynthesislevel in the liver was assessed by measuring FAS, which wasincreased by the HFD [26]. As expected, APP treatment sig-nificantly decreased FAS levels (p < 0.05) at low doses (Fig.3D).

3.5 The effects of APP on liver parameters inHFD-induced obese mice

Lipid peroxidation level in the liver was assessed by measur-ing MDA [5, 27]. In the normal control group, the liver MDAlevel was 8.60 ± 1.30 nM/mg protein. However, the hepaticMDA levels sharply increased to 21.70 ± 4.30 nM/mg protein(p < 0.01) after HFD administration. As expected, this HFD-induced increase was effectively attenuated by pretreatmentwith APP at the tested doses of 200, 400, and 800 mg/kg/dayin a dose-dependent manner (Fig. 4A). The HFD treatmentdramatically elevated MPO levels (Fig. 4B) relative to the con-trol group (p < 0.01). As expected, APP supplementationsuccessfully restored MPO to normal levels, and this pro-tective effect was dose-dependent. The acute administrationof HFD to mice caused characteristic hepatotoxicity that af-fected the antioxidant parameters of liver tissue, as indicatedby a significant decrease in glutathione peroxidase (Fig. 4C),SOD (Fig. 4D), CAT (Fig. 4E), GSH (Fig. 4F), T-AOC (Fig.4G), and ASAFR (Fig. 4H) levels relative to the normal controlmice (p < 0.01). As expected, APP supplementation success-fully restored all these markers to control levels. The HFDtreatment dramatically elevated leptin (Fig. 4I) relative to thecontrol group (p < 0.01). As expected, APP supplementation



Figure 4. Effects of APP on hep-atic parameters in HFD-inducedobese mice. Data are shown asthe mean ± SD (n = 18). **p< 0.01 compared with the nor-mal group. #p < 0.05, ##p <0.01 compared with the high-fatgroup.

reduced leptin to normal levels, especially at low doses. Theenergy production level in the liver was assessed by measur-ing glycogen (Fig. 4J), HK (Fig. 4K), and PK (Fig. 4L). Incontrast to the control mice, the levels of these markers wereobviously reduced after administration of the HFD (p < 0.01),and pretreatment with APP considerably increased the levelof HFD-reduced markers.

3.6 APP protects against palmitate-inducedmitochondrial dysfunction and insulin resistancein HepG2 cells

As shown in Fig. 5A, compared to the cells cultured in theH-DMEM medium with no APP, cell growth was signifi-cantly stimulated by APP over the entire tested concentrationrange from 1 to 200 �g/mL. These results showed that APPwas not toxic to the HepG2 cells. When the polysaccharideconcentration was lower than 25 �g/mL, APP promoted cellproliferation in a dose-dependent manner.

Free fatty acid-induced lipotoxicity plays a pivotal role inthe pathogenesis of hepatic metabolic disorder [20]. To eluci-date the protective effects of APP against FFAs and to deter-

mine whether the effects of APP on liver mitochondrial func-tion were due to amelioration of oxidative stress, we usedan in vitro HepG2 model with palmitate treatment to alterFFAs. After a 24 h treatment, 600 �M palmitate inhibitedinsulin signal transduction by decreasing p-AKT levels underinsulin stimulation, and this inhibition was diminished byAPP treatment (Fig. 5B and C). Additionally, palmitate in-duced significant mitochondrial dysfunction, including cellapoptosis (Fig. 5D), increased ROS production (Fig. 5E–G),and loss of the MMP (Fig. 5H). As expected, treatment withAPP effectively restored mitochondrial function, indicatingthat APP might also be an effective nutrient. Further investi-gation of mitochondrial oxygen consumption and the electrontransport chain complex activities was conducted with APP.The basal OCR levels were less than control in those PAL-treated cells (Fig. 5I). To determine whether the APP affectedmitochondrial respiration, we treated HepG2 cell with APP,PAL, and APP + PAL. Under these conditions, an increase inbasal OCR levels was observed indicating that the cells are us-ing glucose oxidation and consuming more oxygen (Fig. 5I).Subsequent addition of oligomycin showed that the levelsof ATP-linked respiration were attenuated in control cells orcells treated with PAL (Fig. 5J). To determine the maximal



Figure 4. Continued.

respiratory capacity, the mitochondrial uncoupler FCCP wasinjected into the media. The stimulation of mitochondrial res-piration with FCCP after oligomycin was substantially greaterin the presence of 10 �g/mL APP compared to the control.Injection of mitochondrial complex III inhibitor antimycinA significantly inhibited respiration (Fig. 5I). A comparativestudy of effects of APP, PAL, and APP + PAL on the OCRlevels of HepG2 cells showed that palmitate abolished the mi-tochondrial respiratory capacity, including basal respiration,ATP production, maximal respiration, and spare respiratorycapacity, all of which were significantly improved by APPtreatment.

4 Discussion

Several studies have addressed the link within dietary ap-ple, apple product consumption, and benefits in humans[8,28,29]. Apple pomace could be a valuable and easily acces-sible source for bioactive compounds [3, 30], which includechemopreventive agents that can inhibit the developmentof serious chronic diseases and complications [5, 28, 31, 32].Here, we isolated the polysaccharides from the apple po-

mace of “Qinguan.” APP was characterized as an acidicheteropolysaccharide, rich in arabinose (32.52%), galactose(20.60%), and galacturonic acid (29.96%), which accountedfor up to 83.08% of all the quantitative monosaccharides, andwas richer in uronic acids than “Fuji” apple peel polysaccha-rides and apple flesh polysaccharides [5]. Bioactive polysac-charides have been found in a large number of natural re-sources, including plants, fungi, and bacteria, and have at-tracted more and more attention in the biochemical andmedical areas. Among them, apple pectin has demonstrateda better cholesterol-lowering effect than other pectins [31]and polysaccharide of apple pomace on antioxidative stress[3,4,29]. In the current study, we found that APP treatments,significantly inhibited the development of obesity and re-stored the basal serum parameters (Table 2 and Fig. 2), indi-cate that APP had beneficial effects on HFD-induced oxidativestress.

HFD induced severe liver injury [33], as reflected by themarkedly elevated enzymatic activities of serum AST, ALT,and LDH (Fig. 3A–C), while administration of APP signif-icantly reduced the levels of these serum enzymes, whichmay be responsible for its hepatoprotective action by scav-enging and destroying lipid peroxyl radical and ROS such as



Figure 5. Effects of APP on palmitate-induced mitochondrial dysfunctionand insulin resistance in HepG2 cells.APP, HepG2 cells were treated withAPP (10 �g/mL, 24 h); PAL, HepG2cells were exposed to a palmitate chal-lenge (600 �M, 24 h); and APP+PAL,HepG2 cells were pretreated with APP(10 �g/mL, 24 h) and then exposed toa palmitate challenge (600 �M, 24 h).Values are shown as the mean ± SDfrom six independent experiments.*p< 0.05, **p < 0.01, ***p < 0.001, and****p < 0.0001.

the superoxide anion (O2−), the hydrogen peroxide (H2O2),and the hydroxyl radical (OH) [5, 8, 34–36]. Oxidative stressoccurs when there is an imbalance between cellular oxi-dant species production and antioxidant capability [20], andtherefore, antioxidant supplementation to inhibit the freeradical-induced damage has become an attractive therapeu-tic strategy for reducing the risk of liver disease [37]. Theintracellular redox balance depends on both oxidants andthe antioxidant defense system in cells, including GSH-PX,SOD, and CAT, and also small molecular antioxidants, suchas GSH. The results showed that APP possessed a varietyof antioxidant and free radical-scavenging activities, whichexhibited beneficial effects against the oxidative liver dam-age induced by HFD. Other studies have previously shownthat metabolic oxidative stress increases systemic and lep-tin levels over and above the levels found in obesity [38].Increased leptin affects myriad pathways, causing dysregula-tion of immune and metabolic functions [39]. As expected, theHFD-induced increased levels of leptin and markedly reduced

enzymatic activities of PK and HK as well as hepatic glyco-gen concentration (Fig. 4) were completely normalized byAPP.

Cellular defenses that protect the hepatocyte against ox-idative stress have been proposed to be an important way toreduce the liver injury [20]. In this study, APP markedly sup-pressed the decrease of cell viability resulting from palmitateand the cell viability showed significant increased cell via-bility resulting from APP in a dose-dependent manner upto 10 �g/mL compared with media-treated cells. These re-sults indicate that APP exerts a protective effect by inhibitingpalmitate-induced cell death.

There are mounting evidences that HFD-induced oxida-tive stress resulting from the overproduction of ROS is amajor factor contributing to the development and progres-sion of liver damage. The collapse of the MMP results inthe rapid release of cytochrome C into the cytoplasm [40]. Inthe present study, palmitate-treated HepG2 cells showed amarked increased generation of mitochondrial ROS (Fig. 5E



Figure 5. Continued.



and F) and a loss of MMP (Fig. 5H). However, pretreatmentwith APP attenuated the increase in ROS level and preventedthe loss of MMP.

The mitochondria are a major source of ROS, which isparticularly susceptible to oxidative stress [41]. Previous stud-ies have shown that Astragalus and Cornus polysacharidesinhibits mitochondrial injury caused by the continuousproduction of free radicals and selective oxidative damage[42, 43], and Ganoderma atrum polysacharides protect mi-tochondrial by scavenging ROS [40, 44, 45], increasing theactivity of antioxidant enzymes, and Astragalus and gin-seng polysacharides ameliorating mitochondrial dysfunction,which ultimately improved energy metabolism [46–48]. In thepresent study, we found that palmitate-treated HepG2 cellsshowed marked changes in several parameters of the mi-tochondrial function (Fig. 5H–J). Interestingly, APP restor-ing all these parameters to normal levels, as evidenced byenhancing the basal OCR levels, maximal respiration, andmaintaining MMP (Fig. 5H and J), indicating that APP effec-tively improves mitochondrial function in HepG2 cells underpalmitate insult.

Taken together, our results demonstrated that APP suc-cessfully ameliorated the HFD-induced oxidative stress,which may be attributed to APP scavenging mitochondrialROS and improving mitochondrial function. As an abundantand inexpensive compound derived from apple pomace, APPis a promising bioactive food ingredient that may preventhepatic metabolic disorder.

L.C., L.L., and L.H. have carried out animal studies; L.C.,C.L., R.L., M.Z., and D.Z. have carried out cell studies; L.C.,F.S., and R.L. have isolated apple pomace polysaccharides studies;and L.C., J.L., Y.G., and J.L. have written the manuscript.

This study was sponsored by the 973 program (2015CB8-56302, 2015CB553602), the Fundamental Research Funds forthe Central Universities (08143008, 08143101), and the Min-istry of Agriculture of the People’s Republic of China, AgricultureResearch System (CARS-28).

The authors have declared no conflict of interest.

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