Thinned young apple polysaccharide improves hepatic metabolic disorder in high-fat diet-induced obese mice by activating mitochondrial respiratory functions

Journal of Functional Foods 33 (2017) 396–407

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Thinned young apple polysaccharide improves hepatic metabolic disorder in high-fat diet-induced obese mice by activating mitochondrial respiratory functions Lei Chen a, Xi Yang b, Run Liu a, Lei Liu c, Daina Zhao a, Jiankang Liu a, Yurong Guo b,⇑, Jiangang Long a,⇑ a Center for Mitochondrial Biology and Medicine and Center for Translational Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology and Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an, China b College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, China c College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, China

a r t i c l e

i n f o

Article history: Received 29 November 2016 Received in revised form 10 March 2017 Accepted 28 March 2017 Available online 14 April 2017 Chemical compounds studied in this article: Ribose (PubChem CID: 77982) Rhamnose (PubChem CID: 25310) Glucose uronic acid (PubChem CID: 149476) Galacturonic acid (PubChem CID: 439215) Glucose (PubChem CID: 79025) Xylose (PubChem CID: 644160) Galactose (PubChem CID: 6036) Arabinose (PubChem CID: 66308) Fucose (PubChem CID: 17106)

a b s t r a c t Apple polysaccharides have been previously demonstrated to have beneficial effects on hepatic metabolic functions. Approximately 1.9 million tons of young apples are thinned and abandoned annually in China, and whether the polysaccharide derived from these thinned young apples has metabolic benefits lacks evidence. Thinned young apple polysaccharide (TYAP) has been investigated in high fat diet (HFD)induced obese mice for its effect on metabolic disorder. Water-soluble TYAP was isolated from thinned young apples and chemically characterized. TYAP administration at dosages of 400 mg/kg/day and 800 mg/kg/day significantly rescued HFD-induced hepatic metabolic impairment, reduced body weight gain, and ameliorated hepatic oxidative stress induced by HFD. In a palmitate-loaded HepG2 cell model, TYAP protected the cells from palmitate-induced insulin resistance and viability loss, suppressed mitochondrial ROS and improved the mitochondrial respiratory function impaired by palmitate. These findings suggest that TYAP could successfully attenuate obesity-associated hepatic metabolic disorder possibly by activating the hepatic mitochondrial respiratory function. Ó 2017 Published by Elsevier Ltd.

Keywords: Thinned young apple polysaccharide High-fat diet Hepatic metabolic disorder Oxidative stress Mitochondrial respiratory function

1. Introduction Apple thinning is done to remove excessive fruitlets from apple trees after blossom as is an effective measure to improve the quality of apples at harvest and to balance the yield of apples during the following year (Yuan, 2007). Approximately 1.9 million tons of young apples are thinned annually in China, and thinned young apples are usually abandoned on the grounds of the orchard (Dou et al., 2015; Sun, Guo, Fu, Li, & Li, 2013; Zheng, Kim, & Chung, 2012). However, thinned young apples have great value as a source ⇑ Corresponding authors. E-mail addresses: [email protected] (Y. Guo), [email protected] (J. Long). http://dx.doi.org/10.1016/j.jff.2017.03.055 1756-4646/Ó 2017 Published by Elsevier Ltd.

of phytochemicals, which are non-toxic and have certain bioactivities. Polysaccharides are considered dietary free radical scavengers, and they are effective in preventing and treating some oxidative stress-related chronic diseases (Chen et al., 2016; Huang et al., 2016; Li et al., 2010, 2012; Lu et al., 2013; Sun et al., 2014). We have reported the simultaneous separation and purification of water-soluble polysaccharides from thinned young apples and their antioxidant activities (Dou et al., 2015), but whether they can affect hepatic metabolic disorder remains to be explored. In the present study, we analyzed the monosaccharide profiles of TYAP and demonstrated the protective effects of TYAP against HFD-induced hepatic metabolic disorder in mice. We further found that TYAP improved palmitate-induced mitochondrial dysfunction

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and insulin resistance in HepG2 cells. To the best of our knowledge, this is the first report characterizing the chemical profile of TYAP and demonstrating its metabolic benefits in HFD-induced obese mice. 2. Materials and methods 2.1. Chemicals Thinned young apples were collected 30 days after blossom in Liquan, Shaanxi province of China. All samples were stored at
80 °C at Shaanxi Normal University, Xi’an, Shaanxi province, China. Insulin, adiponectin, and leptin were measured using commercial ELISA kits according to the manufacturer’s protocols (Shanghai Guduo Biological Technology Co., Ltd., China). The detection kits for total cholesterol (TC), triacylglycerol (TGs), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), total antioxidant capacity (T-AOC), fungal catalase (CAT), myeloperoxidase (MPO), hexokinase (HK), fatty acid synthase (FAS), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Oligomycin, FCCP, and antimycin A were from Sigma (St. Louis, MO). anti-p-AKT and anti-AKT antibodies were purchased from Santa Cruz Biotechnology (USA). All other reagents were purchased from Invitrogen. 2.2. Polysaccharide extraction from thinned young apples The thinned young apples were collected 30 days after blossom in Liquan country, Shaanxi province, China. All samples were stored at
80 °C at Shaanxi Normal University, Xi’an, Shaanxi province, China. The thinned young apple polysaccharide (TYAP) was extracted according to our previous report (Dou et al., 2015). 2.3. Chemical characterization of TYAP The polysaccharide content of TYAP was estimated by the phenol-sulfuric acid colorimetric method with glucose as a standard (Dou et al., 2015; Yang, Yang, Guo, Jiao, & Zhao, 2013). The total polyphenol content of TYAP was determined based on the Folin-Ciocalteu colorimetric method (L. Sun et al., 2013). The total protein content of TYAP was determined based on the Kjeldahl method. The monosaccharide composition of TYAP was analyzed by HPLC as described by Lv et al. (2009). Briefly, 20 mg of the TYAP sample was hydrolyzed with 2 mL of 3 M trifluoroacetic acid at 100 °C for 8 h in an ampoule (5 mL) sealed in a nitrogen atmosphere to release the constituent monosaccharides, and derivatization was then carried out with PMP. The analysis of PMP-labeled monosaccharides was performed using a reversed-phase HPLC column (4.6 lm i.d.  250 mm, 5 lm, Venusil, USA) on a Shimadzu LC-2010A HPLC system equipped with an UV–Vis detector and a Shimadzu Class-VP 6.1 chromatography workstation (Shimadzu, Japan). The mobile phase A was acetonitrile, and B was 3.3 mM TEA (pH 7.5), with an elution gradient of 95–95–90–90% B by a linear decrease from 0–5–8–30 min, respectively. The wavelength for UV detection was 250 nm. 2.4. Evaluation of in vitro antioxidant activity of TYAP 2.4.1. Determination of DPPH-scavenging activity The DPPH-scavenging activity of TYAP was measured by the method described in our previous reports (Dou et al., 2015; Yang

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et al., 2013). Briefly, 1.0 mL of the tested TYAP at various concentrations was mixed with 3.0 mL of 0.1 mM DPPH in aqueous methanol, followed by vigorous blending. The absorbance at 517 nm was determined after standing in the dark at room temperature for 20 min, and the percentage of inhibition activity was calculated according to the following formula: DPPH-scavenging activity (%) = [1
(Asample
Abackground)/Ablank]  100. When the reaction reached a plateau, the IC50 value was calculated based on the results. 2.4.2. Assay for HO-scavenging effect The HO-scavenging activity of TYAP was measured with an improved Fenton-type reaction (Ghiselli, Nardini, Baldi, & Scaccini, 1998). The reaction mixture contained 1 mL of TYAP or APP samples, 1 mL FeSO4, 1 mL salicylic acid-ethanol and 1 mL H2O2. The reaction mixture was incubated at 37 °C for 60 min, and the absorbance was measured at 510 nm. Mannitol was used as a positive control. The scavenging activity of the TYAP against HO was calculated according to the following equation: scavenging activity against HO(%) = [1
(Asample
Ablank)/Acontrol]  100. 2.5. Animals and experimental design Seventy-two Kunming mice (weight 18–22 g, half of them male and half of them female, and approximately 45 days of age) were provided by the Experimental Animal Center, Lanzhou University (Lanzhou, China). The mice were housed in a temperaturecontrolled (22–28 °C) and humidity-controlled (60 ± 5%) animal room and maintained 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 experiments. After 1 week of acclimatization, the mice were randomly distributed into the following four groups: (1) control mice fed a standard chow diet (12% kcal fat content; KEAO, Beijing, China); (2) mice fed an HFD (45% kcal fat content; KEAO, Beijing, China); (3) mice fed an HFD and given a daily oral gavage of lowdose TYAP (400 mg/kg/day); and (4) mice fed an HFD and given a daily oral gavage of high-dose TYAP (800 mg/kg/day). After 30 days of feeding, the mice were fasted overnight and sacrificed. All procedures were performed in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China. 2.6. Blood samples and biochemical measurements At the end of the experimental period, all mice were fully anesthetized by the ether inhalation, weighed, and then sacrificed to obtain blood and liver samples. The blood samples were centrifuged at 1200g for 20 min and stored at 4 °C, while the livers were frozen at
80 °C. On the basis of the body weight and corresponding liver weights of every mouse, we calculated the hepatosomatic index (HI) according to the following formula: HI = liver weight/body weight  100%. The levels of TG, TC, LDL-C, HDL-C, T-AOC, AST, ALT, MDA, LDH, GSH-PX, SOD, CAT, MPO, HK and PK were analyzed using an automated biochemical analyzer. Serum levels of insulin, adiponectin, and leptin were measured using commercial ELISA kits according to the manufacturer’s protocols. 2.7. HepG2 cell culture and assessment of cell viability HepG2 cells were cultured in DMEM supplemented with 10% fetal calf serum, 100 U/mL penicillin G sodium, and 100 lg/ml streptomycin sulfate at 37 °C in a humidified incubator with 5% CO2, and the experiments were initiated once the cells reached 70% confluence (Yan et al., 2016). The effect of TYAP on the viability of HepG2 cells was analyzed in vitro using MTT assays (Feng et al., 2011). The cells were seeded

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at a density of 5  104 cells/mL in 96-well plates and incubated with TYAP at concentrations of 0, 2, 5, 10, 15, 25, 50, 100, and 200 lg/mL for 24 h. After incubation, 100 lL of MTT-DMEM solution (1:9) was added to each well. The plates were further incubated for 4 h followed by the addition of 100 lL of DMSO to each well. The absorbance was measured at 490 nm on a Bio-Rad model 680 Microplate Reader (PA, USA). 2.8. Oil Red O staining HepG2 were cultured at a density of 1  105 cells/mL in six-well plates. After treatment, the HepG2 cells were washed twice with PBS followed by 15 min of incubation with 0.3% Oil Red O working solution. Then, the HepG2 cells were washed twice with PBS buffer. The cells were observed immediately with a fluorescence microscope (Zeiss Germany), and total lipid accumulation was determined after Oli Red O staining. 2.9. Determination of ROS HepG2 were cultured at a density of 2  105 cells/mL in six-well plates. After treatment, the generation of intracellular reactive oxygen species (ROS) was determined by measuring the fluorescence of 20 , 70 -dichlorofluorescein diacetate (DCFH2-DA). Briefly, DCFH2DA at a final concentration of 10 lM was incubated with the HepG2 cells in serum-free medium for 30 min, and the cells were washed twice and collected with PBS. The cells were centrifuged at 1000g for 1 min at 4 °C and suspended with PBS. The cells were analyzed by flow cytometry (BD Bioscience, Franklin Lakes).

the OCR response to FCCP and dividing that number by the basal respiration to obtain a percentage. After detection, the total protein content of the cells was calculated, and the OCR was adjusted accordingly. 2.13. Western blot analyses The samples were lysed with Western and IP lysis buffer (Beyotime). The lysates were homogenized, and the homogenates were centrifuged at 13,000g for 15 min at 4 °C. The supernatants were collected, and the protein concentrations were determined with a BCA protein assay kit. Equal aliquots (20 lg) of the protein were separated by 10% SDS-PAGE, transferred to pure nitrocellulose membranes (PerkinElmer Life Sciences), and blocked with 5% nonfat milk in TBST buffer. The membranes were incubated with antiAKT and anti-p-AKT (1:1000) antibodies at 4 °C overnight. Then, the membranes were incubated with anti-rabbit secondary antibodies at room temperature for 1.5 h. Chemiluminescent detection was performed using an ECL Western blotting detection kit (Thermo Fisher, Rockford, IL, USA). The results were analyzed with Quantity One software (Bio-Rad, Shanghai, China) to obtain the optical density ratio of the target proteins relative to GAPDH. 2.14. Statistical analysis Data are presented as the mean ± SD. Statistical significance was evaluated with one-way ANOVA followed by LSD post hoc analysis using GraphPad Prism 6. In all comparisons, the level of significance was set at p < 0.05 (see Table 1).

2.10. Measurement of mitochondrial ROS 3. Results HepG2 were cultured at a density of 5  104 cells/mL in six-well plates. The generation of mitochondrial ROS was determined with the MitoSOXTM Red Mitochondrial Superoxide Indicator (Thermo Fisher, USA), and the mitochondria were assessed with MitoTrackerÒ Green FM (Invitrogen, USA). MitoSOXTM and MitoTrackerÒ Green FM at a final concentration of 10 lM were incubated with the HepG2 cells in serum-free medium for 30 min. After washing with PBS, the cells were visualized by confocal microscopy (Zeiss, Jena, Germany). 2.11. JC-1 assay for MMP HepG2 were cultured at a density of 5  104 cells/mL in 96-well plates. The lipophilic cationic probe 5, 50 , 6, 60 -terachloro-1, 10 , 3, 30 tetraethyl-imidacarbocyanine iodide (JC-1) was used to measure the mitochondrial membrane potential (MMP). The cells were washed with PBS once after JC-1 staining and scanned with a microplate fluorometer (Fluoroskan Ascent; Thermo Fisher Scientific, Inc.) at 488 nm excitation and 538 and 590 nm emission wavelengths to measure green and red JC-1 fluorescence, respectively. The red/green fluorescence intensity ratio reflects the MMP (Yan et al., 2016; Zou et al., 2014). 2.12. Measurement of the cell oxygen consumption rate Oxygen consumption was measured using extracellular flux analysis (Seahorse Bioscience) when HepG2 cell density in XF 24well microplates (Seahorse Bioscience, Billerica, MA, USA) had reached 1.5  104 cells/mL. The final concentrations of the mitochondrial inhibitors were 1 lM antimycin A, 0.5 lM FCCP and 1 lM oligomycin. Basal respiration is the baseline oxygen consumption reading before the compounds are injected. Maximal respiration represents the maximum OCR value after the FCCP injection. Spare respiratory capacity is calculated by recording

3.1. Chemical characterization of TYAP TYAP was extracted from thinned young apples using a multistep purification procedure, including hot-water extraction and repeated ethanol precipitation, and the yield of TYAP was approximately 10.8% (w/w) of the dried mass of thinned young apples. The polysaccharide content of TYAP was determined to be 74.30 ± 1.31% by the phenol-H2SO4 assay, and the total protein content of TYAP was determined to be 4.8 ± 0.34% by the Kjeldahl method. In addition, TYAP did not react with the Folin-Ciocalteu reagent, suggesting that the small molecular phenolic compounds in TYAP had been removed by the dialysis process against distilled water during the purification of the macromolecular polysaccharides. Furthermore, the monosaccharide composition of TYAP was also estimated with a validated HPLC-UV technique as described in our previous work (Chen et al., 2016; Dou et al., 2015). Chromatographic analysis was employed to identify and quantify the major monosaccharides present in TYAP. As shown in Fig. 1, 10 PMP-labeled standard monosaccharides were rapidly separated within 38 min. The peaks were identified in the order of mannose, ribose, rhamnose, glucose uronic acid, galacturonic acid, glucose, xylose, galactose, arabinose, and fucose by matching their retention times with those of the monosaccharide standards under the

Table 1 The chemical components of TYAP. Sample

Content (%)

Polysaccharides Protein Polyphenols

74.30 ± 1.31% 4.8 ± 0.34% –

–: Not detected in the sample

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than Vc at all tested concentrations ranging from 0.2 to 1.0 mg/ml. The results implied that TYAP and APP likely contained substances that were hydrogen donors and could react with free radicals to scavenge DPPH. Hydroxyl radicals are among the most reactive and dangerous free radicals among the reactive oxygen species (ROS) and are mainly responsible for the oxidative damage of biomolecules. They are generated by the reaction of Fe2+ complexes with H2O2 in the presence of salicylic acid. As shown in Fig. 2B, the scavenging activity of the compounds could be ranked as Vc > TYAP > APP.

same analytical conditions. The monosaccharide compositions of TYAP are shown in Fig. 1, and the quantified constituents are shown in Table 2. 3.2. In vitro antioxidant activity of TYAP The in vitro antioxidant effects of TYAP and APP were evaluated by testing its scavenging capacity against DPPH and HO. DPPH is a stable free-radical compound that shows maximum absorbance at 517 nm, and it is widely used in assays to evaluate the ability of antioxidants to scavenge radicals. An excellent DPPH scavenging activity indicates superior antioxidant activity. As shown in Fig. 2A, TYAP and apple pomace polysaccharides (APP) showed scavenging effects that were positively correlated with increasing concentrations. The scavenging effect on DPPH of TYAP was higher than that of APP, and all of them showed lower scavenging activity

3.3. The effects of TYAP administration on the body weight of HFDinduced obese mice A HFD-fed mouse model was used to investigate the potential effects of TYAP on obesity-associated hepatic metabolic disorder.

Fig. 1. HPLC chromatograms of 10 standard monosaccharides and the component monosaccharides released from TYAP. Peaks: (1) mannose, (2) ribose, (3) rhamnose, (4) glucose uronic acid, (5) galacturonic acid, (6) glucose, (7) xylose, (8) galactose, (9) arabinose, and (10) fucose.

Table 2 The monosaccharides in TYAP. Monosaccharide Mannose Ribose Rhamnose Glucose uronic acid Galacturonic acid Glucose Xylose Galactose Arabinose Fucose

Retention time (min) 20.99 ± 0.297 22.06 ± 0.239 23.54 ± 0.226 26.08 ± 0.621 28.39 ± 0.701 33.66 ± 0.258 35.62 ± 0.257 36.39 ± 0.349 37.01 ± 0.298 37.68 ± 0.234

Equation of regression (Y = aX + b) 9

Y = 2  10 Y = 1  109 Y = 8  108 Y = 2  109 Y = 2  109 Y = 1  109 Y = 1  109 Y = 1  109 Y = 1  109 Y = 8  108

X–38,234 X–50,073 X–21,008 X–57,784 X–74,755 X–50,945 X–17,896 X–34,606 X + 23,635 X–51,630

R2

Molar contents (%)

0.9992 0.9970 0.9993 0.9966 0.9932 0.9963 0.9995 0.9967 0.9988 0.9988

1.9 1.05 3.92 1.08 3.28 10.32 3.9 25.55 46.72 2.28

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Fig. 2. In vitro antioxidant activity of TYAP and APP determined by using DPPH and HO. (A) DPPH-scavenging assay and (B) HO-scavenging assay. Vc was used as a positive control. Data are shown as the mean ± SD (n = 3).

Table 3 Effects of TYAP on body weight of mice subjected to HFD treatment. Con

Initial body weight (g) Final body weight (g) Body weight gain (g) Food intake (g) Date are shown as means ± SD (n = 18).

High-fat diet

19.51 ± 0.30 32.23 ± 0.25 12.72 ± 0.13 1621.28 4

0

400

800 (mg/kg/day)

19.62 ± 0.40 36.70 ± 0.344 17.08 ± 0.244 1541.57

17.55 ± 0.30 31.49 ± 0.30▲▲ 13.91 ± 0.12▲▲ 1603.68

18.78 ± 0.20 31.12 ± 0.23▲▲ 12.34 ± 0.07▲▲ 1595.85

p < 0.05, as compared with the normal group.

▲

p < 0.05,

▲▲

p < 0.01, as compared with the high fat group.

Table 4 Effects of TYAP on serum parameters of mice subjected to HFD treatment. Con

TC (mmol/L) TG (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L) Leptin (lg/L) Adiponectin (lg/L) Insulin (pmol/L) HOMA-IR Date are shown as means ± SD (n = 18).

0.33 ± 0.04 0.13 ± 0.03 0.04 ± 0.01 0.36 ± 0.02 0.91 ± 0.14 46.63 ± 4.8 32.30 ± 2.7 7.46 ± 1.35 4

High-fat diet 0

400

800 (mg/kg/day)

0.70 ± 0.0144 0.22 ± 0.0244 0.08 ± 0.0144 0.25 ± 0.0444 1.84 ± 0.3244 23.42 ± 2.744 48.9 ± 2.344 13.42 ± 1.5144

0.58 ± 0.07▲▲ 0.19 ± 0.01▲ 0.05 ± 0.01▲▲ 0.51 ± 0.01▲▲ 1.54 ± 0.31▲ 47.31 ± 5.6▲▲ 37.30 ± 1.60▲▲ 9.61 ± 1.32▲▲

0.53 ± 0.02▲▲ 0.15 ± 0.01▲▲ 0.03 ± 0.01▲▲ 0.33 ± 0.01▲ 1.65 ± 0.34 52.31 ± 4.2▲▲ 35.50 ± 3.70▲▲ 8.02 ± 0.76▲▲

p < 0.05, as compared with the normal group.

Obesity was induced with an HFD over a period of 30 days. TYAP was administered by oral gavage at dosages of 400 and 800 mg/ kg/day during the HFD treatment. As shown in Table 3, the HFD significantly (p < 0.05) increased body weight, and the TYAP treatment effectively reduced body weight without affecting food intake. 3.4. The effects of TYAP on serum parameters in HFD-induced obese mice The HFD-induced obesity model is usually accompanied by hyperlipidemia and impaired insulin sensitivity. In the current study, the serum levels of TC, TG, and LDL-C were increased by HFD and effectively blocked by both low- and high-dose TYAP treatments in a dose-dependent manner. The HFD-induced decrease in adiponectin and HDL-C levels were significantly restored by both low- and high-dose TYAP treatments. In addition, a significantly higher level of fasting insulin and the homeostatic model assessment-insulin resistance (HOMA-IR) were induced by HFD and were effectively blocked by both doses of TYAP (Table 4).

▲

p < 0.05,

▲▲

p < 0.01, as compared with the high fat group.

3.5. The effects of TYAP on hepatic injury in HFD-induced obese mice It is well known that liver damage with the leakage of cellular enzymes into the plasma is a sign of hepatic injury (Baldi, Burra, Plebani, & Salvagnini, 1993). The enzymatic activities of serum ALT, AST and LDH are considered sensitive indicators of hepatic function (Hou, Qin, & Ren, 2010; Zhang et al., 2009). As shown in Fig. 3A, 3B, and 3C, the HFD treatment induced acute hepatotoxicity in mice, as indicated by the increase in the serum levels of ALT, AST and LDH relative to the control group (p < 0.01). As expected, TYAP supplementation successfully restored all these enzymatic activities to normal levels. Compared with the normal control group, the HFD-treated mice showed an increased HI (p < 0.05), which could be significantly (p < 0.05) decreased by treatment with TYAP (Fig. 3D). 3.6. The effects of TYAP on liver parameters in HFD-induced obese mice The lipid biosynthesis level in the liver was assessed by measuring FAS, which was increased by the HFD (Kim, Wright, Wright, &

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Fig. 3. TYAP inhibits HFD-induced hepatic injury in mice. (A) HI level, (B) serum ALT level, (C) serum AST level and (D) serum LDH level. Data are shown as the mean ± SD (n = 18), ** p < 0.01 compared with the control group. # p < 0.05, ## < 0.01 compared with the high-fat group.

Spiegelman, 1998). As expected, TYAP treatment significantly decreased FAS levels (p < 0.001) in a dose-dependent manner (Fig. 4A). Lipid peroxidation level in the liver was assessed by measuring MDA and MPO (Cemek et al., 2010; Yang et al., 2013), which were sharply increased by HFD and effectively blocked by both low- and high-dose TYAP treatments in a dose-dependent manner (Fig. 4B and C). The acute administration of HFD to mice caused characteristic hepatotoxicity that affected the antioxidant parameters of liver tissue, as indicated by a significant decrease in GSH-Px (Fig. 4D), SOD (Fig. 4E), CAT (Fig. 4F), GSH (Fig. 4G), T-AOC (Fig. 4H), and ASAFR (Fig. 4I) levels relative to the normal control mice (p < 0.01). As expected, TYAP supplementation successfully restored all these markers to control levels in a dose-dependent manner, with the exception of T-AOC. The energy production level in the liver was assessed by measuring glycogen (Fig. 4J) and HK (Fig. 4K). In contrast to the control mice, the levels of these markers were markedly reduced after administration of the HFD (p < 0.01), and treatment with TYAP considerably increased the level of HFD-reduced markers. 3.7. TYAP attenuates palmitate-induced lipotoxicity and insulin resistance TYAP did not show any toxicity in the HepG2 cells. When the TYAP concentration was 5 lg/mL, TYAP promoted cell proliferation (Fig. 5A). HepG2 cells were co-incubated with 5 lg/mL TYAP and 600 lM palmitate for 24 h. An MTT assay showed that palmitate markedly decreased the viability of HepG2 cells, and TYAP at 5 lg/mL effectively increased the cell viability (Fig. 5B). Additionally, after a 24 h treatment, palmitate inhibited insulin signal

transduction by decreasing p-AKT levels under insulin stimulation, and this effect was diminished by TYAP treatment (Fig. 5C and D). 3.8. TYAP inhibits palmitate-induced cellular lipid accumulation To investigate whether TYAP could affect palmitate-induced cellular steatosis, Oil Red O staining, a simple qualitative method to analyze the amount of TG stored within lipid droplets in the cells, was performed in palmitate-induced HepG2 cells with TYAP. As shown in Fig. 6A and B, palmitate increased the intracellular lipid content. The number and size of the lipid droplets were significantly reduced after TYAP and palmitate treatment in HepG2 cells compared to the palmitate group. 3.9. TYAP attenuates ROS generation induced by palmitate To further characterize the palmitate-induced lipotoxicity, we further evaluated ROS content and found that the palmitateinduced cells exhibited a significant increase in the ROS level, which was normalized by TYAP treatment (Fig. 7A). The mitochondria are the major source of ROS and are particularly susceptible to oxidative stress (Kowaltowski & Vercesi, 1999). We further evaluated mitochondrial ROS and found that the palmitate-induced cells exhibited a significant increase in the mitochondrial ROS level, which was normalized by TYAP treatment (Fig. 7C). 3.10. TYAP protects against palmitate-induced mitochondrial dysfunction in HepG2 cells Palmitate-induced lipotoxicity plays a pivotal role in the pathogenesis of hepatic metabolic disorder (L. Chen et al., 2016; Zou

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Fig. 4. Effects of TYAP on hepatic parameters in HFD-induced obese mice. (A) hepatic fatty acid synthase (FAS) level, (B) hepatic malondialdehyde (MDA) level, (C) hepatic myeloperoxidase (MPO) level and (D) hepatic glutathione peroxidase (GSH-PX) level, (E) hepatic superoxide dismutase (SOD) level, (F) hepatic fungal catalase (CAT) level, (G) hepatic glutathione (GSH) level, (H) hepatic total antioxidant capacity (T-AOC) level, (I) hepatic resistance to total superoxide anion free radicals (ASAFR) level, (J) hepatic glycogen level and (D) hepatic hexokinase (HK) level. Data are shown as the mean ± SD (n = 18). ** p < 0.01 compared with the normal group. # p < 0.05, ## p < 0.01 compared with the high-fat group.

et al., 2014). To elucidate the protective effects of TYAP against palmitate and determine whether the effects of TYAP on liver mitochondrial function were due to the amelioration of oxidative stress, we used an in vitro HepG2 model with palmitate and TYAP treatments. Palmitate induced significant loss of the MMP (Fig. 8A). We found that the treatment with TYAP effectively restored MMP, indicating that TYAP might also be an effective nutrient for mitochondria. Further investigation of mitochondrial oxygen consumption and the electron transport chain complex activities was conducted with apple pomace polysaccharide (APP). The basal OCR levels were lower in the PAL-treated cells than in the control

cells (Fig. 8C). To determine whether TYAP affected mitochondrial respiration, we treated the HepG2 cells with TYAP, PAL, and TYAP + PAL. Under these conditions, an increase in the basal OCR levels was observed, indicating that the cells were using glucose oxidation and consuming more oxygen. Subsequent addition of oligomycin showed that the levels of ATP-linked respiration were attenuated in the control group or PAL-treated cells. To determine the maximal respiratory capacity, the mitochondrial uncoupler FCCP was added to the media. The stimulation of mitochondrial respiration with FCCP after oligomycin treatment was substantially greater in the presence of 5 lg/mL TYAP compared to the control.

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403

Fig. 4 (continued)

Injection of the mitochondrial complex III inhibitor antimycin A significantly inhibited respiration. A comparative study of the effects of TYAP, PAL, and TYAP + PAL on the OCR levels of HepG2 cells showed that palmitate abolished the mitochondrial respiratory capacity, including basal respiration, ATP production, maximal respiration and spare respiratory capacity, all of which were significantly improved by TYAP treatment. 4. Discussion Several studies have addressed the beneficial effects of bioactive polysaccharides in obesity, insulin resistance, nonalcoholic fatty liver disease, inflammation, and diabetes (Chen et al., 2015, 2016; Colin-Henrion, Mehinagic, Renard, Richomme, & Jourjon, 2009; Hu et al., 2016; Huang et al., 2016; Li, Chen, Jin, & Chen,

2009; Li et al., 2010, 2011, 2015; Lv et al., 2009; Yang et al., 2013; Zhang, Nie, Huang, Feng, & Xie, 2014). A growing amount of evidence highlights that ROS produced by metabolic processes have a wide variety of pathological effects, such as causing DNA damage, carcinogenesis, insulin resistance, mitochondrial dysfunction and age-related cellular degeneration (Droese, Hanley, & Brandt, 2009; Kowaltowski & Vercesi, 1999). It is very important to know the monosaccharide profiles of the polysaccharides that are involved in the bioactivity. In this study, we isolated the polysaccharides from thinned young apples, and TYAP was characterized as an acidic heteropolysaccharide rich in arabinose (46.72%), and galactose (25.55%), accounting for up to 72.27% of all the quantitative monosaccharides. We have previously reported that APP had beneficial effects on HFD-induced oxidative stress (Chen et al., 2016). The in vitro data indicated that

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Fig. 5. TYAP attenuates palmitate-mediated lipotoxicity in HepG2 cells. (A) HepG2 cells were treated with TYAP for 24 h; cell viability was measured with the MTT assay. (B) Cell viability was measured with the MTT assay. (C, D) Phosphorylated AKT and total AKT were analyzed by Western blot.TYAP, HepG2 cells were treated with TYAP (5 lg/mL, 24 h); PAL, HepG2 cells were exposed to a palmitate challenge (600 lM, 24 h); and TYAP + PAL, HepG2 cells were pretreated with TYAP (5 lg/mL, 24 h) and then exposed to a palmitate challenge (600 lM, 24 h). Values are shown as the mean ± SD from six independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Fig. 6. TYAP inhibits palmitate-induced cellular lipid accumulation in HepG2 cells. TYAP, HepG2 cells were treated with TYAP (5 lg/mL, 24 h); PAL, HepG2 cells were exposed to a palmitate challenge (600 lM, 24 h); and TYAP + PAL, HepG2 cells were pretreated with TYAP (5 lg/mL, 24 h) and then exposed to a palmitate challenge (600 lM, 24 h). Values are shown as the mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

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Fig. 7. TYAP attenuates ROS generation induced by palmitate in HepG2 cells. (A, B) ROS level was determined by using the H2DCFDA probe. (C) Mitochondrial ROS level was determined using MitoSOXTM. TYAP, HepG2 cells were treated with TYAP (5 lg/mL, 24 h); PAL, HepG2 cells were exposed to a palmitate challenge (600 lM, 24 h); and TYAP + PAL, HepG2 cells were pretreated with TYAP (5 lg/mL, 24 h) and then exposed to a palmitate challenge (600 lM, 24 h). Values are shown as the mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

TYAP had much stronger antioxidant activity (Fig. 2) compared to APP, perhaps due to the presence of uronic acid residues that may alter the properties of polysaccharides (Tian, Zhao, Guo, & Yang, 2011). Our results indicated that TYAP is a more effective freescavenging agent than APP, which could prevent or ameliorate oxidative damage. Polysaccharides are a mixture of different materials with different degrees of polymerization, and their molecular mechanisms in vivo are not fully understood. It has been reported that biopolymers (e.g., polysaccharides) or their active domains can be transferred into the blood after oral administration in experimental animals (Hoshi, Iijima, Ishihara, Yasuhara, & Matsunaga, 2008; Zha et al., 2015). Therefore, it can be assumed that TYAP can reach the intestine and enter the blood in its full form or its degradation products and then act on targets to exert its bioactivities; however, we still cannot exclude the other possible mechanism that the administered polysaccharide may benefit the host by modifying the profile of the gut microbiota (Shi, Li, Wang, & Feng, 2015). The HFD induced severe liver injury (Dhibi et al., 2011), while the administration of TYAP significantly reduced the levels of HI and enzymatic activities of serum AST, ALT and LDH (Fig. 3) An increase in hepatic MDA levels (Fig. 4B) indicates the enhanced lipid peroxidation of the polyunsaturated fatty acids of the biological membranes, leading to tissue damage and failure of the antioxidant-defense mechanisms to prevent the formation of excessive ROS. Oxidative stress occurs when there is an imbalance between the production of cellular oxidant species and the antiox-

idant capability, and therefore, antioxidant supplementation to inhibit the free radical-induced damage has become an attractive therapeutic strategy for reducing the risk of liver injury (Jahn et al., 1985; Karakus et al., 2011; Milic, Lulic, & Stimac, 2014). It is well known that the non-enzymatic antioxidant GSH can provide protection against oxidative stress and that several endogenous antioxidant enzymes, such as SOD and GSH-Px, can also convert ROS into less noxious compounds in living organisms. The present study showed that TYAP exhibited beneficial effects on the maintenance of redox balance by modulating both oxidants and the antioxidant defense system (Fig. 4D–I). In acute HFD-induced liver injury, inflammatory cytokines secreted by macrophages, activate inflammatory responses and downstream signals, resulting in the recruitment of neutrophils, monocytes, and lymphocytes, further amplifying an inflammatory response. Present study showed that HFD-induced a significant increase in TNF-a and IL-6 (Fig. S1) and decreased in anti-inflammatory cytokines (Fig. S2), neutrophils, monocytes, and lymphocytes (Table S1), whereas cotreatment with TYAP and HFD successfully restored all these markers to control levels. There is mounting evidence that HFD-induced oxidative stress is a result of the overproduction of ROS. The mitochondrion is the major source of ROS under HFD, contributing to the development and progression of liver damage. Mitochondrial dysfunction is a consequence of oxidative damage caused by increased oxidant levels. Therefore, decreasing oxidant generation and oxidative damage might be an effective way to inhibit mitochondrial

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Fig. 8. TYAP protects against palmitate-induced mitochondrial dysfunction and insulin resistance in HepG2 cells. (A) MMP was measured with JC-1. (B, C) The oxygen consumption rate of the cells was measured with Seahorse XF-24. TYAP, HepG2 cells were treated with TYAP (5 lg/mL, 24 h); PAL, HepG2 cells were exposed to a palmitate challenge (600 lM, 24 h); and TYAP + PAL, HepG2 cells were pretreated with TYAP (5 lg/mL, 24 h) and then exposed to a palmitate challenge (600 lM, 24 h). Values are shown as the mean ± SD from at least five independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

impairment. Previous studies have shown that certain polysaccharides, such as Astragalus, Cornus, Ganoderma atrum and apple pomace polysaccharides, can protect the mitochondria by scavenging ROS and increasing the activity of antioxidant enzymes, which ultimately ameliorate mitochondrial dysfunction and improve mitochondrial energy metabolism (Bing et al., 2014; Chen et al., 2016; Huang et al., 2016; Li et al., 2009, 2011; Lu et al., 2013). In our study, palmitate-treated HepG2 cells showed a marked increase in mitochondrial ROS (Fig. 7A–C) and a loss of MMP (Fig. 8A). Treatment with TYAP attenuated the increase in ROS and prevented the loss of MMP, thereby effectively improving the mitochondrial respiratory function (Fig. 8B and C). TYAP showed a significant protective effect against palmitate-induced mitochondrial dysfunction and toxicity in HepG2 cells. Dietary modification is considered an alternative strategy to reduce the risk of liver injury, and the increased consumption of apples containing high levels of antioxidant phytochemicals has been recommended to prevent or slow the oxidative stress caused by free radicals. Our results demonstrated that TYAP successfully ameliorated HFD-induced oxidative stress, which could be attributed to the ability of TYAP to scavenge mitochondrial ROS to improve the mitochondrial respiratory function. As an abundant compound derived from otherwise wasted thinned young apples, TYAP is expected to be a promising bioactive food ingredient that can be used as a novel natural supplement in preventing hepatic metabolic disorder.

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