Effects of Dietary Apple Polyphenols Supplementation on Hepatic Fat Deposition and Antioxidant Capacity in Finishing Pigs

Simple Summary Excessive fat deposition (5–10%) in the liver could lead to liver damage and nonalcohol fatty liver disease (NAFLD). However, there is no satisfactory safe and effective measure of preventive and therapeutic treatments so far. Thus, the prevention of excessive fat deposition through diet modification might be a better strategy to protect humans from metabolic diseases. Due to the anatomical and physiological similarities between humans and pigs, the present study took the finishing pig as an animal model to investigate the effects of apple polyphenols on hepatic fat deposition and antioxidant capacity and their mechanisms. The present study indicated that apple polyphenols might be an effective dietary supplementation for decreasing the excessive fat deposition in liver tissue, improving lipid profiles and increasing the antioxidant capacity of finishing pigs. This study provides a better preventive strategy to protect humans from excessive fat deposition in the liver. Abstract Excessive fat deposition in the liver could lead to fatty liver and an increased risk of many metabolic diseases. Apple polyphenols (APPs), the major antioxidants in apples, possess wide-ranging beneficial biological functions. The present study aimed to investigate the effects of APPs on hepatic fat deposition and antioxidant capacity in finishing pigs, and their mechanisms. Results showed that APPs improved lipid profiles, increased antioxidant enzyme activities and reduced the fat deposition in the liver. In the liver, SOD1, CAT, GPX1, GST, NF-E2-related nuclear factor 2 (Nrf2), hormone sensitive lipase (HSL), carnitine palmitoyl transferase-1b (CPT1b), peroxisome proliferator-activated receptor α (PPARα), cholesterol 7α-hydroxylase (CYP7A1) and low-density lipoprotein receptor (LDL-R) mRNA levels were increased by APPs, while Kelch-like ECH-associated protein 1 (Keap1) mRNA level, C16:0 and C20:4n-6 proportions and Δ9-18 dehydrogenase activity were decreased. In conclusion, this study indicated that APPs might be an effective dietary supplementation for improving lipid profiles, increasing antioxidant capacities and decreasing fat deposition in the liver.


Sample Collection
After a 49-day period of feeding trial, blood samples from the external jugular vein of all pigs were collected after 12 h fasting, and rested at room temperature for 30 min. After being centrifuged at 3000× g for 10 min at 4 • C, the serum samples were collected and stored at −20 • C. Eighteen pigs (six per treatment, one per pen close to the average BW of the treatment) were selected and electrically stunned. Fresh liver samples were collected and stored at −80 • C until analysis for hepatic parameters, fatty acid profiles, ether extract content and gene expression.

Fat Deposition Analysis
About 50 g liver samples were sliced up, weighed, placed in a weighing bottle and reweighed. Weighing bottles were placed into a freeze dryer with a temperature of −50 • C for 48 h, and then reweighed. The difference in the weights of initial and dried samples was used to calculated the moisture percentage. Subsequently, dried samples were pulverized using a muller, and then used for fatty acid profiles and ether extract content analysis. The measurement of ether extract content was performed according to the methods of the Association of Analytical Chemists [23].

Serum Antioxidant and Biochemical Analysis
The activities of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and the contents of malonaldehyde (MDA), triacylglycerol (TG), total cholesterol (T-CHO), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) in serum were determined, using corresponding assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China), according to the manufacturer's instructions.

Hepatic Antioxidant and Biochemical Analysis
Liver samples and precooled 0.9% saline were added proportionally at a ratio of 1:9 into a 1.5 mL centrifuge tube and subjected to sonication in an ice bath. After being centrifuged at 2500 rpm for 10 min at 4 • C, the supernatant was collected and used to measure the activities of T-AOC, T-SOD, CAT and GSH-Px, and the contents of MDA, TG and T-CHO in triplicate. The total protein of the supernatant was measured by Coomassie blue staining method, using an assay kit purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).

Analysis of the Fatty Acid Profile
Lipid in the liver was extracted according to the method described by Folch et al. [24]. The extracted lipid was hydrolyzed in 2 mL KOH-methanol (C = 0.5 mol/L) with shaking for 1 min. The mixture was reacted in 95 • C water with continual shaking for 10 min. The free fatty acid mixture was esterified in 2 mL BF 3 -methanol solution (W = 10%) with shaking for 10 s. The mixture was reacted in 80 • C water with continual shaking for 20 min. Then, 1 mL n-hexane and 5 mL saturated NaCl solution were added into the mixture. After shaking for 1 min, the mixture was centrifuged at 3000 rpm for 15 min. 800 µL fatty acid methyl esters were collected and analyzed with a GC-2010 plus gas chromatography (Shimadzu, Japan).

Real-Time Quantitative PCR
Briefly, total RNA extraction, reverse transcription and real-time quantitative PCR were performed according to the method described by Zhang et al. [25], using RNAiso Plus reagent (TaKaRa, Dalian, China), primeScript RT reagent kit with gDNA eraser (TaKaRa) and SYBR ® Premix Ex Taq™ II reagents (TakaRa), respectively, according to manufacturer's directions. The primers of real-time Quantitative PCR are listed in Table 2. Relative mRNA levels of related genes in the liver were calculated by the 2 −∆∆Ct method, with glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA as a reference gene [26].

Statistical Analyses
Data, presented as mean ± SE, were analyzed by the one-way ANOVA and Duncan's multiple range test, using statistical software SPSS 22.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 and 0.05 ≤ P < 0.10 were used to determine statistical significance and tendency among results, respectively.
quantitative PCR are listed in Table 2. Relative mRNA levels of related genes in the liver were calculated by the 2 -ΔΔCt method, with glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA as a reference gene [26].

Statistical Analyses
Data, presented as mean ± SE, were analyzed by the one-way ANOVA and Duncan's multiple range test, using statistical software SPSS 22.0 (SPSS Inc.). P < 0.05 and 0.05 ≤ P < 0.10 were used to determine statistical significance and tendency among results, respectively.

Fat Deposition
As shown in Figure 1, compared with the control group, the content of liver ether extract was significantly decreased (P < 0.05) in the 0.04% APPs diet group. There was no difference (P > 0.10) in fat deposition between 0.04% and 0.08% APPs diet groups (Figure 1). Results of each group are represented as the mean ± SE from six pigs. Within a panel, different letters differ significantly at P < 0.05. CON, basal diet; 0.04% APPs, basal diet with APPs at 0.04%; 0.08% APs, basal diet with APPs at 0.08%. APPs = Apple polyphenols; EE = Ether extract.

Serum Parameters
As shown in Table 3, dietary APPs supplementation had no effect (P > 0.10) on the activities of serum T-SOD and GSH-Px, as well as on the contents of LDL-C, HDL-C and MDA, but resulted in greater (P < 0.05) T-AOC activity and lower TG content. In addition, compared with the control group, the serum T-CHO content was remarkably reduced (P < 0.05) in the 0.04% APPs diet group, and the 0.08% APPs had significantly decreased (P < 0.05) serum CAT activity (Table 3). There was no difference (P > 0.10) in serum parameters between the 0.04% and 0.08% APPs diet groups (Table 3).

Serum Parameters
As shown in Table 3, dietary APPs supplementation had no effect (P > 0.10) on the activities of serum T-SOD and GSH-Px, as well as on the contents of LDL-C, HDL-C and MDA, but resulted in greater (P < 0.05) T-AOC activity and lower TG content. In addition, compared with the control group, the serum T-CHO content was remarkably reduced (P < 0.05) in the 0.04% APPs diet group, and the 0.08% APPs had significantly decreased (P < 0.05) serum CAT activity (Table 3). There was no difference (P > 0.10) in serum parameters between the 0.04% and 0.08% APPs diet groups (Table 3).

Hepatic Parameters
As shown in Table 4, dietary APPs supplementation had no effect (P > 0.10) on hepatic T-AOC, T-SOD and CAT activities, but resulted in lower (P < 0.05) MDA, TG and T-CHO contents. In addition, the hepatic GSH-Px activity was remarkably increased (P < 0.05) in the 0.08% APPs diet group, compared with the basal and 0.04% APPs diet groups (Table 4).

Hepatic Lipid Metabolism-Related Gene mRNA Levels
As shown in Table 6, dietary APPs supplementation had no effect (P > 0.10) on hepatic ACC, FAS and HMG-CoAR mRNA levels, but resulted in higher (P < 0.05) HSL, CPT1b and CYP7A1 mRNA levels. The 0.04% APPs tended to decrease the mRNA level of HMGCoAR (0.05 ≤ P < 0.10) ( Table 6). In addition, the hepatic PPARα and LDL-R mRNA levels were significantly upregulated (P < 0.05) in the 0.08% APPs diet group, compared with the basal and 0.04% APPs diet groups (Table 6).

Discussion
Excessive fat deposition in the liver is one of the manifestations of unhealthy metabolic obesity and increases the risk of NAFLD and cirrhosis [27]. Our study found that dietary APPs supplementation decreased hepatic fat deposition and amounts of TG and T-CHO in finishing pigs. Lipogenesis, Animals 2019, 9, 937 8 of 11 lipolysis and fatty acid oxidation are the three key steps of lipid metabolism. Therefore, we explored the mechanisms of the fat-lowering action of APPs, by detecting the expression of genes related to these three steps. When lipogenesis occurs, ACC catalyzes the carboxylation of acetyl CoA, which is the rate-limiting step of de novo lipogenesis [28]. FAS controls the synthesis of palmitic acid from malonyl CoA and is the decisive factor for de novo lipogenesis in tissue [29]. When lipid mobilization occurs, HSL, as the rate-limiting enzyme for lipolysis, controls the first step in the decomposition of triglyceride, and hydrolyzes the triglyceride to diglyceride and fatty acid [30]. Subsequently, produced fatty acids enter the mitochondria and then are further oxidized to produce energy [31]. Acyl carnitine is the main form of fatty acid transported into mitochondria, and CPT-1b is a rate-limiting enzyme for fatty acid β-oxidation, and catalyzes the formation of long-chain acyl carnitine from free carnitine and acyl CoA [32,33]. PPARα can regulate the mRNA expression level of CPT-1b [31]. Therefore, PPARα and CPT1b are the marker genes for fatty acid β-oxidation. Previous study reported that APPs inhibited the Dex-induced lipogenesis in human sebocytes by decreasing ACC and FAS mRNA levels [34] and increased the mRNA levels of PPARα and CPT-II in the liver [12,35,36]. In this study, we showed that dietary APPs supplementation had no significant effect on ACC and FAS mRNA levels in liver tissue, which was inconsistent with the previous study, suggesting that the fat-lowering effect of APPs might not be mediated by hepatic lipogenesis. Moreover, we found that APPs remarkably increased HSL, PPARα and CPT1b mRNA levels in the liver of finishing pigs, suggesting that the fat-lowering effect of APPs might be mediated by hepatic lipolysis and fatty acid β-oxidation.
It has been reported that obesity indexes were positively correlated with the proportions of serum C16:0, C16:1n-7, C18:0, C18:3n-6, C20:3n-6, C20:4n-6 and C20:5n3, as well as the activities of ∆9 and ∆6 dehydrogenase [37]. ∆9 dehydrogenase can catalyze the dehydrogenation of C16:0 and C18:0 to C16:1 and C18:1n9, and is positively correlated with obesity [37,38]. The work of Ntambi et al. has confirmed that the decrease of ∆9 dehydrogenase activity could improve insulin sensitivity in mice [39]. The work of Zhou et al. has reported that the decrease of ∆9 dehydrogenase activity in the liver helped to inhibit hepatic fat deposition [40]. Moreover, there is a close relationship between fatty acid composition and metabolic syndromes in individuals, such as insulin resistance and diabetes [41]. The proportions of C16:0 and C20:4n-6 were reported to be associated with insulin action [41,42]. In our present study, APPs significantly decreased the proportions of C16:0 and C20:4n-6, as well as the activity of ∆9-18 dehydrogenase, suggesting that APPs could regulate fatty acid composition and increase insulin sensitivity.
As for cholesterol metabolism, HMG-CoAR, LDL-R and CYP7A1 are the marker genes in the synthesis, reabsorption and transformation of cholesterol, respectively. An amount close to 70%~80% of the cholesterol in the body is derived from endogenous biosynthesis, so limiting the synthesis of cholesterol is the most effective measure to reduce the total cholesterol [43]. HMGCoAR is the rate-limiting enzyme in the de novo synthesis of cholesterol. The work of Xu et al. has found that HMGCoAR mRNA level was reduced when ApoE −/− mice were orally treated with APPs [12]. In the present study, we found that dietary 0.04% APPs supplementation tended to decrease the mRNA level of HMGCoAR. LDL-R is located in the hepatocyte membrane and can combine with LDL to reabsorb cholesterol from circulating LDL-C. The work of Tenore et al. reported that apple polyphenolic extracts increased LDL-R binding activity in HepG2 cells [44]. Consistently, this study indicated that APPs could upregulate the mRNA level of hepatic LDL-R in finishing pigs. In hepatocyte, most of the cholesterol has been transformed to bile acid to maintain cholesterol homeostasis and prevent cholesterol over-accumulation in body [45]. The rate-limiting enzyme of this step is CYP7A1, which converts cholesterol to 7α-hydroxycholesterol [43]. The work of Osada et al. reported that, when rats were fed a high-cholesterol diet with 0.2%, 0.5% and 1.0% APPs, APPs could increase the activity of hepatic CYP7A1, thereby increasing the excretion of acidic steroids in feces [46]. In this study, we showed that CYP7A1 mRNA levels in the liver increased when finishing pigs were fed a diet with APPs. Our present study indicated that APPs could increase cholesterol reabsorption and transformation, which might account for the blood cholesterol lowering effect of APPs.
The antioxidant systems are the central defense lines to protect the body from oxidative stress and activated by multifarious bioactive substances and antioxidant related genes, including Nrf2-Keap1 pathway [47]. Nrf2 is a key activator of antioxidant enzymes related genes and suppressed by Keap1 in cells [48]. When the body suffers from oxidative stress, the Nrf2 dissociates from Keap1 and is phosphorylated, to stimulate the expression of antioxidant related enzymes, such as CAT, SOD1, GPX1 and GST [49]. Consistent with previous research in rodents [12,18], our present study indicates that apple polyphenols could significantly increase the activities of CAT, T-AOC, T-SOD, GSH-Px, and the mRNA levels of Nrf2, SOD1, CAT, GPX1, GST, but decrease the content of MDA. In addition, the results of our present study show that the mRNA level of Keap1 in liver tissue was decreased by apple polyphenols, suggesting that the increased Nrf2 expression might be partly mediated by an inhibition of Keap1 expression.

Conclusions
Taken together, our present study indicates that APPs might be an effective dietary supplementation for improving lipid profiles and antioxidant capacities, as well as decreasing excessive fat deposition in liver tissue. As for the mechanisms of this, we provided evidence that the liver fat-lowering effect of APPs might be due to the increase in lipolysis and fatty acid oxidation, and the decrease of T-CHO by APPs might be attributed to an increase in cholesterol reabsorption and transformation. We also provided evidence that its effect on increasing antioxidant capacities might be attributed to the stimulation of the Nrf2-Keap1 system.