Differential effects of EPA, DPA and DHA on cardio-metabolic risk factors in high-fat diet fed mice

Author’s Accepted Manuscript Differential effects of EPA, DPA and DHA on cardio-metabolic risk factors in high-fat diet fed mice Xiao-fei Guo, Andrew J Sinclair, Gunveen Kaur, Duo Li www.elsevier.com/locate/plefa

PII: DOI: Reference:

S0952-3278(17)30120-5 http://dx.doi.org/10.1016/j.plefa.2017.09.011 YPLEF1875

To appear in: Prostaglandins Leukotrienes and Essential Fatty Acids Received date: 10 May 2017 Revised date: 14 September 2017 Accepted date: 19 September 2017 Cite this article as: Xiao-fei Guo, Andrew J Sinclair, Gunveen Kaur and Duo Li, Differential effects of EPA, DPA and DHA on cardio-metabolic risk factors in high-fat diet fed mice, Prostaglandins Leukotrienes and Essential Fatty Acids, http://dx.doi.org/10.1016/j.plefa.2017.09.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Differential effects of EPA, DPA and DHA on cardio-metabolic risk factors in high-fat diet fed mice Xiao-fei Guo1, Andrew J Sinclair2,3, Gunveen Kaur4, and Duo Li1,3,5

1

Department of Food Science and Nutrition, Zhejiang University, Hangzhou,

China, 2School of Medicine, Deakin University, Geelong, Australia, 3Department of Nutrition and Dietetics, Monash University, Melbourne, Australia, 4Institute for Physical Activity and Nutrition (IPAN), School of Exercise and Nutrition Sciences, Deakin University, Melbourne, Australia, 5Institute of Nutrition and Health, Qingdao University, Qingdao, China.

Corresponding Author Professor Duo Li, Department of Food Science and Nutrition, Zhejiang University, Hangzhou, China; Institute of Nutrition & Health, Qingdao University, Qingdao, China E-mail: [email protected]; [email protected]

Running title：n-3 PUFAs and cardio-metabolic risk factors

Funding sources: This study was funded by the National Basic Research Program of China (973 Program: 2015CB553604); by National Natural Science Foundation of China (NSFC: 81773433); and by the Ph.D. Programs Foundation of Ministry of Education of China (20120101110107).

Abstract: The aim of the present study was to assess and compare the effects of eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) supplementation on lipid metabolism in 4 month-old male C57BL/6J mice fed a high-fat diet. The high-fat fed mice showed evidence of fatty liver, obesity and insulin resistance after being on the high-fat diet for 6 weeks compared with the control low-fat diet fed mice. Supplementation of the high-fat diet with either EPA, DPA or DHA prevented the fatty liver, prevented high serum cholesterol and serum glucose and prevented high liver cholesterol levels. DPA (but not EPA or DHA) was associated with a significantly improved homeostasis model assessment of insulin resistance (HOMA-IR) compared with the high-fat fed mice. Supplementation with DPA and DHA both prevented the decreased serum adiponectin levels, compared with EPA and the high-fat diet. In addition, supplementation with DPA and DHA both prevented the increased serum alanine aminotransferase (ALT) levels compared with EPA and the high-fat group, which can be attributed to down-regulation of TLR-4/NF-κB signaling pathway and decreasing lipogenesis in the liver. Therefore, DPA and DHA seem to exert similar effects in cardio-metabolic protection against the high-fat diet and these effects seem to be different to those of EPA.

Keywords: eicosapentaenoic acid; docosapentaenoic acid; docosahexaenoic acid; glucose and lipid metabolism; inflammatory markers

Abbreviations: ACC1, acyl CoA carboxylase 1; ALT, alanine aminotransferase; AMPK, AMP-activated protein kinase; AST, aspartate aminotransferase; CVD, cardiovascular disease; CYP7A1, cholesterol 7a-hydroxylase; DHA, docosahexaenoic acid; DHA group, mice fed a high-fat chow plus DHA-olive oil mixture; DPA group, mice fed a high-fat chow plus DPA-olive oil mixture; Elovl, elongation of very long chain fatty acids protein; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; EPA group, mice fed a high-fat chow plus EPA-olive oil mixture; Fads, fatty acid desaturase; Fas, fatty acid synthase; GPR120, G protein-coupled receptor 120;

HDL-C, high-density lipoprotein cholesterol; homeostasis model assessment of insulin resistance, HOMA-IR; IL-1β, interleukin-1β; IL-6, interleukin-6; LDL-C, low-density lipoprotein-cholesterol; MCP-1, monocyte chemoattractant protein-1; NAFLD, non-alcoholic fatty liver disease; n-3 PUFAs, omega-3 polyunsaturated fatty acids; HF-OO group, mice fed a high-fat chow plus olive oil; PPAR-α, peroxisome proliferator activated receptor-a; SCD, stearoyl-CoA desaturate; SREBP-1c, sterol regulatory element binding protein-1c; T2DM, type 2 diabetes mellitus; TAG, triglyceride; TC, total cholesterol; TLR-4/NF-κB, toll-like receptor-4/nuclear factor-κB; TNF-α, tumor necrosis factor-α.

1. Introduction Cardio-metabolic disorder, formerly called metabolic syndrome, is widely defined as the combination of risk factors responsible for type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) [1]. With changes in lifestyles, the prevalence of metabolic syndrome is predicted to exceed 50% in the U.S population over 50 years of age, owing to obesity [2]. Interactions of genetics and environmental factors are responsible for the initiation and development of metabolic syndrome. Regarding environmental factors, a dietary pattern of excess calories, rich in fat and simple carbohydrates contributes to the development of obesity. Excessive caloric intake increases adipose tissue and fat storage, causing increases in plasma free fatty acids (FFA) and accumulation of triglycerides in liver and muscle which eventually leads to decreased capacity of glucose and lipid metabolism in peripheral tissues in response to insulin[3,4], resulting in insulin resistance and metabolic syndrome. Increased adipose tissue also leads to increases in cytokine production causing inflammation which further exacerbates the insulin resistance. There is a substantial literature which has revealed that supplementation with long-chain omega-3 polyunsaturated fatty acids (n-3 PUFAs) is associated with improving glucose and lipid metabolism in subjects with metabolic syndrome [2,5,6]. Long-chain n-3 PUFAs; eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) have been widely researched, and shown to have cardio-protective effect by the anti-inflammatory effects of their

meditators, reductions in plasma TAG levels, reducing platelet aggregation, lowering blood pressure [7], and activating peroxisome proliferator activated receptors (PPARs) and G protein-coupled receptor 120 (GPR120) signaling pathways [2,8-10].

DPA n-3 is the predominant n-3 PUFA in beef, goat and lamb flesh and certain marine species such as abalone and menhaden [11]. There have been few studies on the biological effects of docosapentaenoic acid (DPA) owing to the difficulty of its purification from marine oils [12,13]. Recent reviews have summarized the possible role of DPA intake related to cardiovascular health benefits, mental health, and cancers [14,15]. There is only human trial to date with pure DPA showing differential effects compared with pure EPA, in terms of chylomicronemia [16], incorporation into red cell membranes [17] and production of lipid mediators [18]. Current evidence suggests EPA, DPA and DHA may have independent and shared effects for health benefits [5,15].

Although EPA and DHA have been studied in relation to prevention or reversal of metabolic syndrome in rodent models [19-21], there have been no published studies in this area on DPA. Therefore, the objective of the present study was to evaluate and compare the three long-chain n-3 PUFAs on serum biochemical parameters and inflammatory biomarkers and molecular mechanisms related to glucose and lipid metabolism in mice with cardio-metabolic risk factors induced by a high-fat diet.

2. Materials and Methods 2.1. Fatty acids EPA (98%; W/W), DPA (72%; W/W), and DHA (97%; W/W), as ethyl esters, were kindly provided by Bizen Chemical Co., LTD (Okayama, Japan). The PUFAs were diluted in olive oil for administration to the mice. Thirteen grams of each of EPA, DPA and DHA were mixed into 30 grams of olive oil in 50 mL tubes. Then the solutions were blended and sub-packaged into 1.5 ml tubes sealed with nitrogen and stored at -20 oC for later use.

2.2 Animals Male C57BL/6J mice aged 2 months were purchased from Slacom Co., LTD (Shanghai, China). The mice were housed at 23 ± 2 oC under 12 hours light/dark schedule in specific pathogen-free conditions, and were allowed free access to chow and water. Low-fat chow (3.52 kcal per gram; 10, 20 and 70 % of energy from fat, protein and carbohydrate, respectively) and high-fat diet (4.59 kcal per gram; 45, 20 and 35% of energy from fat, protein and carbohydrate, respectively) were purchased from Slacom Co., LTD (Shanghai, China). The low-fat diet contained 5.28% soybean oil (Supplementary Table 1). The high-fat diet contained by weight: 41.1% carbohydrates (including 7% sucrose), 18.7% lard oil, 3.2% soybean oil, 21.7% crude protein and 1.2% cholesterol, as shown in Supplementary Table 2. The % energy contribution from fat in high fat diet was 45% and in the low-fat diet 10% of the total energy.

Prior to starting the high-fat diets, the mice were allowed free access to water and normal chow for a one week period of acclimatization. After 1 week of acclimatization mice in the control group received chow diet while other groups received high fat diet. After 1 week on respective diets, the mice were divided into 5 groups with randomized block design, according to body weight (n = 10 each group): (1) control group was fed a low-fat chow accompanied by gavage with 0.1 ml olive oil daily (control group); (2) high-fat group was fed a high-fat diet accompanied by daily oral feeding with 0.1 ml olive oil (HF-OO group); (3) EPA group was fed a high-fat diet accompanied by daily gavage with 0.1 ml EPA-olive oil mixture (1:2.3; W/W) (HF-EPA group); (4) DPA group was fed a high-fat diet accompanied by daily oral feeding with 0.1 ml DPA-olive oil mixture (1:2.3 W/W) (HF-DPA group), and (5) DHA group was fed a high-fat diet accompanied by daily gavage with 0.1 ml DHA-olive oil mixture (1:2.3 w/w) (HF-DHA group). The dose of n-3 PUFAs was based on a previous study, in which the purified EPA/EPA/DHA were added to the diet at 1 gram per 100 g diet [12]. Since a mouse eats approx. 5 g food per day, this

equates to 50 mg of DPA per day. In the present 6-week study each mouse was administered 0.1 ml of each n-3 fatty acid by oral feeding, with oil containing 27.2 mg per day of each n-3 PUFAs. The food was replaced every 3 days and the weight of the remaining food was recorded. The body weights of the mice were measured weekly throughout the experiment. The study was approved by Ethics Committee of the College of Biosystems Engineering and Food Science at Zhejiang University, China (ZJU-BEFS-2015016).

2.3. Collection of tissues After administration of n-3 PUFAs for 6 weeks, the animals were fasted for 12 hours and then humanely euthanized by decapitation. Blood samples were collected and serum was obtained by centrifugation at 350 g for 15 minutes at 4 oC, and immediately stored at -80 oC freezer until further use. Next, the liver, epididymal, and perirenal adipose tissues were collected according to defined anatomical landmarks. The weights of the tissues were recorded, and the tissues were frozen in liquid nitrogen and transferred to -80 oC freezer until further analysis.

2.4. Determination of serum biochemical parameters The concentrations of serum glucose, triglyceride (TAG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), alanine transaminase (ALT), and aspartate transaminase (AST) were determined by an automated analyser (Model XE-2100; Sysmex Kobe, Japan). In addition, the concentrations of serum adiponectin, insulin, uric acid and tumor necrosis factor-α (TNF-α) were measured by ELISA kits, in accordance with manufacturer’s instructions (ShangHai Lengton Bioscience Co., LTD. Shanghai, China). Homeostasis model assessment of insulin resistance (HOMA-IR, calculated as fasting (insulin (mU/L) × fasting glucose (mmol/L) / 22.5) was used as a surrogate biomarker indicating insulin resistance [22]. Low density lipoprotein-cholesterol (LDL-C) was calculated based on Friedwald formula [23]: LDL-C = TC – (HDL-C) – TAG / 2.2

2.5. Determination of liver TAG and TC After homogenizing 0.1 gram of liver in methanol, the liver solutions were centrifuged at 2500 rpm for 10 minutes at 4 oC. Then the supernatants were used for the measurement of TAG and TC levels with colorimetric methods in accordance with the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.6. Determination of fatty acid composition in liver phospholipid The fatty acid composition was analyzed by gas-liquid chromatography as described previously [24]. Briefly, 100 mg of liver tissue per mouse was used and the lipids were extracted in chloroform/methanol (1:1) solution at 4 oC for 24 hours, and the phospholipid fraction was obtained by separating these from the other lipids by thin-layer chromatography. The methyl esters of the fatty acids of the phospholipids were prepared using transmethylation reagent (concentrated sulfuric acid in methanol (0.9 mol/L) and then the fatty acid methyl esters were separated by gas-liquid chromatography. The composition of individual fatty acids was expressed as mole percentages of total phospholipid fatty acid methyl esters.

2.7. RNA extraction and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) The total RNA was extracted from mouse livers using the UNIQ-10 Colum Trizol Total RNA Isolation Kit (Sangon Biotech Co., Ltd., Shanghai China), and the cDNA was then synthesized using Prime Script RT Reagent kit (Takara Bio Co., Ltd., Dalian, China) in accordance with the manufacturer’s protocols. Quantitative RT-PCR was conducted in ABI ViiA 7 real-time PCR system (Applied Biosystems, Foster, CA, USA) using SYBR Premix Ex Taq (Tli RNaseH Plus) kit (Takara Bio Co., Ltd., Dalian, China). The thermal cycling program was implemented as described previously [25]. The abundance of mRNA was normalized to β-actin, and the relative quantification of gene expressions was calculated using 2-△△CT method. The primer sequences that were used in the present study are listed in Supplementary Table 3.

2.8. Western blot analysis of liver tissue The effects of n-3 PUFAs supplementation on protein expression of liver were assessed using immunoblot. The methodology of immunoblot analysis was conducted as described previously [26]. In brief, 30 mg of liver was separated and homogenized with liquid nitrogen. Then the protein of liver was extracted using lysate solution (Goodbio Technology Co., Wuhan, China), and the protein concentration was determined using bovine serum albumin as a standard. Total of 30 μg of protein was loaded into the SDS-PAGE gels and transferred to PDVF membranes (Millipore, Darmstadt, Germany). The membrane sheets were then incubated with antibodies overnight, including total anti-AMP-activated protein kinase (AMPK) (1:1000 dilutions, Proteintech Group, Inc, American), anti-SREBP (sterol regulatory element binding protein)-1c (1:1000 dilutions, Bioss Group, Inc, American), anti-TLR-4 (1:1500 dilutions, Proteintech Group, Inc, American) and anti-β-actin (1:1000 dilutions,

Goodbio

technology

Co.,

Wuhan)

followed

by

horseradish

peroxidase-conjugated secondary antibody (1:3000 dilutions, Goodbio technology Co., Wuhan, China) for 2 hours. After washing, the immunoreactivity of protein expression was visualized using the chemiluminescence (ECL) (GE Health Care UK, Ltd., Buckinghamshire, England).

2.9. Histological analysis of liver and epididymal adipose tissue Samples of liver and epididymal adipose tissue (approximately 100 milligrams per tissue) were fixed with 4% formalin solution for 1 day, then the tissues were dehydrated and embedded in paraffin. Liver samples were stained with Oil Red O, washed and counterstained with hematoxylin to detect cellular structure and lipid accumulation. The liver and epididymal adipose tissues were also stained with hematoxylin and eosin (H&E) to visually observe the size of adipocytes and macrovesicular steatosis of them, respectively. The image acquisition and analysis system incorporated in an inverted fluorescence microscope (Nikon ECLIPSE TI-SR, Japan) was used to capture the tissue sections at 200 × magnification in 3 fields per

liver and adipose tissue sections.

2.10. Statistical analysis The data of the present study were expressed as mean ± standard deviation (SD). One way ANOVA was used to compare the differences among experimental groups, following by Bonferroni’s multiple-comparison tests. Statistical analysis was performed using Stata 13.0 for Windows (Stata Corp, College Station, TX, USA). A p-value of < 0.05 was considered statistically significant.

3. Results 3.1. Bodyweight and caloric intake The animals on the high-fat diets showed significantly increased cumulative weight gain compared with the control group after 6 weeks of intervention (Fig. 1). In addition, although not significant, the cumulative weight gain was decreased by 12.3%, 11.0% and 11.1% in the HF-EPA, -DPA and -DHA groups, respectively, compared with the HF-OO group. The low-fat control group had a significantly higher food intake per day compared with the other groups (Fig. 2A). Mice given DHA had a significantly decreased caloric intake compared with the low-fat control group (Fig. 2B). There were no other significant differences in caloric intakes between the groups. The HF-OO group showed a significantly higher liver weight compared with the other groups. There were no significant effects of diet on the weight of epididymal and perirenal adipose tissue (Table 1).

3.2. Effects of n-3 PUFAs on serum cardio-metabolic risk factors The HF-OO group had significantly increased serum concentrations of LDL-C, TC, glucose, insulin and ALT, compared with the control group (Table 2). Administration of EPA, DPA and DHA showed significantly decreased serum concentrations of LDL-C, TC, glucose and ALT, compared with the HF-OO group. In addition, the DPA supplemented group showed a significantly decreased serum AST concentration compared with HF-OO- and EPA-groups (p<0.05). A surrogate marker of insulin

resistance, HOMA-IR was calculated based on fasting glucose and insulin levels. It was observed that HOMA-IR was significantly increased in HF-OO group compared with the low-fat control group. DPA supplementation, but not EPA and DHA, significantly reduced the HOMA-IR (p<0.05). No significant differences were observed in the serum concentrations of uric acid.

3.3. Influence of n-3 PUFAs on inflammatory biomarkers Serum TNF-α levels were significantly increased in the HF-groups compared with the low-fat control group, with no significant differences between the HF-groups. In contrast, serum adiponectin was significantly decreased in the HF-OO group compared with the low-fat control group, but was significantly higher in HF-DPA and -DHA groups compared with the control, HF-OO and HF-EPA groups, respectively (Table 2).

3.4. Effects of n-3 PUFAs on hepatic TAG and TC contents Mice fed the high-fat diet had a significantly increased hepatic TC content compared with the low-fat control mice. Administration of EPA, DPA and DHA prevented the increase in hepatic TC concentration, compared with the HF-OO group (p<0.05) (Table 1). The concentrations of hepatic TAG in all the HF-groups was not significantly different from the low-fat control group, but the concentrations of hepatic TAG were significantly lowered in HF-EPA and -DHA groups compared with HF-OO group (Table 1).

3.5. Effects of n-3 PUFAs on liver and epididymal adipose tissues morphology A dramatic hypertrophy of adipocytes in the adipose tissue was observed in the HF-OO group, compared with the control group (Fig. 3A). Administration of EPA, DPA or DHA prevented the increase in the size of adipocytes seen in the HF-OO group. Extensive macrovesicular and microvesicular steatosis, based on H & E straining, were observed in the liver of mice fed the HF-OO diet (Fig. 3B). Supplementation with n-3 PUFAs showed an evident decrease in hepatic steatosis,

especially for the HF-DPA group. Furthermore, significant liver lipid accumulation was observed for the HF-OO group compared with the low-fat control group using Oil Red O staining (Fig. 3C). The morphological visual inspection demonstrated that the lipid droplet size was the largest in the HF-OO group, and the size was dramatically less in the HF-DPA and HF-DHA groups.

3.6. Effects of n-3 PUFAs supplementation on liver phospholipid fatty acid composition The liver phospholipid levels of EPA and DHA were increased significantly by supplementation with each of the three different n-3 PUFAs, with the highest EPA level being found in the EPA supplemented group and the highest DHA level seen in the DHA group (Fig. 4). The proportion of DPA was significantly increased by EPA and DPA supplementation, but not by DHA supplementation. The proportion of arachidonic acid (AA) was significantly lowered with n-3 PUFAs supplementation. The administration of the three different n-3 PUFAs significantly increased the ratio of EPA+DPA+DHA/AA, with the highest ratio being found in the HF-DHA group.

3.7. Effects of n-3 PUFAs supplementation on mRNA expression and western blot analysis The effects of n-3 PUFAs supplementation on mRNA expression of lipid metabolism and inflammatory cytokines are shown in Table 3. The findings of the present study showed that HF-OO group significantly upregulated the toll-like receptor (TLR)-4/nuclear factor (NF)-κB signaling pathway. The mRNA expression levels of TLR-4 and MyD88 were significantly increased in the HF-OO-group, as compared with low-fat control group. Conversely, administration of n-3 PUFAs significantly lowered the mRNA expression levels of these genes. Accordingly, the mRNA expression levels of inflammatory cytokines regarding TNF-α, interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1 were also significantly increased in the HF-OO group compared with the control and were lowered with n-3 PUFAs supplementation compared with the HF-OO group. Furthermore, administration of

DPA and DHA significantly decreased mRNA expression levels of IL-6 compared with the HF-diet. The HF-OO group showed a significantly reduced expression of adiponectin compared with the control group, and in contrast, DPA and DHA groups showed significantly raised adiponectin expression levels compared with the HF-OO-group and the low-fat control group. The mRNA expression levels of acyl-CoA carboxylase 1 (ACC1), stearoyl-CoA desaturate (SCD) and Fas (fatty acid synthase) were significantly reduced by the n-3 PUFAs supplementation compared with the HF-OO-group. Conversely, mRNA expression levels of cholesterol 7a-hydroxylase (CYP7A1) were significantly elevated in the DPA and DHA groups compared with the HF-OO group. Administration of DHA showed significantly decreased mRNA expression level of SREBP-1c compared with the HF-diet. It was observed that the n-3 PUFAs supplementation did not exert significant effect on mRNA expression levels of the genes involved in β-oxidation compared with HF-OO-group, including UCP2 (uncoupling protein 2), CPT1A (carnitine palmitoyltransferase 1A), PPAR-α and AOX (acyl-CoA oxidase). There were no significant effects of the three n-3 PUFAs on mRNA expression levels of Fads1 (fatty acid desaturase 1), Fads2, Elovl4 (elongation of very long chain fatty acids protein 4) and Elovl6, compared with HF-OO-group. The effects of n-3 PUFAs supplementation on western blot analysis are shown in Fig. 5. The results showed that DHA supplementation attenuated TLR-4 expression compared with the HF-OO, HF-EPA and HF-DPA-groups. DHA supplementation also reduced SREBP-1c expression compared with the HF-OO, and HF-EPA groups. The western blot analysis indicated that n-3 PUFAs supplementation dramatically enhanced the expression of AMPK compared with HF-OO-group and low fat control group.

4. Discussion Obesity is defined by excess body fat, and is generally associated with a low-grade and chronic inflammatory state [27-30]. Mounting evidence has revealed that the accumulation of white adipose tissue is associated with elevation of macrophage infiltration of the adipose tissue, circulating free fatty acids and cytokines such as

TNF-α, which would trigger TLR-4 signaling pathways and inhibit insulin signaling pathways [28,31,32], leading to various kinds of cardio-metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD), T2DM and CVD [3,4,29,30].

It has been reported that a high-fat diet in mice induces weight gain, insulin resistance and cardio-metabolic syndrome [21,33,34]. In the present study, the HF-OO group exhibited characteristics of the cardio-metabolic syndrome compared with the low-fat control group, such as significantly increased weight gain, increased weight of liver and epididymal adipose tissue, fatty liver, raised hepatic TC content, hypertrophy of epididymal adipocytes, raised serum ALT, TC, glucose, insulin, HOMA-IR and TNF-α and lowered adiponectin levels. In addition, in the HF-OO group there were significant increases in the mRNA expression of the inflammatory markers in the TLR-4/NF-κB pathway.

The present study is the first to evaluate the effects of DPA supplementation on cardio-metabolic risk factors and inflammatory markers in mice fed a high-fat diet and compare them to those of EPA and DHA supplementation. As a whole, the results demonstrated that administration of n-3 PUFAs protected against the high-fat diet-induced disturbances in glucose and lipid metabolism and inflammatory factors, however there were some significant differences in the effectiveness of the 3 different n-3 PUFAs, as shown in Table 4. Overall, supplementation with n-3 PUFAs prevented the increased liver weight, prevented accumulation of fatty droplets in the liver, prevented the hypertrophy of adipocytes and was associated with reduced hepatic TC concentrations, reduced serum TC, glucose and ALT levels, compared with the HF-OO group. In addition, supplementation with n-3 PUFAs prevented the significant increases in inflammatory markers in the TLR-4/NF-κB pathway seen in the HF-OO group.

Supplementation with the three n-3 PUFAs significantly reduced serum fasting glucose concentrations, compared with the HF-OO group. However, no change was

observed in the serum insulin levels in the omega-3 groups compared with HF-OO group. HOMA-IR is generally regarded as a surrogate biomarker of insulin resistance and is calculated (see methods) based on fasting glucose and insulin levels [21]. Our data indicated that the high-fat diet induced insulin resistance in the mice as indicated by significantly increased HOMA-IR levels and that only supplementation with DPA prevented the rise in the HOMA-IR, while all three n-3 PUFAs significantly prevented the increased plasma glucose levels (p<0.001). It has been previously reported that supplementation with EPA could protect against high-fat diet-induced HOMA-IR and glucose intolerance in C57BL/6J mice [21] and in rats [35]. Our study did not show significant reduction in HOMA-IR with EPA and DHA supplementation possibly due to high variability seen for insulin measurements which prevented detecting any possible differences between groups.

White adipose tissue is an active endocrine and paracrine organ. An accumulation of white adipose tissue results in a release of inflammatory cytokines, including MCP-1, TNF-α, IL-1β and IL-6, and other cytokines, which may contribute to disturbances in glucose and lipid metabolism. Unlike the above cytokines, the secretion of adiponectin from adipose tissue, with opposing effects to other inflammatory cytokines, is associated with improved insulin resistance, and glucose homeostasis, and has anti-inflammatory effects [21]. In addition, preventing excess weight gain and lowering insulin resistance are thought to be mediated by adiponectin secretion [36]. The present results showed that the high-fat diet (HF-OO group) significantly reduced the plasma adiponectin levels, suggesting reduced secretion of adiponectin from the adipose tissue. DPA and DHA supplementation prevented the decline in the adiponectin levels in the plasma, which has been reported to be PPAR-γ-dependent [36]. The elevated adiponectin levels could have contributed to the significant improvements in glucose metabolism in these groups. In accordance with our finding, Flachs et al. also reported that partial replacement of vegetable oil with marine fish oil triggered adiponectin secretion in C56BL/6J mice fed a high-fat diet, independent of energy intake [33]. In addition, a significant association has been observed between

plasma n-3 PUFAs and circulating adiponectin in healthy subjects [37]. Perez-Matute et al [38], who fed a cafeteria diet to male Wistar rats also showed that EPA administration was associated with an increased adiponectin level. In the present study, the administration of n-3 PUFAs did not significantly decrease serum TNF-α levels compared with the HF-OO group, in contrast to Perez-Matute et al [38]. In contrast, in the liver there were significant decreases in mRNA expression levels of TNF-α in the n-3 PUFAs supplemented groups compared with the HF-OO group.

High-fat diets are associated with accumulation of visceral adipose depots, leading to increased lipolytic rate. The release of free fatty acids from visceral adipose depots contributes to impaired fat oxidation, and stimulating fatty acid esterification into TAG in the liver [2]. Moreover, free fatty acids can bind TLR-4, and trigger the TLR-4 signaling pathway. The triggering of the signaling results in the activation of the transcription factors NF-κB and activator protein-1 (AP-1), which upregulates gene expression of inflammatory mediators, contributing to the increase in inflammatory cytokines that can lead to insulin resistance and metabolic dysfunction [28,31]. It has been shown that TLR-4 mutant mice had decreased markers of insulin resistance and gene expression levels of inflammatory cytokines induced by fructose, compared with wild-type mice [39]. In the present study, n-3 PUFAs supplementation inhibited the HFD induced TLR-4/NF-κB signaling pathway including the mRNA expression of TLR-4. The western blot analysis demonstrated reduced TLR-4 protein expression in the liver with DHA supplementation but not with EPA and DPA supplementation. The mRNA expression levels can be influenced by post transcriptional events and are not always seen to correlate with protein expression levels. This could be the reason for differences in the reduced mRNA levels of TLR-4 in EPA and DPA group but no difference in the protein expression. DHA supplementation also reduced the mRNA as well as the protein expression of SREBP-1c in the liver tissue. In contrast, there was an increased AMPK expression observed with n-3 PUFAs supplementation. In line with our findings, supplementation with fish oil has been shown to increase hepatic AMPK in rats [40]. Moreover, it has

been reported that stimulation of adiponectin may be responsible for the activation of AMPK [41]. Therefore, the activation of AMPK by n-3 PUFAs supplementation might play pivotal roles regulating lipid synthesis and glucose and lipid metabolism.

We also investigated whether administration of n-3 PUFAs could protect against fatty liver injury in mice induced by the high-fat diet. In clinical settings, elevated serum concentrations of ALT and AST have been generally regarded as surrogate biomarkers associated with NALFD and hepatic steatohepatitis. The high-fat fed mice in this study had a fatty liver and elevated serum concentrations of ALT and AST, and the individual n-3 PUFAs (EPA, DPA and DHA) prevented the rise in ALT with DPA and DHA being the most potent. In combination with histological analysis, supplemental n-3 PUFAs were strongly associated with decreased hepatic steatitis and lipid accumulation, especially for DPA and DHA supplementation. These changes were supported by chemical analysis of the livers which showed that the n-3 PUFAs supplementation significantly reduced the total amount of TAG and TC in the liver compared with the HF-OO-supplemented group. Similar findings showed that EPA and DHA reduced hepatic lobular inflammation, and AST and ALT levels, consistent with findings reported for the treatment of NAFLD in mice fed a high-fat diet [42]. Moreover, in another study histological analysis revealed that supplementation with EPA protected against a high-fat diet induced macrovesicular and microvesicular steatosis [21]. As the primary regulator of lipogenesis in the liver [43], fatty acid synthesis-related gene expression, such as Fas and ACC1, were significantly reduced following the n-3 PUFAs supplementation in the present study.

The possible mechanisms by which n-3 PUFAs supplementation could protect against the high-fat induced fatty liver associated with cardio-metabolic risk factors are shown in Fig. 6 and could include: 1) Suppressing the release of free fatty acids from visceral adipose which can attenuate TLR-4/NF-κB signaling pathway, so the expression levels of inflammatory cytokines would be inhibited. 2) Inhibition of liver lipid synthesis, including SREBP-1c, Fas and ACC1. These changes would result in

decreased liver TAG and cholesterol synthesis. Increased fatty acid β-oxidation would also contribute to reduced accumulation of liver TAG [8]; however in the present study, there were no effects of the n-3 PUFAs on the mRNA expression of important fat oxidation genes. As a transcription factor, PPAR-α is mainly expressed in the liver and is responsible for fatty acid oxidation and lipoprotein metabolism. There are several possible explanations why there were no significant difference observed between groups with respect to mRNA expression level of PPAR-α. First, differences may have been observed if there had been a larger number of animals per treatment group. Second, if protein expression had been measured rather than just gene expression, differences may have been found. Third, the dose of n-3 PUFA may not have been sufficiently high to induce changes in expression in this study.

The n-3 PUFAs supplementation increased the levels of all 3 long-chain n-3 PUFA in the liver phospholipid fraction, rather than specifically enriching the liver in EPA, DPA and DHA, respectively. This suggests rapid conversion of EPA and DPA to DHA, as well as retroconversion of DPA to EPA in this animal model. Associated with the increase in the n-3 PUFAs, there were highly significant reductions in AA. These reciprocal changes would alter the balance between towards an anti-inflammatory environment in the n-3 PUFAs treated groups, which might contribute to decreased risk factors associated with metabolic syndrome [44].

There were some limitations of this study including that the DPA was only 72% pure (Supplementary Table 4), compared with EPA and DHA which were 98% and 97% pure, respectively. The DPA contained other PUFAs including 4.8% EPA, 4.8% 21:5n-3, 6% 22:5n-6 and 8% DHA. Thus, we cannot exclude these other PUFAs might have played some role in the bioactivity of the DPA reported here. Another limitation was that supplementation with n-3 PUFAs lasted only for 6 weeks. A longer period of supplementation might have contributed to significant differences on biomarkers pertaining to glucose and lipid metabolism and inflammatory biomarkers.

In conclusion, EPA, DPA and DHA were equally effective in reducing the fasting blood glucose levels, while DPA was most effective in reducing the HOMA-IR increased by the HFD. The findings of the present study also indicated that DPA as well as DHA groups significantly trigged adiponectin secretion, compared with the EPA group. DPA was equivalent to DHA in protecting against liver injury by decreasing serum concentrations of AST and ALT. Therefore, DPA and DHA seem to exert similar effects in cardio-metabolic protection against the high-fat diet and these effects seem to be different to those of EPA. Whether these differences relate to the different lipid mediators formed from each of these 3 n-3 PUFA is not known [18]. The protective mechanisms of n-3 PUFAs supplementation against the high-fat diet induced cardio-metabolic syndrome in this study can be attributed to down-regulating TLR-4/NF-κB signaling pathway and decreasing lipogenesis in the liver.

Conflict of interest None of the authors declared a conflict of interest.

Acknowledgement We thank Bizen Chemical Co., LTD for the generous provision of the pure supplements. This study was funded by the National Basic Research Program of China (973 Program: 2015CB553604); by National Natural Science Foundation of China (NSFC: 81773433); and by the Ph.D. Programs Foundation of Ministry of Education of China (20120101110107). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contribution X.G, AJS and D.L conceived the study. X.G conducted the experiment and data analysis. X.G, AJS, GK and D.L made significant contribution to interpretation of the data and drafting of the manuscript.

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Fig. 1. Cumulative weight gain for male C57BL/6J mice fed high fat diets with HF-OO, EPA, DPA and DHA supplementation. Values were presented as mean ± standard deviation (SD) (n = 10 animals/groups). Abbreviations: Control, a low-fat chow accompanied by oral feeding with olive oil group; HF-OO, a high-fat chow accompanied by gavage with olive oil group; EPA, a high-fat chow accompanied by gavage with EPA-olive oil mixture group; DPA, a high-fat chow accompanied by gavage with DPA-olive oil mixture group; DHA, a high-fat chow accompanied by gavage with DHA-olive oil mixture group. In the first week the mice were fed high-fat diet, and for the next 6 weeks the mice were fed the high-fat diet accompanied with the fatty acid interventions (+OO, +OO+EPA, +OO+DPA, +OO+DHA). There were no significant differences in cumulative weight gain among the five groups. Fig. 2. Food intake (A) and caloric intake (B) for male C57BL/6J mice fed high fat diets with HF-OO, EPA, DPA and DHA supplementation. Values were presented as mean ± SD (n = 10 animals/group). The means marked with different superscript letters indicated significantly different value relative to others. Fig. 3. Histological analysis in liver and epididymal adipose tissues. A: H&E stained epididymal adipose tissues of male C57BL/6J mice fed high fat diets with HF-OO, EPA, DPA and DHA supplementation. B: H&E stained liver of male C57BL/6J mice. C: Oil Red O was used to stain liver sections of male C57BL/6J mice. Representative sections were from three mice with different kinds of supplementation. Fig. 4. Fatty acid composition of liver phospholipid fed high fat diets with HF-OO, EPA, DPA and DHA supplementation. LCn-3 / LCn-6 = [EPA+DPA+DHA] /AA. * p < 0.05 versus control group; # p < 0.05 versus HF-OO-group. Data were expressed as mean ± SD (n = 10 per group). Fig. 5. Western blot analysis from liver tissue for male C57BL/6J mice fed high fat diets with HF-OO, EPA, DPA and DHA supplementation. Abbreviations: AMPK, AMP-activated protein kinase; TLR-4, toll-like receptor-4; SREBP-1c, sterol responsive element binding protein-1c (n = 3 animals/group).

Fig. 6. The protective mechanisms of n-3 PUFAs supplementation on high fat diets induced cardio-metabolic risk. Abbreviations: ACC1, acyl-CoA carboxylase 1; Fans, fatty acid synthase; FFA, free fatty acids; IL-1 β, interleukin il-1 β; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; MyD88, myeloid differentiation primary response gene 88; TNF-α, tumor necrosis factor-α.

Cumulative weight gain (g)

6

Control HF-OO EPA DPA DHA

4

2

0

0

2

4 Weeks

4.5

a

8

b

b

b b

1.5

0.0

B

ab

ab ab

Calorie intake (g/day)

Food intake (g/day)

a

3.0

15

A

6

b

10

5

0 Control HF-OO EPA

DPA

DHA

Control HF-OO EPA

DPA

DHA

Fatty acid levels (% of total liver phospholipid fatty acids)

30

24

# *

*

#

18

### * **

# ## ***

#

12

# ## ***

6

0

Control HF-OO EPA DPA DHA

****

## **

# ## ***

6:0 7:0 8:0 n-9 n-6 n-6 n-3 n-3 n-3 n-6 C1 C1 C1 18:1 18:2 20:4 20:5 22:5 22:6 3/LC C C C C C C nLC

Table 1 Tissue weight and hepatic contents of TAG and TC for the male C57BL/6J mice fed high fat diets with HF-OO, EPA DPA and DHA supplementation Variable

Control

HF-OO

EPA

DPA

DHA

p-value

Liver wt (g) Epididymal adipose tissue wt (g) Perirenal adipose tissue wt (g) Liver TAG content (mg/g) Liver TC content (mg/g)

1.81 ± 0.174b 1.84 ± 0.459 1.26 ± 0.132 5.10 ± 0.602ab 3.49 ± 1.62b

2.41 ± 0.211a 2.73 ± 0.401 1.63 ± 0.225 6.08 ± 0.699a 7.12 ± 1.11a

1.97 ± 0.201b 2.42 ± 0.595 1.38 ± 0.320 4.66 ± 0.558b 4.14 ± 0.816b

1.92 ± 0.193b 2.50 ± 0.687 1.38 ± 0.309 5.01 ± 0.425ab 4.33 ± 0.480b

1.94 ± 0.173b 2.39 ± 0.222 1.24 ± 0.223 4.06 ± 0.621b 4.21 ± 0.924b

<0.001 0.054 0.326 <0.001 0.001

Values were presented as mean ± SD (n = 10 animals/group). Different letters indicated significant difference between groups.

Table 2 Serum cardio-metabolic parameters of male C57BL/6J fed high fat diets with HF-OO, EPA, DPA and DHA supplementation Variable

Control

HF-OO

EPA

DPA

DHA

p-value

TC (mmol/L) HDL (mmol/L) LDL (mmol/L) TAG (mmol/L) Glucose (mmol/L) Insulin (mU/L) HOMA-IR Adiponectin (mg/L) TNF-α (ng/L) Uric acid (μmol/ml) ALT (U/L) AST (U/L)

1.26±0.15c 1.09±0.17b 0.407±0.003c 0.510±0.090b 2.95±0.47b 8.76±1.89b 1.16±0.49b 7.07±1.01b 91.7±8.9b 51.3±3.48 17.4±2.5c 69.8±9.3a

2.97±0.38a 1.18±0.18ab 1.420±0.108a 0.680±0.062a 3.85±0.35a 13.40±1.88a 2.28±0.49a 4.59±0.91c 138.3±11.8a 71.7±10.0 31±2.9a 71.5±6.7a

2.25±0.38b 1.36±0.10a 0.780±0.108b 0.582±0.057ab 2.98±0.14b 12.00±2.87ab 1.61±0.45ab 6.38±1.7bc 135.2±14.2a 69.7±17.2 24.2±2.1b 67.6±6.0a

2.19±0.10b 1.21±0.02ab 0.642±0.022b 0.583±0.026ab 2.59±0.17b 10.50±2.85ab 1.32±0.35b 9.25±1.14a 123.3±11.5a 67.2±14.7 18.3±1.7c 53.6±7.6b

2.27±0.20b 1.35±0.09a 0.623±0.083b 0.537±0.054b 2.82±0.06b 10.80±1.90ab 1.40±0.31ab 9.68±1.28a 124.5±10.5a 68.2±5.0 17±1.4c 59.8±5.5ab

<0.001 0.008 <0.001 0.012 <0.001 0.032 0.010 <0.001 <0.001 0.283 <0.001 0.001

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, homeostasis model assessment of insulin resistance; LDL-C, low-density lipoprotein-cholesterol; TAG, triglyceride; TC, total cholesterol; Values were presented as mean ± SD (n = 10 animals/group). Different letters indicated significant difference between groups.

Table 3 mRNA expression levels as relative to control in liver Genes

Control

HF-OO

EPA

DPA

DHA

p-value

ACC1 Adiponectin AOX CPT1A CYP7A1 Elovl4 Elovl6 Fas Fads1 Fads2 G6P IL-1 β IL-6 MCP-1 MyD88 PPAR-α SCD SREBP-1c TLR-4 TNF-α UCP2

1.02±0.24b 1.00±0.04b 1.00±0.08 1.00±0.11 1.01±0.10b 1.03±0.30 1.00±0.05 1.00±0.12b 1.09±0.18 1.01±0.18 1.00±0.14a 1.37±0.34b 1.08±0.50c 1.07±0.37b 1.03±0.28b 1.07±0.17a 1.19±0.23b 1.00±0.10b 1.01±0.20b 1.07±0.40b 1.02±0.23

2.94±0.87a 0.60±0.06c 0.79±0.15 0.85±0.19 1.40±0.28b 0.73±0.17 0.83±0.12 2.50±0.47a 1.07±0.20 0.88±0.21 0.50±0.06b 99.24±18.28a 9.26±2.40a 4.18±1.14a 6.77±2.12a 1.02±0.11ab 6.77±1.49a 4.18±1.33a 1.38±0.35a 6.46±1.19a 1.06±0.20

1.64±0.19b 0.84±0.08b 0.87±0.17 0.71±0.16 2.11±0.35ab 0.96±0.12 0.77±0.11 1.29±0.07b 0.93±0.10 0.94±0.10 0.60±0.12b 18.45±3.12b 6.93±1.48ab 1.46±0.27b 2.08±0.16b 0.69±0.17b 1.51±0.31b 2.62±0.52ab 0.64±0.17b 1.19±0.20b 0.79±0.12

1.89±0.25b 1.35±0.09a 0.95±0.06 0.75±0.08 3.23±0.50a 0.83±0.20 0.76±0.16 1.56±0.28b 0.93±0.13 0.77±0.05 0.62±0.11b 2.42±1.50b 5.68±1.49b 0.95±0.17b 1.38±0.20b 0.81±0.06b 1.75±0.97b 2.65±0.52ab 0.74±0.21b 1.39±0.22b 0.81±0.13

1.60±0.30b 1.52±0.12a 0.90±0.08 0.78±0.18 2.65±0.84a 0.78±0.09 0.76±0.06 1.51±0.24b 0.73±0.16 0.65±0.09 0.61±0.13b 10.33±0.94b 5.83±0.66b 0.98±0.16b 1.87±0.75b 1.02±0.10b 0.78±0.11b 1.49±0.48b 0.43±0.08b 2.07±0.20b 0.87±0.09

<0.001 <0.001 0.185 0.096 <0.001 0.344 0.086 <0.001 0.098 0.064 <0.001 <0.001 <0.001 <0.001 <0.001 0.018 <0.001 <0.001 <0.001 <0.001 0.101

Abbreviations: ACC1, acyl-CoA carboxylase 1; AOX, acyl-coenzyme A oxidase; CPT1A, Carnitine palmitoyltransferase 1A; CYP7A1, Cholesterol 7a-hydroxylase; Elovl4, elongation of very long chain fatty acids protein 4; Elovl6, elongation of very long chain fatty acids protein 6; Fas, fatty acid synthase; Fads1, fatty acid desaturase 1; Fads2, fatty acid desaturase 1; G6P, glucose-6-phosphatase; PPAR-α, Peroxisome proliferator-activated receptor alpha; SCD, stearoyl-CoA desaturate; UCP2, uncoupling protein 2.

Table 4 Comparison of the effects of EPA, DPA and DHA on cardio-metabolic parameters, mRNA levels and protein expression Parameter Plasma Glucose HOMA-IR Plasma adiponectin Plasma-ALT Plasma-AST Liver TAG concentration Lipid droplets Liver Adiponectin mRNA expression Liver IL-6 mRNA expression Liver TLR-4 mRNA expression Liver SREBP-1c mRNA expression Liver TLR-4 protein expression Liver SREBP-1c protein expression Liver AMPK protein expression

Effect of HF-OO (compared to Control) Increased Increased Decreased Increased No change No change Increased Decreased Increased Increased Increased Increased No change No change

Comparative effects of n-3 PUFAs Reduced by EPA = DPA = DHA Reduced by DPA Increased by DPA = DHA Reduced by DPA = DHA > EPA Reduced by DPA Reduced by EPA = DHA Reduced by DPA = DHA Increased by DPA = DHA>EPA Reduced by DPA = DHA Reduced by EPA = DPA = DHA Reduced by DHA Reduced by DHA Reduced by DHA Increased by EPA = DPA = DHA

Highlights 1. The present study was the first to three long-chain n-3 PUFAs (eicosapentaenoic acid, EPA; docosapentaenoic acid, DPA; docosahexaenoic acid, DHA) on serum biochemical parameters and inflammatory biomarkers in mice with cardio-metabolic syndrome induced by a high-fat diet. 2. Supplementation with DPA was shown to be more superior to decrease blood glucose and improve insulin resistance compared with EPA and DHA. DPA as well as DHA groups significantly trigged adiponectin secretion, compared with the EPA group. DPA was equivalent to DHA in protecting against liver injury. 3. The protective mechanisms of n-3 PUFAs supplementation were attributed to down-regulating toll-like receptor (TLR)-4/nuclear factor (NF)-κB signaling pathway and decreasing lipogenesis in the liver.