Effect of Increasing Doses of Linoleic and α-Linolenic Acids on High

Jan 8, 2016 - ABSTRACT: Doses and ratio of linoleic acid (LA) and α-linolenic acid (ALA) preventing metabolic syndrome (MS) were investigated. SD rat...
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Effect of Increasing Doses of Linoleic and α‑Linolenic Acids on HighFructose and High-Fat Diet Induced Metabolic Syndrome in Rats Jianan Zhang,† Ou Wang,† Yingjian Guo,† Tuo Wang,‡ Siyi Wang,† Guopeng Li,† Baoping Ji,*,† and Qianchun Deng*,§,⊗ †

Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science & Nutritional Engineering and ‡College of Engineering, China Agricultural University, Beijing 100083, People’s Republic of China § Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, People’s Republic of China ⊗ Hubei Key Laboratory of Lipid Chemistry and Nutrition, Wuhan 430062, People’s Republic of China S Supporting Information *

ABSTRACT: Doses and ratio of linoleic acid (LA) and α-linolenic acid (ALA) preventing metabolic syndrome (MS) were investigated. SD rats were fed (i) basal diet, (ii) high-fructose and high-fat diet (HFFD), (iii) HFFD with increasing-dose LA (0.75 energy-% ALA + 3, 6, 9, 12, 15, and 30 energy-% LA), and (iv) HFFD with increasing-dose ALA (6 energy-% LA + 0.3, 0.5, 0.75, 1.5, 2.25, and 3.75 energy-% ALA) for 18 weeks. Results showed 6, 12, 15, and 30 energy-% LA significantly ameliorated central obesity, hyperlipidemia, glucose homeostasis, and leptin status; 0.5 and 0.75 energy-% ALA significantly improved insulin sensitivity, adiponectin, and anti-inflammatory status. Moreover, high intakes of ALA (1.5, 2.25, and 3.75 energy-%) presented a pro-oxidant activity. In conclusion, dose instead of ratio determines the prevention of MS. The optimal doses are 6 energy-% LA and 0.75 energy-% ALA; high intakes of ALA may have side effects. KEYWORDS: linoleic acid, α-linolenic acid, dose, ratio, metabolic syndrome

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INTRODUCTION Metabolic syndrome (MS) is a common metabolic disorder, detectable both systemically and focally within related tissues, and predisposes patients to type II diabetes mellitus, atherosclerosis, cardiovascular disease (CVD), and certain cancers.1,2 A state of chronic low-grade inflammation probably contributes to the syndrome.2 Studies have shown that dietary pattern with low carbohydrate and saturated fatty acids (SFA) and high fiber and polyunsaturated fatty acids (PUFA) could reduce the risk of MS.3,4 Beneficial effects of PUFA in maintaining lipid homeostasis have been well documented; nevertheless, the proper application of PUFA as nutritional supplements is still under investigation. In the past few decades, the role of the ratio between n-6 and n-3 PUFA has generated long-standing interest in the prevention or slowing the progression of low-grade inflammation chronic diseases. Some researchers suggested that decreasing the ratio of n-6 to n-3 PUFA by reducing the intake of n-6 PUFA would be protective, as n-6 PUFA theoretically leads to increased production of proinflammatory products.5,6 However, in most of those studies, the ratio decrease was achieved by changing the intake doses of n-6 and n-3 PUFA simultaneously.7−9 Thus, this “optimal ratio” remains to be further explored. Additionally, there are controversial results about whether to decrease the intake of n-6 PUFA or to increase that of n-3 PUFA.10−12 Of note, these two approaches can both reduce the ratio of n-6 to n-3 PUFA. Studies also indicated increasing intakes of either n-6 PUFA or n-3 PUFA would be health-beneficial, whereas decreasing the intake of n-6 PUFA would not be.10,11 This may indicate that the dose is critical instead of the ratio. Moreover, the “optimal ratio” may not be available with excessive intakes of PUFA. © XXXX American Chemical Society

Evidence indicates that excess intakes of n-3 PUFA would not only cause increased bleeding13 and decreased immune function14 but would also induce inefficiency effects.15 Evidence showed that 1 energy-% (2.2 g/day) intakes of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) modestly increased but did not decrease the plasma low-density lipoprotein-cholesterol (LDL-C) level.14 Thus, addressing the dose rather than the ratio of n-6 to n-3 PUFA may be beneficial for appropriate use of PUFA as dietary supplements and also contribute new prophylaxis methods to low-grade inflammation diseases. Studies have obscured the concept of n-3 PUFA to marinederived n-3 PUFA (EPA + DHA). The beneficial effects of plant-derived α-linolenic acid (ALA) have been credited with functions the same as those of EPA and DHA exerted in vivo,16 as ALA is the precursor of n-3 PUFA.5 However, recent studies demonstrated that compared with EPA and DHA, ALA has selective and potentially independent effects on the inhibition of chronic low-grade inflammatory-related diseases.17,18 Health organizations (FAO/WHO) recently proposed that the research of PUFA should emphasize absolute intakes and PUFA should be studied individually but not as congeneric groups.19 On the other hand, the current recommended doses of dietary linoleic acid (LA) and ALA vary. Governments (United Kingdom, United States, The Netherlands, Australia, and New Zealand) and health organizations (FAO/WHO, Received: October 12, 2015 Revised: December 30, 2015 Accepted: January 8, 2016

A

DOI: 10.1021/acs.jafc.5b04715 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry American Dietetic Association, American Heart Association) recommend dietary intakes of LA varying from 3 to 10 energy% (6.6−22.0 g/day) and for ALA from 0.5 to 1.2 energy-% (1.1−2.7 g/day).19−21 The lack of agreement on the intake of PUFA influences its efficient use as nutritional supplements. Herein, we focus our research on refining the optimal absolute intakes of LA and ALA individually within the range of the recommended intakes. Apart from the issues of recommended protective doses, there are a number of concerns regarding the potential side effects produced by excess doses of PUFA. Current recommendations are based on preventing symptoms of PUFA deficiency, but the potential toxic effects of excess intakes are neglected. Researchers implied a note of caution for high intakes of LA (2 energy-%) showed decreased plasma high-density lipoprotein-cholesterol (HDLC) level and increased ratio of LDL-C to HDL-C in CVD patients.14 Contradictory results might be raised by the excess intake of LA and ALA on lipid homeostasis. It has been well documented that the benefits of PUFA may rely on the antioxidant activity exerted by the double bonds.15 Nevertheless, recent studies indicated that excess intakes of double bonds may result in converting their predominant antioxidant activity into potential pro-oxidant activity, especially for n-3 PUFA.22 Some researchers indicate the great importance of studying both antioxidant and pro-oxidant effects of these fatty acids by increasing the doses gradually.22 Therefore, determining the individual optimal doses of LA and ALA as well as the upper limits for dietary intakes is urgent and tantalizing. Our aim was to investigate the dose-related effect of LA and ALA on central obesity, lipid homeostasis, glucose intolerance, insulin resistance (IR), inflammation, and systemic oxidative stress in a high-fructose and high-fat diet (HFFD) induced rat model. The potential mechanism was also primarily investigated. Besides, this study was also designed to determine whether the parameters of MS were influenced by intake doses of LA and ALA or by the LA-to-ALA ratio. Furthermore, the possible detrimental effects of excess doses of LA and ALA were explored.

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Table 1. Fatty Acid Supplements in the Diets of the LA and ALA Groups LA groups LA1 ALA (energy-%) LA (energy-%)

LA3

LA4

LA5

LA6

15

30

ALA4

ALA5

ALA6

1.5

2.25

3.75

0.75 3 ALA1

LA (energy-%) ALA (energy-%)

LA2

a

6 ALA2

9 12 ALA groups ALA3a 6

0.3

0.5

0.75

Doses of linoleic acid (LA) and α-linolenic acid (ALA) in LA2 and ALA3 groups were the same; thus, they were performed as one group in this study. a

average body weight and fed (i) basal diet (NG; 13.75 kJ/g, 4.5% fat, w/w) + control gavage; (ii) HFFD (MG; 19.65 kJ/g, 15% fructose and 25% lard) + control gavage; (iii) HFFD + increasing dose of LA gavage (LA groups; 95.7%); and (iv) HFFD + increasing dose of ALA gavage (ALA groups; 85.0% ALA, 14.8% LA) for 18 weeks with ad libitum access to water and diet (Experimental Animal Center, Beijing, China). The ingredients of basal diet and HFFD are shown in Table 2. In this research, the increasing gavage doses of LA and ALA were chosen and calculated on the basis of the recommended dose ranges and the consideration of the ratio of LA to ALA. The gavage doses of LA groups (LA1, LA2, LA3, LA4, LA5, and LA6) were designed as 0.75 energy-% ALA + 3, 6, 9, 12, 15, and 30 energy-% LA (27.5 mg/kg bw/day ALA + 110, 220, 330, 440, 550, and 1100 mg/kg bw/day LA), respectively; the gavage doses of ALA groups (ALA1, ALA2, ALA3, ALA4, ALA5, and ALA6) were designed as 6 energy-% LA + 0.3, 0.5, 0.75, 1.5, 2.25, and 3.75 energy-% ALA (220 mg/kg bw/day LA + 11.0, 18.3, 27.5, 55.0, 82.5, and 137.5 mg/kg bw/day ALA, shown in Table 1 in detail), respectively. The fatty acid compositions of LA, ALA, basal diet, and HFFD are described in Table 2. Compositions of LA and ALA were of no significant difference between basal diet and HFFD. Gavage solutions were prepared. LA or ALA was dissolved in normal saline with 5% (w/v) purified delipidated BSA, and the pH was adjusted with sodium hydroxide to 7.3−7.4.23 The control gavage solution (NG and MG) was normal saline with an equivalent dose of delipidated BSA as in the LA and ALA groups. To minimize the difference of gavage total volume, additional normal saline was added. The prepared LA and ALA were stored at −20 °C to minimize lipid oxidation. Gavage doses of LA or ALA were calculated on the basis of the body weights (w/w) of the rats. During the experiment, the body weights of the rats were measured twice per week and food consumption was monitored daily. The experiment design was approved by the Animal Ethics Committee of the Beijing Key Laboratory of Functional Food from Plant Resources (Permit A330-5) and strictly conducted in accordance with the guidelines for animal care of the National Institute of Health.24 Fatty Acid Analysis. The dietary lipids were extracted with a mixture of chloroform and methanol (2:1, v/v). Butylated hydroxytoluene (0.1%) was used as the antioxidant in the extraction solvent.18 Extracted dietary fatty acid and gavage fatty acids were measured as fatty acid methyl esters and analyzed on a Supelco SP2560 column (100 m × 0.25 mm × 0.20 μm; Supelco, Bellefonte, PA, USA) by a Shimadzu GC-2010 (Japan) equipped with a flame ionization detector. For identification purposes, an 11 fatty acid methyl ester mixture standard was used for retention time calibration. Organ Weights. At the end of the study, animals were fasted overnight and sacrificed under deep anesthesia. After abdominal circumference measurement, liver, spleen, kidney, and epididymal and perirenal fat pads were removed and weighed immediately. Organ weights were normalized to body weight as organ indices. Plasma Biochemistry. Blood samples were taken from orbital venous by capillary tube under anesthesia after overnight fasting (12 h) at weeks 5, 11, and 18. Collected blood samples were kept at 4 °C for 2 h and centrifuged at 4000g for 10 min to obtain plasma. Plasma was

MATERIALS AND METHODS

Chemicals. LA (95.7%) and ALA (85.0% ALA, 14.8% LA) were purchased from Jingzhu Biotechnology Co., Ltd. (Nanjing, China). Delipidated BSA and 11 fatty acid methyl esters were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Total triglyceride (TG), total cholesterol (TC), LDL-C, and HDL-C commercial assay kits were purchased from Biosid Biotechnology and Science Inc. (Beijing, China). A plasma insulin radio-immunological assay kit was purchased from Kemei Tech Co., Ltd. (Beijing, China). Plasma adiponectin, leptin, tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), interleukin-6 (IL-6), and 8-F2-iso-protane (8-F2-IsoP) commercial enzyme-linked immunosorbent assay (ELISA) kits were purchased from Abcam (Cambridge, MA, USA). Other chemicals used were all of analytical grade. Animals and Diets. The experiment groups consisted of 130 male Sprague−Dawley rats (SD, 8 weeks old) supplied by Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) [Certificate SCXK (Beijing) 2014-0001] and housed in a controlled environment (23 ± 1 °C, relative humidity 50 ± 20%, 12 h light/dark cycle). After 1 week of acclimatization, the rats were randomly divided into 13 groups (n = 10 each, instead of 14 groups, shown in Table 1) with equal B

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Journal of Agricultural and Food Chemistry Table 2. Compositions and Fatty Acid Profiles of Diets and Fatty Acid Supplements (LA, ALA) diets composition

fatty acid supplements

basal dieta

total energy density, kJ/g 13.75 macronutrient composition, g/kg total carbohydrate 520.00 total fat 45.00 total protein 200.00 total fiber 35.00 calcium 11.20 phosphorus 6.97 total vitaminsc 0.29 total mineralsd 2.28 choline bitartratee 0.00 ash 63.00 total moisture 77.00 fatty acid (g/100 g of total recovered fatty acid) (n = 3/group) C4:0 0.00 ± 0.00 C6:0 0.00 ± 0.00 C8:0 0.00 ± 0.00 C10:0 0.00 ± 0.00 C12:0 0.00 ± 0.00 C14:0 0.22 ± 0.00 C14:1 0.00 ± 0.00 C15:0 0.00 ± 0.00 C16:0 16.16 ± 0.04 C16:1 0.22 ± 0.00 C17:0 0.00 ± 0.00 C17:1 0.00 ± 0.00 C18:0 2.60 ± 0.00 C18:1 n-9t 0.00 ± 0.00 C18:1 n-9c 20.07 ± 0.09 C18:2 n-6c 55.86 ± 0.01 γ-C18:3 n-6 0.00 ± 0.00 α-C18:3 n-3c 4.23 ± 0.06 C20:0 0.22 ± 0.00 C20:1 0.43 ± 0.00 C20:2 0.00 ± 0.00 C24:1 0.00 ± 0.00 total SFA 19.20 ± 0.05 total MUFA 20.72 ± 0.09 total PUFA 60.09 ± 0.05 trans FA 0.00 ± 0.00

HFFDb 19.65

LA

ALA

37.00

37.00

0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.09 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 3.87 ± 0.02 95.70 ± 0.06 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.35 ± 0.07 0.09 ± 0.01 4.22 ± 0.09 95.70 ± 0.06 0.00 ± 0.00

0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.57 ± 0.01 14.19 ± 0.01 0.24 ± 0.00 85.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.57 ± 0.01 99.43 ± 0.01 0.00 ± 0.00

421.40 254.21 181.16 22.60 10.40 6.70 0.29 2.28 2.00 59.30 75.00 0.27 ± 0.00 0.21 ± 0.00 0.16 ± 0.00 0.37 ± 0.00 0.48 ± 0.00 2.75 ± 0.00 0.16 ± 0.00 0.24 ± 0.02 24.93 ± 0.03 1.92 ± 0.01 0.35 ± 0.02 0.16 ± 0.00 12.63 ± 0.03 0.48 ± 0.00 36.06 ± 0.04 16.15 ± 0.02 0.80 ± 0.00 0.27 ± 0.00 1.01 ± 0.01 0.59 ± 0.00 0.27 ± 0.00 0.00 ± 0.00 42.66 ± 0.11 39.32 ± 0.02 17.54 ± 0.03 0.48 ± 0.00

Basal diet ingredients: corn, soy meal, fish meal, flour, wheat bran, salt, calcium phosphate dibasic, plant oil, mineral mixture, vitamin mixture, amino acids. Because of trade secret, the proportion of the materials could not be offered. bHFFD ingredients (by weight): on the base of basal diet, 15% fructose and 25% lard were added. cTotal vitamins: 10.70 KIU/kg VA, 1.50 KIU/kg VD, 103.05 IU/kg VE, 6.14 mg/kg VK, 16.00 mg/kg VB1, 16.03 mg/kg VB2, 10.43 mg/kg VB6, 91.92 mg/kg niacin, 30.09 mg/kg pantothenic acid, 7.45 mg/kg folic acid, 0.28 mg/kg biotin, 0.03 mg/kg VB12. d Total minerals: 0.26% magnesium, 0.64% potassium, 0.32% sodium, 180.00 mg/kg iron, 123.49 mg/kg manganese, 19.80 mg/kg copper, 58.20 mg/ kg zinc, 0.61 mg/kg iodine, 0.16 mg/kg selenium. eCholine bitartrate was supplied to help digestion in HFFD and PUFA groups. a

transferred to Eppendorf tubes and stored at −80 °C for further analysis. The concentrations of plasma TG, TC, LDL-C, and HDL-C were measured using corresponding commercial assay kits on an Alcyon 300 automatic analyzer (Alcyon, USA) at weeks 5, 11, and 18. Plasma insulin levels were analyzed with a radio-immunological assay kit at 18 weeks. Hepatic tissue was homogenized, and the lipids were extracted as previously described.25 The hepatic TC and TG levels were measured as the plasma lipid profiles. Oral Glucose (OGTT) and Insulin (ITT) Tolerance Tests. OGTT and ITT were measured 2 days apart at weeks 6, 12, and 18. For OGTT, basal blood glucose levels were measured via tail blood of fasting rats (12 h) using a One Touch Ultra test strips and glucometer (Life Scan Inc., Milpitas, CA, USA). The tail vein blood samples were taken before (0 min) and at subsequent time intervals of 30, 60, 90,

and 120 min following glucose (2 g/kg bw) administration. For ITT, basal blood glucose levels were carried out after 4−5 h of food deprivation following intraperitoneal injection of insulin (0.5 U/kg bw) as for OGTT. Total areas under the curve (AUC) of OGTT and ITT were calculated using a trapezoidal method. HOMA-IR index (week 18) was determined as previously described.26 Adipocytokines, Inflammation, and Oxidative Stress Status. The plasma levels of adiponectin, leptin, TNF-α, CRP, IL-6, and 8-F2IsoP were detected by commercial ELISA kits. All measurements were carried out according to the manufacturer’s protocols. Histological Analysis. Hepatic tissue, pancreatic tissue, and epididymal fat tissue were fixed in 10% neutral buffered formalin for 3 days and embedded in paraffin wax after dehydration.26 Then they were sliced (3 μm) and stained with hematoxylin and eosin (HE) for C

DOI: 10.1021/acs.jafc.5b04715 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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a

LA groups: diet with HFFD with increasing dose of LA and constant dose of ALA solution gavage. Each value is the mean ± SEM. Means within a row without a common letter are significantly different, n = 10 per group, P < 0.05. bNG, diet with basal diet with control solution gavage. cMG, diet with HFFD with control solution gavage.

0.7 ± 0.1b 1.1 ± 0.2d 0.7 ± 0.1b 1.3 ± 0.1cd 0.9 ± 0.1ab 1.4 ± 0.2bcd 0.9 ± 0.1a 2.2 ± 0.2a 0.7 ± 0.0b 1.2 ± 0.1cd 1.0 ± 0.2a 1.7 ± 0.2abc 0.3 ± 0.0c 0.4 ± 0.1e

0.9 ± 0.1a 1.9 ± 0.3ab

24.2 ± 0.7a 2.5 ± 0.2a 3.6 ± 0.5a 2.5 ± 0.0a 0.10 ± 0.01b 0.47 ± 0.02a 21.9 ± 0.4bc 1.6 ± 0.2bc 2.0 ± 0.2b 2.1 ± 0.1c 0.12 ± 0.01a 0.49 ± 0.01a

LA6 LA5

280.7 ± 4a 262.7 ± 14.1c 17.62 ± 0.40cd 81.35 ± 1.89cd 21.4 21.1 ± 0.3c 2.1 ± 0.2abc 3.2 ± 0.2ab 2.4 ± 0.1a 0.11 ± 0.01ab 0.50 ± 0.01a 281.3 ± 2.7a 283.4 ± 19.7bc 18.71 ± 0.58c 86.39 ± 2.71bc 17.3 22.4 ± 0.6bc 2.1 ± 0.2abc 3.2 ± 0.3ab 2.5 ± 0.1a 0.10 ± 0.01b 0.52 ± 0.02a

LA4 LA3

281.7 ± 3.8a 339.1 ± 17.3b 18.43 ± 0.46cd 85.08 ± 2.17bcd 13.2 23.3 ± 0.6ab 2.6 ± 0.2a 3.6 ± 0.3a 2.4 ± 0.1a 0.11 ± 0.01ab 0.48 ± 0.01a 280.4 ± 3.6a 295.5 ± 16.2bc 18.63 ± 0.47cd 87.28 ± 2.21abc 9.2 22.1 ± 0.6bc 2.0 ± 0.2abc 3.3 ± 0.4a 2.1 ± 0.0bc 0.11 ± 0.01ab 0.50 ± 0.01a

LA2 LA1

281.8 ± 3.3a 323.5 ± 18.8b 19.20 ± 0.35bc 88.82 ± 1.93abc 5.1 22.3 ± 0.5bc 2.3 ± 0.3ab 3.5 ± 0.4a 2.4 ± 0.1ab 0.10 ± 0.01b 0.48 ± 0.01a ± ± ± ± ± ± ± ±

initial bw, g bw gain, g food intake, g/day energy intake, kJ/day fatty acids intake, kJ/day abdominal circumference, cm epididymal fat index, g/100 g bw perirenal fat index, g/100 g bw liver index, g/100 g bw spleen index, g/100 g bw kidney index, g/100 g bw hepatic lipid accumulation hepatic TC, mmol/L hepatic TG, mmol/L

4.6a 16.7a 0.35b 1.83ab

MGc NGb variable

Table 3. Dietary Intake, Weight Gain, Body Composition, Hepatic Function, and Lipid Accumulation in LA Groupsa D

280.1 396.4 20.67 95.72

RESULTS Body Weight, Dietary Intake, and Organ Indices. The body weight of MG rats was significantly increased by 32.0% at week 18 (compared to NG, P < 0.05, Tables 3 and 4). Food intake of NG was significant higher than that of MG (P < 0.05, Tables 3 and 4), but energy intake showed no significant difference (Tables 3 and 4), indicating the difference between normal diet and HFFD could be ignored. LA groups showed a significant inhibition in body weight gains, with a decreasing tendency observed as the doses of LA increased (compared to MG, P < 0.05, Table 3). A significant decreasing trend was shown in LA groups in food intake; also, the energy intakes of LA5 and LA6 were markedly reduced (compared to MG, P < 0.05, Table 3). These findings indicated that high intakes of LA may suppress the appetite of the rats. ALA1, ALA2, ALA3, ALA4, and ALA5 groups exhibited significantly inhibitory effects in body weight gains (compared to MG, P < 0.05, Table 4), with an increasing tendency observed as the doses of ALA increased (Table 4). With the increasing doses of ALA, no significant difference was shown in either food intake or energy intake (compared to MG, Table 4). Abdominal circumference and indices of epididymal fat, perirennal fat, and liver in MG rats were significantly increased (compared to NG, P < 0.05, Tables 3 and 4), indicating the consumption of HFFD induced central obesity. All LA groups presented significantly lower abdominal circumference, as ALA 2 and ALA 3 groups showed marked decreases (compared to MG, P < 0.05, Tables 3 and 4). Epididymal fat indices of LA6 and ALA2 were reduced by 37.9 and 31.2%, respectively (compared to MG, P < 0.05, Tables 3 and 4). The liver index of LA2 rats was decreased by 15.7%, as those of all ALA groups were reduced significantly (compared to MG, P < 0.05, Tables 3 and 4). Plasma Lipid Profiles. After 5 weeks of HFFD consumption, concentrations of plasma LDL-C, TC, and TG were significantly increased in MG rats (compared to NG, P < 0.05, Tables S1 and S2), suggesting dyslipidemia under HFFD induction. LA2, LA4, LA5, and LA6 groups (week 11) showed significantly reduced LDL-C levels; all ALA groups (week 18) were normalized (compared to MG, P < 0.05, Tables S1 and S2). Significant reduction was shown in plasma TC and TG levels in LA2, LA4, LA5, and LA6 groups at week 5 (compared to MG, P < 0.05, Table S1). ALA2, ALA3, ALA5, and ALA6 treatments significantly suppressed TC throughout the study; only the ALA3 group showed a significantly decreased TG level (compared to MG, P < 0.05, Table S2). Hepatic Lipid Accumulation. In the hepatic lipid profile analysis, the TC content in the MG group was about triple that in NG rats and the TG level was about quadruple (P < 0.05, Tables 3 and 4), indicating the 18 weeks of HFFD consumption induced ectopic lipid accumulation in liver. Hepatic TC and TG were markedly suppressed in LA2, LA4,

6.1a 18.8bc 0.37a 1.21a

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280.7 300.4 27.95 91.96

determination of cellular necrosis, fat vacuole, and inflammatory cell infiltration. For the Oil red O staining, hepatic tissue was frozen in liquid nitrogen, sliced (10 μm), and stained with Oil red O solution (0.5 g/100 mL, dissolved in isopropanol) for determination of fat accumulation. All observations were carried out with a DM-3000 microscope (Leica, Germany). Statistical Analysis. All data are presented as the mean ± SEM. Differences were evaluated with a one-way analysis of variance (ANOVA) followed by Duncan’s multiple-comparisons test performed by SAS v8.2 (SAS Institute Inc., Cary, NC, USA), and differences were considered significant at P < 0.05.

280.6 ± 5.3a 258.0 ± 23.9c 16.81 ± 0.41d 77.14 ± 1.80d 41.7 22.0 ± 0.6bc 1.6 ± 0.2c 3.3 ± 0.6a 2.3 ± 0.1abc 0.10 ± 0.00b 0.47 ± 0.01a

Journal of Agricultural and Food Chemistry

DOI: 10.1021/acs.jafc.5b04715 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 4. Dietary Intake, Weight Gain, Body Composition, Hepatic Function, and Lipid Accumulation in ALA Groupsa NGb

variable

MGc

initial bw, g 280.7 ± 6.1a bw gain, g 300.4 ± 18.8b food intake, g/day 27.95 ± 0.37a energy intake, kJ/day 91.96 ± 1.21a fatty acid intake, kJ/day abdominal 21.9 ± 0.4c circumference, cm epididymal fat index, 1.6 ± 0.2c g/100 g bw perirenal fat index, 2.0 ± 0.2b g/100 g bw liver index, 2.1 ± 0.1b g/100 g bw spleen index, 0.12 ± 0.01a g/100 g bw kidney index, 0.49 ± 0.01a g/100 g bw hepatic lipid accumulation hepatic TC, mmol/L 0.3 ± 0.0d hepatic TG, mmol/L 0.4 ± 0.1d

280.1 396.4 20.67 95.72

± ± ± ±

ALA1

4.6a 16.7a 0.35b 1.83a

280.6 300.4 19.06 87.78 8.5

± ± ± ±

ALA2

4.8a 16.6b 0.48b 2.34a

280.6 295.4 18.90 87.26 8.8

± ± ± ±

2.7a 25.6b 0.44b 2.11a

ALA3 280.4 295.5 18.63 87.28 9.2

± ± ± ±

3.6a 16.2b 0.47b 2.21a

ALA4 281.8 327.7 19.57 90.29 10.2

± ± ± ±

2.7a 16.8b 0.54b 2.51a

ALA5 281.8 334.5 19.97 93.23 11.2

± ± ± ±

5.4a 27.9b 0.56b 2.76a

ALA6 280.2 356.3 20.68 94.22 13.2

± ± ± ±

3.5a 20.6ab 0.52b 2.50a

24.2 ± 0.7a

22.9 ± 0.4abc

22.1 ± 0.5bc

22.1 ± 0.6bc

22.8 ± 0.5abc

22.5 ± 0.4abc

23.6 ± 0.7ab

2.5 ± 0.2a

2.2 ± 0.2abc

1.7 ± 0.1bc

2.0 ± 0.2abc

2.2 ± 0.1abc

2.3 ± 0.3ab

2.3 ± 0.2ab

3.6 ± 0.5a

3.6 ± 0.4a

3.5 ± 0.5a

3.3 ± 0.4a

4.0 ± 0.5a

3.5 ± 0.5a

4.0 ± 0.4a

2.5 ± 0.0a

2.2 ± 0.0b

2.2 ± 0.1b

2.2 ± 0.0b

2.2 ± 0.1b

2.2 ± 0.1b

2.2 ± 0.1b

0.10 ± 0.01ab

0.10 ± 0.00b

0.12 ± 0.01ab

0.11 ± 0.01ab

0.10 ± 0.01b

0.10 ± 0.01ab

0.11 ± 0.01ab

0.47 ± 0.02a

0.49 ± 0.02a

0.48 ± 0.01a

0.5 ± 0.01a

0.5 ± 0.01a

0.48 ± 0.02a

0.48 ± 0.02a

1.0 ± 0.2ab 1.7 ± 0.2ab

1.1 ± 0.1a 2.0 ± 0.2a

0.9 ± 0.1abc 1.2 ± 0.1c

0.7 ± 0.0c 1.2 ± 0.1c

0.9 ± 0.1abc 1.8 ± 0.1abc

0.8 ± 0.1bc 1.5 ± 0.2bc

0.7 ± 0.1bc 1.5 ± 0.2bc

ALA groups: diet with HFFD with increasing dose of ALA and constant-dose of LA solution gavage. Each value is the mean ± SEM. Means within a row without a common letter are significantly different, n = 10 per group, P < 0.05. bNG, diet with basal diet with control solution gavage. cMG, diet with HFFD with control solution gavage.

a

Table 5. Levels of Plasma Fasting Glucose, AUC of OGTT, and ITT in LA Groupsa at Weeks 6, 12, and 18 variable

NGb

fasting glucose, mmol/L 6 weeks 3.9 ± 0.2c 12 weeks 4.1 ± 0.1b 18 weeks 4.1 ± 0.2c OGTT, mmol/L × min 6 weeks 13.3 ± 0.3bc 12 weeks 12.7 ± 0.2b 18 weeks 13.1 ± 0.1bcd ITT, mmol/L × min 6 weeks 8.0 ± 0.3bc 12 weeks 6.8 ± 0.5c 18 weeks 7.4 ± 0.5cd

MGc

LA1

LA2

LA3

LA4

LA5

LA6

4.5 ± 0.1a 4.9 ± 0.2a 5.1 ± 0.2a

4.6 ± 0.1a 4.3 ± 0.3b 4.6 ± 0.1b

4.6 ± 0.1a 4.0 ± 0.2b 4.2 ± 0.1bc

4.1 ± 0.1bc 4.1 ± 0.2b 4.6 ± 0.2b

3.9 ± 0.2cd 4.1 ± 0.2b 4.3 ± 0.1bc

3.4 ± 0.1de 4.0 ± 0.1b 4.4 ± 0.1bc

3.3 ± 0.1e 3.8 ± 0.2b 4.5 ± 0.1bc

14.7 ± 0.4a 14.4 ± 0.6a 14.2 ± 0.5a

14.6 ± 0.3ab 13.4 ± 0.5ab 13.7 ± 0.1ab

14.7 ± 0.3a 13.1 ± 0.5ab 13.1 ± 0.3bcd

14.4 ± 0.3ab 13.6 ± 0.2ab 13.6 ± 0.1abc

14.6 ± 0.4a 13.5 ± 0.2ab 13.1 ± 0.3bcd

14.8 ± 0.4a 13.8 ± 0.4ab 12.8 ± 0.2d

12.9 ± 0.4c 12.7 ± 0.3b 12.8 ± 0.3cd

10.1 ± 0.8a 11.2 ± 0.7a 10.1 ± 0.6a

8.8 ± 0.6ab 9.4 ± 0.6ab 9.5 ± 0.5ab

7.2 ± 0.4bc 8.3 ± 0.4bc 7.0 ± 0.4d

8.7 ± 0.7ab 9.5 ± 0.7ab 9.0 ± 0.6abc

8.0 ± 0.7bc 9.1 ± 0.6b 8.4 ± 0.6bcd

7.1 ± 0.5bc 9.3 ± 0.5b 7.4 ± 0.4cd

6.7 ± 0.3c 9.1 ± 0.5b 7.5 ± 0.6cd

LA groups, diet with HFFD with increasing dose of LA and constant-dose of ALA solution gavage. Each value is the mean ± SEM. Means within a row without a common letter are significantly different, n = 10 per group, P < 0.05. bNG, diet with basal diet with control solution gavage. cMG, diet with HFFD with control solution gavage.

a

Table 6. Levels of Plasma Fasting Glucose, AUC of OGTT, and ITT in ALA Groupsa at Weeks 6, 12, and 18 variable

NGb

fasting glucose, mmol/L 6 weeks 3.9 ± 0.2b 12 weeks 4.1 ± 0.1b 18 weeks 4.1 ± 0.2b OGTT, mmol/L × min 6 weeks 13.3 ± 0.3b 12 weeks 12.7 ± 0.2c 18 weeks 13.1 ± 0.1a ITT, mmol/L × min 6 weeks 8.0 ± 0.3b 12 weeks 6.8 ± 0.5e 18 weeks 7.4 ± 0.5cd

MGc

ALA1

ALA2

ALA3

ALA4

ALA5

ALA6

4.5 ± 0.1a 4.9 ± 0.2a 5.1 ± 0.2a

4.8 ± 0.1a 4.4 ± 0.3ab 4.6 ± 0.2ab

4.8 ± 0.1a 4.3 ± 0.2b 4.1 ± 0.2b

4.6 ± 0.1a 4.0 ± 0.2b 4.1 ± 0.1b

4.9 ± 0.1a 4.5 ± 0.1ab 4.3 ± 0.1b

4.5 ± 0.2a 4.3 ± 0.2b 4.2 ± 0.2b

4.7 ± 0.1a 4.0 ± 0.1b 4.1 ± 0.1b

14.7 ± 0.4a 14.4 ± 0.6ab 14.2 ± 0.5a

14.8 ± 0.3a 14.1 ± 0.2ab 14.3 ± 0.5a

15.2 ± 0.2a 14.5 ± 0.3a 13.2 ± 0.4a

14.8 ± 0.3a 13.1 ± 0.5bc 13.1 ± 0.3a

15.0 ± 0.3a 14.9 ± 0.3a 13.4 ± 0.3a

15.0 ± 0.3a 14.9 ± 0.5a 14.2 ± 0.5a

15.2 ± 0.2a 14.1 ± 0.3ab 13.2 ± 0.4a

10.1 ± 0.8a 11.2 ± 0.7a 10.1 ± 0.6a

8.7 ± 0.3ab 10.3 ± 0.3abc 9.1 ± 0.5ab

9.0 ± 0.4ab 9.0 ± 0.6cd 7.2 ± 0.4d

7.2 ± 0.4b 8.3 ± 0.4de 7.0 ± 0.4d

8.4 ± 0.6ab 9.4 ± 0.4bcd 8.5 ± 0.3bcd

8.4 ± 0.5ab 10.7 ± 0.5ab 8.9 ± 0.6abc

7.5 ± 0.5b 9.2 ± 0.5bcd 8.1 ± 0.5bcd

ALA groups, diet with HFFD with increasing dose of ALA and constant-dose of LA solution gavage. Each value is the mean ± SEM. Means within a row without a common letter are significantly different, n = 10 per group, P < 0.05. bNG, diet with basal diet with control solution gavage. cMG, diet with HFFD with control solution gavage.

a

E

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Journal of Agricultural and Food Chemistry LA5, and LA6 groups (compared to MG, P < 0.05, Table 3). In the ALA groups, the level of hepatic TC showed no difference (compared to MG, Table 4), and the hepatic TG content was significantly decreased in the ALA3 group (compared to MG, P < 0.05, Table 4). Fasting Glucose Level, OGTT, ITT, Fasting Insulin Level, and HOMA-IR Index. The fasting glucose levels as well as the AUC of OGTT and ITT in MG rats were significantly higher; also, the plasma insulin level and HOMA-IR index of MG were significantly elevated by 20.1 and 56.9%, respectively (compared to NG, P < 0.05, Tables 5 and 6 and Figure 1).

Figure 2. Levels of plasma adiponectin [(A) ALA groups; (B) ALA groups] and leptin [(C) LA groups; (D) ALA groups]. Data are represented as the mean ± SEM. Means without a common letter are significantly different, P < 0.05. n = 10 per group. NG, normal group; MG, model group.

was shown among LA2, LA3, LA4, LA5, and LA6 groups in significantly suppressed plasma leptin elevation (compared to MG, P < 0.05, Figure 2). Meanwhile, a dose−response manner was shown in ALA groups in elevating the adiponectin level, and ALA1, ALA2, ALA3, and ALA4 significantly decreased the leptin level (compared to MG, P < 0.05, Figure 2). Inflammatory cytokines were significantly increased by HFFD consumption as the TNF-α level was increased by 27.2%, and IL-6 and CRP levels were even doubled and quadrupled, respectively (compared to NG, P < 0.05, Figure 3). The plasma TNF-α level was significantly alleviated in LA2, LA3, LA4, LA5, and LA6 groups, and the level of IL-6 was significantly decreased in LA2 and LA6 groups (compared to MG, P < 0.05, Figure 3). ALA2, ALA3, ALA4, ALA5, and ALA6 groups showed markedly decreased TNF-α level, and ALA2 and ALA3 groups also showed significantly reduced IL-6 level (compared to MG, P < 0.05, Figure 3). CRP level was markedly reduced by all PUFA treatments, with a dose−effect within ALA but not LA groups (compared to MG, P < 0.05, Figure 3). Oxidative Stress Status. Oxidative stress was remarkably stimulated by HFFD as the 8-F2-IsoP level was increased by 21.4% (compared to NG, P < 0.05, Figure 4). Significant suppression of 8-F2-IsoP was shown in LA2, ALA1, ALA2, and ALA3 groups; moreover, significant promotion of 8-F2-IsoP was observed in the ALA5 group (compared to MG, P < 0.05, Figure 4). Histology. Hepatic lipid deposition, hepatocellular necrosis, and inflammatory cell infiltration were observed in MG rats compared to NG in histopathology analysis (Figure 5A,B). LA2, LA4, LA5, and LA6 groups and ALA2 and ALA3 groups showed alleviation effects in hepatic histopathology compared to MG as hepatocellular necrosis and fat vacuoles were obviously attenuated (Figure 5C,F−H,J). These results were consistent with the hepatic function analysis (Tables 3 and 4). Inflammatory cell infiltration was observed in ALA5 (Figure 5), which was in line with the oxidative stress status (Figure 4). Hepatic tissue of MG rats showed more red-dyed fatty droplets in Oil red O staining of hepatic tissue than that of NG rats (Figure 6A,B), indicating a higher hepatic lipid accumulation.

Figure 1. Levels of plasma fasting insulin [(A) LA groups; (B) ALA groups] and HOMA-IR [(C) LA groups; (D) ALA groups]. Data are presented as the mean ± SEM. Means without a common letter are significantly different, n = 10 per group. P < 0.05. NG, normal group; MG, model group.

These data indicated that HFFD impaired glucose tolerance and IR in vivo. LA3, LA4, LA5, and LA6 groups (week 6) and LA1 and LA2 groups (week 12) showed significantly alleviated fasting glucose; also, groups LA6 (week 6) and LA2, LA4, and LA5 (week 18) significantly reduced glucose tolerance (compared to MG, P < 0.05, Table 5). Insulin tolerance was also markedly alleviated in groups LA2, LA4, LA5, and LA6 (week 6) as the AUC of ITT showed a significant decrease (compared to MG, P < 0.05, Table 5). Significantly lower fasting glucose levels were observed in ALA2, ALA3, ALA5, and ALA6 groups (week 12),and in ALA1 and ALA4 groups (week 18, compared to MG, P < 0.05, Table 6). ALA2, ALA3, ALA4, and ALA6 could significantly attenuate insulin tolerance, but no obvious changes in all ALA groups were observed in OGTT determined (compared to MG, P < 0.05, Table 6). In LA2, ALA1, and ALA2 groups, plasma insulin levels were decreased significantly (compared to MG, P < 0.05, Figure 1). IR was significantly improved in LA2, LA4, LA5, and LA6 groups and in all ALA groups as the HOMA-IR was decreased significantly (compared to MG, P < 0.05, Figure 1). Adipocytokines and Inflammation Status. The 18 week period of HFFD consumption resulted in a significant decrease of adiponectin level and a significant increase of leptin level (compared to NG, P < 0.05, Figure 2). The levels of adiponectin in all of the LA groups were significantly increased with no difference among doses, as a dose−response manner F

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Figure 3. Levels of inflammatory cytokine of TNF-α, IL-6, and CRP in LA groups (A−C) and in ALA groups (D−F). Data are represented as the mean ± SEM. Means without a common letter are significantly different, P < 0.05. n = 10 per group. NG, normal group; MG, model group.

of hepatic TG analysis (Tables 3 and 4). In the histopathology analysis of pancreas sections, both the inflammatory cell and adipocyte infiltrations were observed in MG rats but not in NG (Figure S1A,B), suggesting pathological changes of pancreas tissue by HFFD. LA3, ALA5, and ALA6 groups did not attenuate the side effect of HFFD as infiltrated inflammatory cells and deformed pancreas isles were observed (Figure S1E,L,M). Hypertrophy of adipocytes was observed in MG rats in the histopathology analysis of adipose tissue section (compared to NG, Figure S2A,B). The prevention of adipotytes hypertrophy was observed in LA2, LA4, LA5, and LA6 groups and ALA1, ALA2, and ALA3 groups as the adipocytes were smaller (compared to MG, Figure S2B,C,F−J). These results were in line with the analysis of epididymal index and body weight gains (Tables 3 and 4).

Figure 4. Levels of plasma 8-F2-IsoP: (A) LA groups; (B) ALA groups. Data are represented as the mean ± SEM. Means without a common letter are significantly different, P < 0.05. n = 10 per group. NG, normal group; MG, model group.

The lipid accumulation level could be inhibited in LA2, LA4, LA5, and LA6 groups and ALA2 and ALA3 groups as the red fat droplets were obviously reduced (compared to MG, Figure 6C,F−H,J). These findings were in accordance with the results

Figure 5. Hematoxylin and eosin staining of hepatic tissue in rats (original magnification, ×200): (A) normal group; (B) model group; (C) LA2/ ALA3 group; (D) LA1 group; (E) LA3 group; (F) LA4 group; (G) LA5 group; (H) LA6 group; (I) ALA1 group; (J) ALA2 group; (K) ALA4 group; (L) ALA5 group; (M) ALA6 group. The bar represents 200 μm. The arrow pointer indicates fat vacuoles. G

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Figure 6. Oil red O staining of hepatic tissue in rats (original magnification, ×200): (A) normal group; (B) model group; (C) LA2/ALA3 group; (D) LA1 group; (E) LA3 group; (F) LA4 group; (G) LA5 group; (H) LA6 group; (I) ALA1 group; (J) ALA2 group; (K) ALA4 group; (L) ALA5 group; (M) ALA6 group. The bar represents 200 μm. The arrow pointer indicates fat droplets.

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DISCUSSION Epidemiological, clinical, and animal studies suggest that increased intake of PUFA may decrease the incidence of MS.15 However, current studies rarely focused on absolute intake of individual PUFA.11,12 Thus, increasing doses were designed to study the effects of absolute intakes of LA and ALA on HFFD-induced MS in rat. Results showed that the mitigation of MS was determined by doses of LA and ALA. Although LA and ALA both have potential effects to protect against central adiposity and dyslipidemia,14 we found LA to have a greater effect compared to ALA in regulating body weight gains and increasing abdominal circumference and adipose tissues (Tables 3 and 4 and Figure S2). Part of the difference is due to the ability to just induce lipid redistribution away from the abdominal area by ALA, but with little effect on suppressing body weight gains.18 Another part of the difference, however, may related to the anorexic or orexigenic effects of PUFA. We found the food intakes of 15 and 30 energy-% LA groups were significantly decreased, whereas that of 3.75 energy-% ALA were increased (compared to MG, P < 0.05, Tables 3 and 4). These trends were similar to body weight gains and abdominal circumference increase (Tables 3 and 4). Previous study suggested that orexigenic effects of ALA-rich oil failed to oppose visceral obesity in n-3-depleted rats.27 Additionally, the doses of PUFA actually influence the appetite. Reports indicated endocannabinoids 2-arachidonoylglycerol, and anandamide related to appetite were improved in 8 energy-% LA rats (compared to 1 energy-% LA), resulting in a higher adiposity.28 Thus, our data confirmed the view that LA and ALA intake may also affect the appetite, and it may be closely dose-determinate. LA and ALA have effects of improving dyslipidemia by modulating lipoproteins.14,15 We found doses of 6, 12, 15, and 30 energy-% LA alleviated hyperlipidemia by inhibiting the production of LDL-C, TC, and TG; however, doses of 3 energy-% LA indicated poor effect on improving dyslipidemia (Table S1). This may be explained by the “18-2 threshold” hypothesis, which indicates that dyslipidemia would be greatly exaggerated if LA intake did not reach the value of threshold.14,29 In agreement with the hypothesis above,14,15 3 energy-% may be the threshold. Studies have implied ALA was

similar to LA in lowering plasma TC level; nevertheless, it was not as effective as LA in regulating LDL-C production or clearance.14 Consistent with this study, ALA groups showed a similar effect (Tables S1 and S2). LA and ALA are inversely associated with the incidence of impaired glucose tolerance and type II diabetes.17 We found LA showed a greater effect than ALA on attenuation of hyperglycemia (Tables 5 and 6), which may due to the regulation of glucose-mediated insulin release or the enhancement of hepatic insulin uptake.30 No improvements of OGTT were found in ALA groups (Table 5). These confirmed the view that diabetic diets with n-3 PUFA exhibited lower insulin response to blood.30 The insulin resistance theory was accepted widely as the hypothesis in the pathophysiology of MS.2 We found ALA showed greater effects than LA in attenuation of IR, as the HOMA-IR value and fasting insulin level were both numerically lower (Tables 5 and 6). Other studies also indicated ALA was associated with decreased fasting glucose and a lower prevalence of IR.31 An enhancement of insulin-mediated glucose disposal was shown because of the substitution of dietary LA with ALA in sucrose-fed rats.32 In addition, ALA is considered to be the endogenous ligand of insulin secretion receptors G protein-coupled receptor 40 (GPR40) and GPR120.31 The direct and indirect glucagon-like peptide-1 (GLP-1) mediated insulinotropic effects of ALA are mediated by GPR40 in the pancreatic β-cells and enteroendocrine L-cells, respectively.31 This effect occurred only with ALA but not LA.31 This might explain the different IR attenuations of LA and ALA in our study. Adiponectin and leptin regulate energy expenditure and lipid and carbohydrate metabolism33 and also play anti-inflammatory roles to improve IR and obesity-related diseases.31 Reports showed that plasma adiponectin level was negatively correlated with body weight and visceral adiposity.34,35 It has been suggested that ALA could decrease the secretion of inflammatory cytokines via the phosphorylation of AMPK and regulate the effect on adiponectin release and IR.31 Surprisingly, a dose-dependent manner of plasma adiponectin levels was shown in ALA but not LA groups (Figure 2). This suggests the inactivation of LA in adiponectin regulation, and H

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Figure 7. Analysis of the effects of the absolute doses of dietary linoleic acid (LA) and α-linolenic acid (ALA) and the ratio LA-to-ALA in groups LA2/ALA3, LA1, LA3, LA5, ALA1, ALA2, and ALA4.

oxidant effect has not been observed in the LA groups, and a dose-dependent correlation with F2-IsoP level was shown in ALA but not LA (Figure 4). Thus, the “pro-oxidant activity” might be generated due to the extra double bond from ALA.22 Consistently, previous study showed that diets enriched with ALA can either prevent or trigger the activity of antioxidant enzymes in normal tissues in vivo.22 Moreover, lipid peroxidation is an autoxidation process or self-propagating, capable of extensive tissue damage until substrate is consumed or termination occurs.41 This suggests that lipid peroxidation may not be inhibited by extraneous antioxidant. Certainly, an upper limit of ALA intake should be considered to thwart lipid peroxidation, although cardioprotective effects have been highly underscored. It has been suggested a lower ratio of LA to ALA is of benefit to attenuate low-grade inflammatory diseases.5,6 In this study, reducing the ratio of LA to ALA was achieved by two approaches: (i) decreasing LA in groups of LA5, LA3, LA2, and LA1 and (ii) increasing ALA in groups of ALA1, ALA2, ALA3, and ALA4 (Figure 7). We found the most effective ratio in attenuating MS is that of the LA2 (ALA3) group (8:1), but not those (LA1 or ALA4, 4:1) with lower ratio. This indicates the “optimal ratio” of LA to ALA may not be an optimal approach to evaluate the balance of PUFA (Figure 7). Moreover, groups with identical ratio but diverse doses (LA1 and ALA4; LA3 and ALA2; LA5 and ALA1) indicate different results (Figure 7). This also confirms the LA-to-ALA ratio is not the main factor to determine the application of PUFA. Hence, our findings indicate that the LA-to-ALA ratio is of little value under the condition of high intake of PUFA. Harris also suggested the “optimal ratio” has been abused and is of little use on both theoretical and evidential grounds.10 Similarly, it has been suggested that intake of PUFA was a more optimal approach to dietary n-6 and n-3 PUFA balance than a simple ratio.14 FAO/ WHO also stated that there was no rational recommendation for n-6 to n-3 ratio or LA-to-ALA ratio if intakes of n-6 and n-3 PUFA are within the recommendations.19 Herein, it is the individual dose of LA and ALA rather than their ratio that should be considered discretely. In conclusion, the protective effects of LA and ALA on HFFD-induced MS in rats were mainly dependent on their doses. Doses of the 6, 12, 15, and 30 energy-% LA were able to significantly prevent MS, due to the ability to modulate central obesity, glucose metabolism, and plasma leptin. Meanwhile, doses of 0.5 and 0.75 energy-% ALA were efficacious in MS inhibition, and the underlying mechanism might be closely related with the alleviation of IR and regulation of adiponectin,

the elevation may be credited to the constant dose of (0.75 energy-%) ALA. However, a previous study also suggested the inaction of ALA (3.68 energy-%) on adiponectin regulation.36 Considering our results, this inaction may due to the excess dose of ALA. Meanwhile, we found the level of plasma leptin was significantly lowered by LA (compared to MG, P < 0.05, Figure 2). A 6 energy-% LA dose showed great influence on suppressing plasma leptin level as 3 and 9 energy-% doses were not that efficient. These results are consistent with those of hypertrophy of adipocytes and lipid homeostasis (Figure 2 and Figure S2C−E). Evidence indicated the plasma leptin level was directly correlated with body fat mass and adipocyte size.37 Thus, the inactivation of 3 and 9 energy-% LA in leptin level may be related to the 18-2 threshold theory and appetite effects discussed before. Increasing evidence indicates that IR is associated with the abundance of pro-inflammatory cytokines as well.15,33 Plasma levels of TNF-α, IL-6, and CRP are predictive of CVD and type II diabetes.38 It has been reported that the level of plasma CRP was negatively correlated with dietary intake of ALA.14 We found that treatments of LA or ALA significantly inhibited the elevation of TNF-α and CRP levels, indicating anti-inflammatory effects in both LA and ALA (P < 0.05, Figure 3). A dosedependent manner could be observed in CRP level in ALA but not in LA, suggesting the dose of 0.75 energy-% ALA exerted favorable influence (Figure 3). The anti-inflammatory effect of n-3 PUFA was assumed to be based on multiple mechanisms, such as inhibition of macrophage activation, potent inflammation-resolving effects, and inherent competition of n-3 PUFA with n-6 PUFA for incorporation into phospholipid membranes.38 By contrast, high doses of LA in our study reduced the plasma pro-inflammatory cytokine level (Figure 3). This strongly contradicted the comment of reducing the intakes of LA to inhibit pro-inflammation state.5,6 Thus, the postulated pro-inflammation effect of n-6 PUFA in low-grade inflammation chronic diseases remains to be elucidated in vivo. Produced primarily from oxidized arachidonic acid, F2-IsoP has been widely regarded as a reference marker to evaluate the in vivo oxidative stress status.39 Studies indicated that the stimulatory effect of exogenous fructose on isoprostanes release would exacerbate lipid accumulation and adipocyte dysfunction in mice40 and, thus, was also shown in our HFFD rats (Tables 3 and 5; Figure S2). The level of F2-IsoP has been alleviated by 0.3, 0.5, and 0.75 energy-% but promoted by 2.25 energy-% ALA treatment (compared to MG, P < 0.05, Figure 4), indicating the antioxidant ability of ALA, however, might convert to pro-oxidant ability in vivo. Of note, such a proI

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TNF-α, and CRP secretion. It was also found that high intakes of ALA could provoke oxidative stress in vivo. By contrast, high intakes of LA did not induce oxidative stress, and the potential anorexic effect of LA may result in MS alleviation. Of note, the ratio of LA to ALA is insignificant in the prevention of MS. The absolute dose of PUFA may be a more optimal approach to estimate dietary n-6 and n-3 PUFA balance.

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REFERENCES

(1) Ruderman, N. B.; Carling, D.; Prentki, M.; Cacicedo, J. M. AMPK, insulin resistance, and the metabolic syndrome. J. Clin. Invest. 2013, 123, 2764−2772. (2) Romeo, G. R.; Lee, J.; Shoelson, S. E. Metabolic syndrome, insulin resistance, and roles of inflammation − mechanisms and therapeutic targets. Arterioscler., Thromb., Vasc. Biol. 2012, 32, 1771− 1776. (3) De Caterina, R. n-3 fatty acids in cardiovascular disease. N. Engl. J. Med. 2011, 364, 2439−2450. (4) Kopel, E.; Sidi, Y.; Kivity, S.; Estruch, R.; Ros, E. Mediterranean diet for primary prevention of cardiovascular disease. N. Engl. J. Med. 2013, 369, 672−677. (5) Simopoulos, A. P. The importance of the ratio of omega-6/ omega-3 essential fatty acids. Biomed. Pharmacother. 2002, 56, 365− 379. (6) Simopoulos, A. P. The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases. Exp. Biol. Med. 2008, 233, 674−688. (7) Riediger, N. D.; Azordegan, N.; Harris-Janz, S.; Ma, D. W.; Suh, M.; Moghadasian, M. H. ‘Designer oils’ low in n-6: n-3 fatty acid ratio beneficially modifies cardiovascular risks in mice. Eur. J. Nutr. 2009, 48, 307−314. (8) Yamashita, T.; Oda, E.; Sano, T.; Ijiru, Y.; Giddings, J.; Yamamoto, J. Varying the ratio of dietary n-6/n-3 polyunsaturated fatty acid alters the tendency to thrombosis and progress of atherosclerosis in apoE−/− LDLR−/− double knockout mouse. Thromb. Res. 2005, 116, 393−401. (9) Enos, R. T.; Velázquez, K. T.; McClellan, J. L.; Cranford, T. L.; Walla, M. D.; Murphy, E. A. Reducing the dietary omega-6:omega-3 utilizing α-linolenic acid; not a sufficient therapy for attenuating highfat-diet-induced obesity development nor related detrimental metabolic and adipose tissue inflammatory outcomes. PLoS One 2014, 9, e94897. (10) Harris, W. S. The omega-6/omega-3 ratio and cardiovascular disease risk: Uses and abuses. Curr. Atheroscler. Rep. 2006, 8, 453−459. (11) Deckelbaum, R. J. n-6 and n-3 fatty acids and atherosclerosis ratios or amounts ? Arterioscler., Thromb., Vasc. Biol. 2010, 30, 2325− 2326. (12) Harris, W. S.; Mozaffarian, D.; Rimm, E.; Kris-Etherton, P.; Rudel, L. L.; Appel, L. J.; Engler, M. M.; Engler, M. B.; Sacks, F. Omega-6 fatty acids and risk for cardiovascular disease a science advisory from the American Heart Association Nutrition Subcommittee of the Council on Nutrition, Physical Activity, and Metabolism; Council on Cardiovascular Nursing; and Council on Epidemiology and Prevention. Circulation 2009, 119, 902−907. (13) Harris, W. S. Expert opinion: omega-3 fatty acids and bleedingcause for concern? Am. J. Cardiol. 2007, 99, S44−S46. (14) Wijendran, V.; Hayes, K. Dietary n-6 and n-3 fatty acid balance and cardiovascular health. Annu. Rev. Nutr. 2004, 24, 597−615. (15) Poudyal, H.; Panchal, S. K.; Diwan, V.; Brown, L. Omega-3 fatty acids and metabolic syndrome: effects and emerging mechanisms of action. Prog. Lipid Res. 2011, 50, 372−387. (16) Rajaram, S. Health benefits of plant-derived α-linolenic acid. Am. J. Clin. Nutr. 2014, 100, S443−S448. (17) David, W. The role of n-6 and n-3 polyunsaturated fatty acids in the manifestation of the metabolic syndrome in cardiovascular disease and non-alcoholic fatty liver disease. Food Funct. 2014, 5, 426−435. (18) Poudyal, H.; Panchal, S. K.; Ward, L. C.; Brown, L. Effects of ALA, EPA and DHA in high-carbohydrate, high-fat diet-induced metabolic syndrome in rats. J. Nutr. Biochem. 2013, 24, 1041−1052. (19) World Health Organization. Fats and Fatty Acids in Human Nutrition, report of an expert consultation; FAO Food and Nutrition Paper, Geneva, Switzerland, 2008. (20) Capra, S. Nutrient Reference Values for Australia and New Zealand: Including Recommended Dietary Intakes; Commonwealth of Australia, 2006

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04715. Figure S1. Hematoxylin and eosin staining of pancreatic tissues in rats. Figure S2. Hematoxylin and eosin staining of epididymal adipose tissue in rats. Table S1. Levels of plasma lipids in LA groups at weeks 5, 11, and 18. Table S2. Levels of plasma lipids in ALA groups at weeks 5, 11, and 18 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*B.J. and Q.D. are regarded as joint corresponding authors. (B.J.) E-mail: [email protected]. Mail: China Agricultural University, P.O. Box 294, No. 17 Tsinghua East Road, Haidian District, Beijing 100083, P. R. China. Phone: +86-10-62736628. Fax: +86-10-62347334. *B.J. and Q.D. are regarded as joint corresponding authors. (Q.D.) E-mail: [email protected]. Mail: Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, No. 2 Xudong Two Road, Wuchang District, Wuhan 430062, P. R. China. Phone: +86-027-86827874. Fax: +86-02786815916. Author Contributions

B.J. and J.Z. designed the experiments; J.Z., Y.G., S.W., T.W., O.W., and G.L. conducted research; J.Z. analyzed the data; Q.D. provided reagents/materials/analysis tools; J.Z and O.W. wrote the paper; B.J. and Q.D. had primary responsibility for final content. All authors read and approved the final manuscript. Funding

This work was financially supported by the National Natural Science Foundation of China (General Program, 31371766) and the Director Fund of Oil Crops Research Institute (1610172014006). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ALA, α-linolenic acid; AUC, total area under the curve; CRP, C-reactive protein; CVD, cardiovascular disease; DHA, docosahexaenoic acid; ELISA, enzyme-linked immunosorbent assay; EPA, eicosapentaenoic acid; F2-IsoP, F2-iso-prostanes; GLP-1, glucagon-like peptide-1; GPR, G protein-coupled receptor; HDL-C, high-density lipoprotein-cholesterol; HE, hematoxylin and eosin; HFFD, high-fructose and high-fat diet; IL-6, Interleukin-6; ITT, insulin tolerance test; IR, insulin resistance; LA, linoleic acid; LDL-C, low-density lipoproteincholesterol; MS, metabolic syndrome; OGTT, oral glucose tolerance test; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; TC, total cholesterol; TG, total triglyceride; TNF-α, tumor necrosis factor-α J

DOI: 10.1021/acs.jafc.5b04715 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.5b04715 J. Agric. Food Chem. XXXX, XXX, XXX−XXX