Influence of a Dietary Vegetable Oil Blend on Serum Lipid Profiles in

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Influence of dietary vegetable oil blend on serum lipid profiles in large yellow croaker (Larimichthys crocea) Si Zhu, Peng Tan, Renlei Ji, Xiaojun Xiang, Zuonan Cai, Xiaojing Dong, Kangsen Mai, and Qinghui Ai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03382 • Publication Date (Web): 10 Aug 2018 Downloaded from http://pubs.acs.org on August 12, 2018

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Journal of Agricultural and Food Chemistry

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Influence of dietary vegetable oil blend on serum lipid profiles in large yellow croaker (Larimichthys crocea)

Si Zhu 1, Peng Tan 1, Renlei Ji 1, Xiaojun Xiang 1, Zuonan Cai 1, Xiaojing Dong 1, Kangsen Mai 1,2, Qinghui Ai 1,2* 1. Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture) & Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, Qingdao, Shangdong 266003, China 2. Laboratory for Marine Fisheries and Aquaculture, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shangdong, China

Corresponding author’s contact information: Address correspondence to Qinghui Ai, Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture) & Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, Qingdao, Shangdong 266003, China (E-mail: [email protected]).

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Abstract

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Serum lipid metabolic responses are associated with certain metabolic disorders

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induced by dietary habit in mammals. However, such associations have not been

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reported in fish. Lipidomic analyses were performed to investigate fish lipid

5

metabolic responses for dietary vegetable oil (VO) blend, and to elucidate the

6

mechanism of how dietary VO blend affects serum lipid profiles. Results showed

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that dietary VO blend strongly affects serum lipid profiles, especially ratio of

8

triglyceride:phosphatidylcholine (TAG:PC), via inhibiting hepatic PC biosynthesis

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and facilitating hepatic and intestinal lipoprotein assembly. Studies in vitro suggested

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that changes of serum TAG:PC ratio may be partially attributed to altered fatty acids

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composition in diets. Additionally, the reduction of 16:0/18:1-PC induced by dietary

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VO blend may play a role in abnormal lipid deposition through inhibiting

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PPARA-mediated activation of β-oxidation. These findings suggested that serum

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TAG:PC ratio might be a predictive parameter for abnormal lipid metabolism

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induced by dietary nutrition in fish.

16 17 18 19

Keywords: vegetable oil blend, serum lipid profiles, phosphatidylcholine,

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triglyceride, β-oxidation , large yellow croaker

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Introduction

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Substitution of fish oil by various other lipid sources has become an imperative issue

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since the rise and continued expansion of aquaculture, and the decreasing production

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of fish oil .1, 2 Vegetable oils, with relatively acceptable prices, large production

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volumes, and relatively low organic contaminants, are promising alternatives to fish

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oil. Considerable success in partially or totally replacing fish oil with vegetable oils

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without inhibiting fish growth performance have been reported in some commercial

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fish species 3, 4-6. However, long-term intake of vegetable oils often leads to changes

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in lipid metabolism of fish. Such changes include induced abnormal lipid deposition

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in the liver and decreased long chain polyunsaturated fatty acid (LC-PUFA) levels in

33

the tissue, which diminish the health benefits of fish for human consumption.3, 4-6

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The effects of replacing fish oil with vegetable oils on genes expression related to

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lipid metabolism in fish tissues have been widely investigated, although, studies

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were mainly focused on the regulation of fatty acids and triglyceride (TAG)

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mechanism by dietary vegetable oils.7, 8-9 Possible effects of dietary vegetable oils on

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other lipid classes such as phospholipids and sphingolipids, and further, the

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mechanisms in regulating these lipid classes are poorly studied. Only a few reports

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have investigated whether fatty acid distribution of phospholipid fraction reflects

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dietary nutrition.10, 11 It is well known that phospholipids and sphingolipids play a

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pivotal role in regulating lipid, lipoprotein and energy metabolism.12, 13-14 In addition,

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despite the extensive studies on the liver and intestine, the effects of dietary

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vegetable oils on lipid metabolic responses in the blood of fish are still quite



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fragmentary.

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Blood is a major carrier of lipids which bathes tissues and organs in the entire body

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and functions as a liquid highway for lipids that are being secreted, excreted, and

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discarded from different tissues. Studies have reported that levels of lipids in blood

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and other organs are closely correlated, and changes in blood lipid profiles reflect the

50

metabolic perturbations in response to metabolic alteration at a systemic level. 15, 16-17

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In mammals, the associations between serum lipid metabolic responses and certain

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metabolic disorders under excessive dietary nutrition are well demonstrated, and

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genes expression related to lipid metabolism were detected to explain changes in

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response to diets.18-19 However, such associations have not been reported in fish,

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where comprehensive data is still quite deficient.

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The large yellow croaker (Larimichthys crocea) is an economically important marine

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fish species that is widely cultured in southeast China due to its delicious taste and

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high nutritional value.20 The production of this fish species was 165496 metric tons

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in China in 2016, earning it first place in mariculture output which it continuous to

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hold.21 Vegetable oils have been commonly used in the culture of large yellow

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croaker due to the limited production of fish oil. Our previous study showed that

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partially or totally replacing fish oil with a vegetable oil blend (soybean oil: linseed

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oil=1:1) induced abnormal lipid metabolism in large yellow croaker in the form of

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decreased tissue levels of LC-PUFA and increased hepatic lipid accumulation.22

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Additionally, it was found that this fish species shares a strikingly similar regulation

66

in lipid metabolism with those of other fish species and mammals,23,


24

which

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suggested that large yellow croaker is an appropriate fish species to investigate the

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mechanism of how dietary vegetable oils influence serum lipid profiles.

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In the present study, our main objective was to investigate the lipid metabolic

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responses in the serum of fish under the replacement of fish oil by a vegetable oil

71

blend (soybean oil: linseed oil=1:1), and to elucidate the mechanism of how dietary

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vegetable oil blend affects serum lipid profiles. The knowledge of biochemical

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changes of fish will be helpful in seeking effective measures to prevent and alleviate

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abnormal lipid metabolism induced by long-term use of vegetable oils.

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Materials and Methods

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Materials

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Acetonitrile, isopropanol, tetrahydrofuran, formic acid (LC-MS grade) were

78

purchased from Sigma-Aldrich (St. Louis, Mo). Fatty acids (Purity ≥ 99%) including

79

docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3),

80

linoleic acid (LA, 18:2n-6) and linolenic acid (LNA, 18:3n-3) were obtained from

81

Sigma-Aldrich (St. Louis, Mo). Lipid standards including phosphatidylcholine (PC;

82

18:1/14:0), phosphatidylethanolamine (PE; 16:0/18:1), lysophosphatidylcholine

83

(LPC; 20:0), lysophosphatidylethanolamine (LPE; 17:1), sphingomyelin (SM;

84

d18:1/18:0) and diacylglycerol (DAG; 16:0/18:1) were purchased from the Avanti

85

Polar Lipids (Alabaster, AL, USA). TAG (14:0/18:2/18:1) was purchased from

86

Larodan (Limhamn, Sweden).

87

Diet preparation and feeding trial

88

Diet preparation and fish rearing were reported in our previous studies.22 In brief,



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three isoproteic (41% crude protein) and isolipidic diets (12% crude lipid) were

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formulated with 0%, 50%, and 100% of fish oil replaced by a vegetable oil blend

91

(soybean oil: linseed oil=1:1) named “FO (fish oil), FV (fish oil: vegetable oil

92

blend=1:1) and VO (vegetable oil blend)” respectively. The formula and fatty acids

93

composition of the experimental diets are shown in Supporting Information Table 1

94

and Table 2. Disease-free and similar sized large yellow croaker were obtained and

95

reared at Xihu harbor (Ningbo, China). A total of 270 fish with an average initial

96

weight of 8.93±0.20 g were fasted for 24h and randomly allocated to nine floating

97

net cages (1.5×1.5×2.0 m) corresponding to triplicate cages of the three dietary

98

treatments. Fish were fed twice per day to apparent satiation for 70 days (5 August to

99

15 October). Water temperature ranged between 23.5 and 30.0 °C and dissolved

100

oxygen ranged between 6.0 and 7.0 mg/L during the feeding trial. All diets were

101

stored at -20 °C until used to maintain good quality on the farm. The protocols used

102

for fish husbandry and handing in this study were approved by the Institutional

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Animal Care and Use Committee of Ocean University of China.

104

Sample collection

105

At the end of the experiment, fish were fasted for 24 h to empty their guts before

106

sampling. Nine fish per treatment (three fish per cage) were randomly selected and

107

anaesthetized with MS222 (Sigma, USA). 1mL of blood from each individual fish

108

was phlebotomized and stored at 4 °C for 3-4 h. After centrifugation (3000× g for

109

10min at 4 °C), the serum was collected and stored at -80℃ before analysis. The liver

110

and intestine samples were collected from those bloodless fish, immediately frozen


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in liquid nitrogen and stored at -80 °C prior to analysis.

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Primary hepatocytes culture and treatment

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Cultivation of primary hepatocytes was performed according to our previous

114

report.25 Briefly, liver was isolated from large yellow croaker and washed in cold

115

sterile PBS. Liver tissue was then dissected into 1 mm3 pieces and digested with

116

0.25% Trypsin-EDTA (Gibco) at room temperature for 10 min. Cell suspension was

117

filtered and centrifuged at 800 g for 10 min. Primary hepatocytes were resuspended

118

and cultured (1×106 cells/mL) in six well plates with complete medium (DMEM/F12

119

medium containing 10% FBS, 1×streptomycin and 1×penicillin) and then maintained

120

at 28 °C in 5% CO2. After 2 d cultivation, primary hepatocytes were starved with

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FBS free DMEM/F12 overnight. In consideration of the major fatty acids

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composition in fish oil and vegetable oil blend, the cells were respectively incubated

123

with 100 µM DHA , EPA, LA and LNA conjugated with 2% fatty acid free bovine

124

serum albumin (BSA) (Equitech-Bio, USA) in DMEM/F12 for 6 h. Cells in the

125

control group were incubated with 2% BSA in DMEM/F for 6 h.

126

UPLC analysis

127

Chromatographic separation was carried out on an ACQUITY® UPLC BEH C8

128

analytical column (2.1×100 mm, 1.7 µm) using a ACQUITY® Ultra Performance LC

129

system (Waters). The serum samples were dissolved in 1000 µL water/acetonitrile

130

(1:4, by vol), and centrifuged at 12,000 ×g for 10 min at 4 °C. The supernatant was

131

filtered through a 0.22 µm ultrafiltration membrane (Millipore, USA). An aliquot (3

132

µL) of each sample was injected into the column for analyzing. The elution was



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performed

134

acetonitrile/isopropanol/tetrahydrofuran (1:1:1, by vol) and mobile phase B

135

composed of water/ acetonitrile (1:2, by vol), both containing 0.1% formic acid and

136

0.01% lithium acetate. The gradient elution was programmed as follows: 0-3 min,

137

2% A; 3-5 in, 2-30% A; 5-12 min, 30-50% A; 12-22 min, 50-80% A; 22-25 min,

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80% A; 25-27 min, returning to initial 2% A; 27-30 min, maintaining at 2% A. The

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total gradient duration was 30 min at a flow rate of 0.5 mL/min.

140

Mass spectrometric analysis

141

Mass spectrometric analysis was conducted on a Q-TOF Premier® (Waters)

142

operating in both positive and negative ion modes. For positive mode, the capillary

143

voltage was to 3.0 kV and the sampling cone voltage was set to a ramp of 25-40 V.

144

Whilst for negative mode, the capillary voltage was to 2.6 kV and the sampling cone

145

voltage was set to a ramp of 25-40 V. MSE analysis was performed on the mass

146

spectrometer at 6 KV for low collision energy and ramp of 25-50 kV for high

147

collision energy. Data was collected in a centroid mode from 100 to 1200 m/z. The

148

instrument was previously calibrated with sodium formate, and the lock mass spray

149

for precise mass determination was set by leucine encephalin.

150

Identification and semi-quantitative analysis of lipids

151

The precise molecular weight (the mass difference 0.05, Tukey’s test), and the relative mRNA expression of target genes in FO group was selected as the calibrator. ppara, peroxisome proliferator-activated receptor alpha; cpt1a, carnitine palmitoyltransferase 1A; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend. Figure 9: Protein amounts of hepatic PPARA (A) and CPT1A (B) in large yellow croaker fed three different diets. Protein bands density were quantified by NIH Image 1.63 and normalized to GAPDH. Values (means ± SD, n=9) in bars that have the same superscript letter are not significantly different (P>0.05, Tukey’s test). PPARA, peroxisome

proliferator-activated

receptor

alpha;

CPT1A,

carnitine

palmitoyltransferase 1A; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend. Figure 10: Phosphatidylcholine (PC) and triglyceride (TAG) metabolism related genes expression in primary hepatocytes of large yellow croaker incubated with different fatty acids, including pcyt1a, chpt1, pemt, dgat2, mttp and apoB100 (A-F). Values



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34 (means ± SD, n=9) in bars that have the same superscript letter are not significantly different (P>0.05, Tukey’s test), and the relative mRNA expression of target genes in control group was selected as the calibrator. pcyt1a, phosphate cytidylyltransferase 1, choline, alpha; chpt1, choline phosphotransferase 1; pemt, phosphatidylethanolamine N-methyltransferase;

mttp,

microsomal

triglyceride

transfer

protein;

dgat2,

diacylglycerol O-acyltransferase 2; apoB100, apolipoprotein B100; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend. Figure 11: Protein amounts of PCYT1A (A), CHPT1 (B) and DGAT2 (C) in primary hepatocytes of large yellow croaker incubated with different fatty acids. Protein bands density were quantified by NIH Image 1.63 and normalized to GAPDH and then normalized to GAPDH. Values (means ± SD, n=9) in bars that have the same superscript letter are not significantly different (P>0.05, Tukey’s test). PCYT1A, phosphate cytidylyltransferase 1, choline, alpha; CHPT1, choline phosphotransferase 1; DGAT2, diacylglycerol O-acyltransferase 2; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend.


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35 Table 1. Contents of significantly differential lipid species in the serum of large yellow croaker response to different diets (nmol/mg dry total lipids)

RT(min)

7.88 7.44 9.04 8.14 9.11 8.56 6.66 6.28 6.9 7.95 7.68 6.24 8.21 7.42 7.57 8.43 9.35 6.88 16.66 16.9 17.49 16.82 18.06 18.63 16.2 17.43 18.57 18 11.32 12.26 9.65 8.7 7.81 9.32 10.05 10.84 0.96 1.14

adduct ion +

[M+H] [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+Li]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+Li]+ [M+Li]+ [M+Li]+ [M+Li]+ [M+Li]+ [M+Li]+ [M+Li]+ [M+Li]+ [M+Li]+ [M+Li]+ [M+Li]+ [M+Li]+ [M-H][M-H][M-H][M-H][M+H]+ [M+H]+ [M+H]+ [M+HCOO]-

m/z

806.5 780.5 834.5 832.5 760.5 808.5 834.5 802.5 804.5 732.5 806.5 826.5 758.5 756.5 782.5 784.5 786.5 780.5 883.7 885.7 887.7 859.7 889.7 891.7 857.7 861.7 865.7 863.7 623.5 625.5 742.5 740.5 738.5 790.5 811.5 813.5 520.3 540.3

VIP

Compound name

12.1547 9.90225 7.2255 6.72161 6.55482 5.8556 4.0676 3.61018 2.85906 2.66549 2.43415 2.34405 10.6848 9.02633 8.9109 8.33704 7.05093 5.64222 4.76413 3.92789 3.78364 3.48175 3.44651 3.21677 2.68155 2.67359 2.58059 2.31799 3.6849 3.51251 1.74221 1.68854 1.55678 1.84996 2.59086 2.53297 2.48434 1.67047

16:0/22:6-PC 16:0/20:5-PC 18:0/22:6-PC 18:1/22:6-PC 16:0/18:1-PC 18:0/20:5-PC 18:3/22:6-PC 18:3/20:5-PC 18:2/20:5-PC 16:1/16:0-PC 18:1/20:5-PC 20:5/20:5-PC 16:0/18:2-PC 16:0/18:3-PC 18:2/18:2-PC 18:1/18:2-PC 18:0/18:2-PC 18:3/18:2-PC 18:2/16:0/20:5-TAG 18:1/18:2/18:3-TAG 18:2/18:2/18:1-TAG 18:2/16:0/18:3-TAG 18:1/18:1/18:2-TAG 18:1/18:1/18:1-TAG 18:3/18:2/16:1-TAG 18:2/18:2/16:0-TAG 18:1/18:1/16:0-TAG 16:0/18:2/18:1-TAG 18:2/18:2-DAG 18:1/18:2-DAG 18:0/18:2-PE 18:1/18:2-PE 18:2/18:2-PE 22:6/18:0-PE d18:2/24:1-SM d18:1/24:1-SM 18:2-LPC 16:0-LPC

FO

Content (nmol/mg) FV a

33.96±2.66 27.48±1.73a 11.01±0.79a 13.37±0.89a 8.80±0.42a 10.33±0.62a 0.55±0.11b 0.23±0.03b 2.35±0.19a 2.16±0.13a 8.40±0.56a 0.34±0.04a 17.76±0.78c 1.99±0.13b 1.43±0.11c 5.33±0.45c 6.21±0.38b nd 1.72±0.11a 1.30±0.08c 1.98±0.08c 1.14±0.07c 2.06±0.08c 2.26±0.11c 0.19±0.02c 2.33±0.12c 3.31±0.09a 3.27±0.10a nd 0.50±0.03b nd nd nd 5.79±0.25a 1.19±0.11a 1.62±0.17a 0.49±0.05c 2.63±0.24a


b

21.02±1.02 13.81±0.77b 7.79±0.36b 6.84±0.16b 7.33±0.20b 6.32±0.30b 0.78±0.07a 0.54±0.07a 1.25±0.08b 1.49±0.04b 3.93±0.07b 0.11±0.01b 23.70±1.04a 8.47±0.54a 8.24±0.35b 12.47±0.94b 11.18±0.38a 2.50±0.11b 0.95±0.04b 2.80±0.09b 3.07±0.11b 3.18±0.13b 2.91±0.11b 2.62±0.12b 1.12±0.08b 3.63±0.12b 2.80±0.09b 3.43±0.23a 0.59±0.08b 0.87±0.08b 1.45±0.06b 0.97±0.08b 0.50±0.03b 2.66±0.38b 0.61±0.04b 1.09±0.06b 0.80±0.08b 2.33±0.12a

VO

1.93±0.07c 1.40±0.07c 0.80±0.02c 0.49±0.01c 4.39±0.17c 0.89±0.03c 0.09±0.01c 0.14±0.01b nd 0.33±0.01c 0.52±0.02c nd 21.26±0.92b 8.05±0.42a 10.99±0.55a 15.18±0.41a 12.18±0.51a 4.42±0.27a 0.24±0.01c 5.29±0.11a 5.08±0.19a 4.12±0.13a 4.19±0.24a 3.43±0.12a 2.22±0.05a 4.10±0.10a 2.27±0.03c 3.63±0.11a 4.37±0.22a 4.59±0.39a 3.04±0.17a 2.01±0.07a 1.19±0.04a 1.69±0.04c 0.14±0.01c 0.34±0.03c 1.33±0.11a 0.80±0.05b


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36 1.03 1.66 1.14

[M+HCOO][M-H][M-H]-

612.3 480.3 452.3

1.11454 1.0873 1.03385

22:6-LPC 18:0-LPE 16:0-LPE

1.10±0.12a 2.83±0.12a 1.58±0.10a

0.53±0.04b 1.71±0.07b 0.76±0.04b

Values are means ± SD, n=9. Different superscript letter within the same row represents statistical significant difference (P0.05, Tukey’s test). DAG, diglyceride; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; TAG, triglyceride; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend. 39x20mm (600 x 600 DPI)


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Figure 4: Phosphatidylcholine (PC) and triglyceride (TAG) metabolism related genes expression in the liver of large yellow croaker fed three different diets, including pcyt1a, chpt1, pemt, dgat2, mttp and apoB100 (A-F). Values (means ± SD, n=9) in bars that have the same superscript letter are not significantly different (P>0.05, Tukey’s test), and the relative mRNA expression of target genes in FO group was selected as the calibrator. pcyt1a, phosphate cytidylyltransferase 1, choline, alpha; chpt1, choline phosphotransferase 1; pemt, phosphatidylethanolamine N-methyltransferase; dgat2, diacylglycerol O-acyltransferase 2; mttp, microsomal triglyceride transfer protein; apoB100, apolipoprotein B100; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend. 69x60mm (600 x 600 DPI)



Figure 5: Protein amounts of PCYT1A (A), CHPT1 (B) and DGAT2 (C) in the liver of large yellow croaker fed three different diets. Protein bands density were quantified by NIH Image 1.63 and normalized to GAPDH. Values (means ± SD, n=9) in bars that have the same superscript letter are not significantly different (P>0.05, Tukey’s test). PCYT1A, phosphate cytidylyltransferase 1, choline, alpha; CHPT1, choline phosphotransferase 1; DGAT2, diacylglycerol O-acyltransferase 2; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend. 96x115mm (600 x 600 DPI)


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Figure 6: Triglyceride (TAG) metabolism related genes expression in the intestine of large yellow croaker fed three different diets, including dgat1, mttp and apoB48 (A-C). Values (means ± SD, n=9) in bars that have the same superscript letter are not significantly different (P>0.05, Tukey’s test), and the relative mRNA expression of target genes in FO group was selected as the calibrator. dgat1, diacylglycerol Oacyltransferase 1; mttp, microsomal triglyceride transfer protein; apoB48, apolipoprotein B48; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend. 33x14mm (600 x 600 DPI)



Figure 7: Content of serum PC (16:0/18:1) in large yellow croaker fed three different diets. Values (means ± SD, n=9) in bars that have the same superscript letter are not significantly different (P>0.05, Tukey’s test). PC, phosphatidylcholine; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend. 41x44mm (600 x 600 DPI)


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β-oxidation related genes expression in the liver of large yellow croaker fed three different diets, including ppara (A) and cpt1a (B). Values (means ± SD, n=9) in bars that have the same superscript letter are not significantly different (P>0.05, Tukey’s test), and the relative mRNA expression of target genes in FO group was selected as the calibrator. ppara, peroxisome proliferator-activated receptor alpha; cpt1a, carnitine palmitoyltransferase 1A; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend. 49x30mm (600 x 600 DPI)



Figure 9: Protein amounts of hepatic PPARA (A) and CPT1A (B) in large yellow croaker fed three different diets. Protein bands density were quantified by NIH Image 1.63 and normalized to GAPDH. Values (means ± SD, n=9) in bars that have the same superscript letter are not significantly different (P>0.05, Tukey’s test). PPARA, peroxisome proliferator-activated receptor alpha; CPT1A, carnitine palmitoyltransferase 1A; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend. 83x86mm (600 x 600 DPI)


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Figure 10: Phosphatidylcholine (PC) and triglyceride (TAG) metabolism related genes expression in primary hepatocytes of large yellow croaker incubated with different fatty acids, including pcyt1a, chpt1, pemt, dgat2, mttp and apoB100 (A-F). Values (means ± SD, n=9) in bars that have the same superscript letter are not significantly different (P>0.05, Tukey’s test), and the relative mRNA expression of target genes in control group was selected as the calibrator. pcyt1a, phosphate cytidylyltransferase 1, choline, alpha; chpt1, choline phosphotransferase 1; pemt, phosphatidylethanolamine N-methyltransferase; mttp, microsomal triglyceride transfer protein; dgat2, diacylglycerol O-acyltransferase 2; apoB100, apolipoprotein B100; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend. 86x92mm (600 x 600 DPI)



Figure 11: Protein amounts of PCYT1A (A), CHPT1 (B) and DGAT2 (C) in primary hepatocytes of large yellow croaker incubated with different fatty acids. Protein bands density were quantified by NIH Image 1.63 and normalized to GAPDH and then normalized to GAPDH. Values (means ± SD, n=9) in bars that have the same superscript letter are not significantly different (P>0.05, Tukey’s test). PCYT1A, phosphate cytidylyltransferase 1, choline, alpha; CHPT1, choline phosphotransferase 1; DGAT2, diacylglycerol Oacyltransferase 2; FO, fish oil; FV, fish oil and vegetable oil blend; VO, vegetable oil blend. 99x125mm (600 x 600 DPI)


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Table of contents 59x44mm (300 x 300 DPI)


Influence of a Dietary Vegetable Oil Blend on Serum Lipid Profiles in

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