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Bioactive Constituents, Metabolites, and Functions
Folic Acid Reduced Triglycerides Deposition in Primary Chicken Hepatocytes Yanli Liu, Jing Shen, Xin Yang, Qingzhu Sun, and Xiaojun Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05193 • Publication Date (Web): 28 Nov 2018 Downloaded from http://pubs.acs.org on December 4, 2018
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Journal of Agricultural and Food Chemistry
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Folic Acid Reduced Triglycerides Deposition in Primary Chicken Hepatocytes
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Yanli Liu, Jing Shen, Xin Yang, Qingzhu Sun, Xiaojun Yang*
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College of Animal Science and Technology, Northwest A&F University, Yangling, China
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*Corresponding Author:
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Xiaojun Yang: College of Animal Science and Technology, Northwest A&F University,
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No.22 Xinong Road, Yangling, Shaanxi, China; Postal code:712100.
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Tel: 86 029 87092102;
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Fax: 86 029 87092341;
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E-mail:
[email protected] (Xiaojun Yang);
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ABSTRACT
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Abdominal fat or fatty liver cause huge economic loss in poultry industry and nonalcoholic
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fatty liver disease (NAFLD) is also a global health issue in human. More than 90% of de novo
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lipogenesis in human and chickens is undertaken by the liver which is proved to be full of lipid
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in new-born chickens. Folic acid was thought to have correlation with lipid metabolism.
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Primary hepatocytes from new-born chickens were employed as a natural model of early stage
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fatty liver in vitro and further to explore whether folic acid could prevent fatty liver in the
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current study. We found that folic acid addition reduced triglyceride deposition by suppressing
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de novo fatty acid synthesis and coordinately promoting triglyceride hydrolysis and exportation
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in primary chicken hepatocytes from new-born chickens. In addition, lipogenesis suppression
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was through PI3K/AKT/SREBP pathway mediated by weakening insulin/IGF signal. Our data
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suggested that folic acid may be considered as a precautionary strategy for abdominal fat
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deposition in broilers or fatty liver in laying hens and humans. In addition, mechanism
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regulation also implied that IGF2 inhibitor and PI3K inhibitor may be used for the NAFLD
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precautionary measure to reduce TG deposition.
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KEYWORDS: folic acid; hepatocytes; lipid metabolism; insulin-like growth factor 2
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Nowadays, commercial genetic breeding selection of meat-type chickens significantly
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increased body weight and breast meat yield while higher abdominal fat was also accompanied
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because there were positive inheritance and genetic relationships between body weight and
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abdominal fat 1. The fatness negatively affected ketone body production, meat quality and feed
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utilization efficiency 2. In addition, fatty liver is a metabolic disease and widespread in laying
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hens, but it is often ignored because its symptoms are not obvious as infectious diseases 3.
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Nevertheless, fatty liver can decrease egg production, fertilization rate and hatching rate,
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ultimately causing huge economic losses in poultry industry 4. Unlike rodents and rabbits whose
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adipose tissues and livers contributed equally to de novo lipogenesis 5, more than 90% of de
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novo lipogenesis in human and chickens is undertaken by the liver 6. Therefore, chickens have
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been widely accepted and used as an animal model for human disease research about lipid
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metabolism 7, 8. Adult laying hens have been used to identify biomarkers as potential diagnostic
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indicators for human nonalcoholic fatty liver disease (NAFLD) 9, which is a global health issue
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with an estimated prevalence between 20%-68% in different countries 10. Hence, it is necessary
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to find ways to avoid fatty liver for human health and reducing poultry industry loss.
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Hepatic triglyceride accumulation results from the imbalance among de novo lipogenesis,
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free fatty acid uptake, fatty acid oxidation, triglyceride (TG) hydrolysis and export 11, and it is
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a complex disorder involving in genetic predisposition and environmental factors such as diets.
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It was reported that methyl donor deficient diets might contribute to hepatic lipid accumulation
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by suppression of very low-density lipoprotein (VLDL) assembly machinery
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methyl-donors could increase fatty acid utilization and prevent hepatic lipid accumulation in 3 ACS Paragon Plus Environment
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Dietary
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NAFLD by activating AMPK 13. Paternal dietary B vitamins difference could cause the variance
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in hepatic total cholesterol, triglycerides and lipid metabolism genes expression of female
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offspring 14. Folic acid, as a water soluble B vitamin, is involved in one-carbon metabolism and
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has been also reviewed to have many potential mechanisms linking its deficiency to fatty liver
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disease15. Yu et al found that folic acid addition could reduce FAS and C/EBPα expression by
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changing methylation level of gene promoter 16. Low serum folic acid levels were identified as
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an independent risk factor for NAFLD in the Chinese population 17. Hence, folic acid may be
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likely to alleviate fatty liver to some extent, but the mechanism is less known.
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It was reported that 80% lipid content of the yolk was absorbed into the embryonic liver
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during last seven days of chicken embryo development and hepatic lipid contents reached the
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higher level in new-born chickens 18. Hematoxylin-eosin (HE) and oil red O staining we did for
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the liver of new-born chicken also showed that it is full of lipid (as shown in Figure 1A and
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1C). In addition, hepatocytes from newborn chicken were proved to be abundant in lipid
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droplets by Oil Red O staining (Figure 1E) and electron microscopy observation (Figure 1F).
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Therefore, primary chicken hepatocytes from new-born chicken could be a better model for the
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fatty liver of early stage in vitro to some extent. Thus, primary hepatocytes from new-born
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chickens were isolated in the current study and employed to ascertain the regulation mechanism
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of folic acid on fatty liver in vitro, aiming to provide theoretical basis for improving the current
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situation of abdominal fat deposition in broilers or NAFLD in poultry and in human.
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MATERIALS AND METHODS
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Lipid analysis for the liver and hepatocytes from new-born chicken
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Liver samples were fixed for 24 h with 4% formaldehyde at room temperature. Fixed liver 4 ACS Paragon Plus Environment
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samples were processed for sectioning and then stained with HE or oil red O by Wuhan goodbio
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technology Co., Ltd. (Wuhan, China). In addition, Hepatocytes from newborn chicken was
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collected for electron microscopy analysis by Wuhan goodbio technology Co., Ltd. (Wuhan,
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China). The animal protocol was performed in sterile conditions and approved by the Animal
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Care and Use Committee of the College of Animal Science and Technology of the Northwest
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A&F University (Shaanxi, China).
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Isolation and culture of primary chicken hepatocytes
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Hepatocytes were isolated from male new-hatched chickens by collagenase digestion and
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filtration according to previous description 19-23 with some modifications, and specific isolation
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process were described in the previous report 24. After 12 h attachment incubation in culture
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medium containing 10% FBS (Biowest, Nuaile, France), 10 μg/mL human transferrin (Sigma),
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5 μg/mL bovine insulin (Sigma), 10-6 mol/L dexamethasone (Sigma), 100 U/mL penicillin, 100
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mg/mL streptomycin (Gibco) at 37 oC in a humidified atmosphere with 5% CO2, hepatocytes
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were washed with PBS and replaced with fresh culture medium. When reaching about 80%
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confluence, cells were washed and changed with treatment culture mediums for another12 h.
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RPMI 1640 Medium (Cat No. 11875) used in the current study was purchased from Gibco Co.
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Ltd which contained 1 mg/L folic acid.
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For treatments with folic acid, there was two groups, one for the control and another one
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for folic acid addition group with 15 mg/L folic acid in the culture medium. This dosage was
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based on the study from Yu et al 25 and our preliminary experiment 26, and data were not shown
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here. The folic acid from Sigma (St. Louis, MO) was dissolved with the aid of a minimal volume
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of 10% ammonium hydroxide and adjusted to 500 mg/L in deionized water, then filtered using 5 ACS Paragon Plus Environment
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0.22-μm filters 25. The stock solution was diluted further in RPMI 1640 medium to reach the
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final 15 mg/L concentration.
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For treatments with IGF2 and IGF2 inhibitor, there are three groups: one for the control,
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one for IGF2 (recombinat human IGF2 protein, R&D Systems) with 20 ng/mL in the medium
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and one for chromeceptin (Sigma-Aldrich) with 5μg/mL in the medium. The dosage of IGF2
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and chromeceptin used in the study was referred the previous study
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LY294002 (50 μM) was used as a pharmacological inhibitor of the PI3K/AKT pathway, and
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cells were pretreated with the compound 2 h prior to stimulation with 20 ng/mL IGF2. Optimal
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inhibition was obtained with this concentration as previous study 30.
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Metabolomic analysis
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Metabolites extraction, derivatization and GC-MS analysis
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cells were washed with PBS twice, and 300 μL ice-cold methanol were added to collect cells
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into tubes using wipers, then same volume ice water were mixed with methanol. Preliminary
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treatment about metabolites extraction and derivatization for samples was performed according
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to the previous method 24. Metabolomics instrumental analysis was performed on an Agilent
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7890A gas chromatography system coupled to an Agilent 5975C inert MSD system (Agilent
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Technologies Inc., CA, USA). A HP-5ms fused-silica capillary column (30 m × 0.25 mm ×
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0.25μm; Agilent J&W Scientific, Folsom, CA) was utilized to separate the derivatives. Detailed
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separation conditions and data collection were based on the previous report 24.
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Identification of differential metabolites
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further processing of raw GC-MS data were referred to the previous published protocols
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Multivariate statistical analysis was performed using SIMCA (version 13.0, Umetrics AB,
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PI3K inhibitor
After appropriate treatments,
The peak picking, alignment, deconvolution, and
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Umeå, Sweden) after normalizing against total peak abundance. The quality of the models is
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described by the R2X or R2Y and Q2 values. The differential metabolites were determined by
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the combination of the variable importance in the projection (VIP) value (>1) of orthogonal
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partial least squares discriminant analysis (OPLS-DA) model and the p values (
