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Brain Natriuretic Peptide Stimulates Lipid Metabolism through Its Receptor NPR1 and the Glycerolipid Metabolism Pathway in Chicken Adipocytes HuaYun Huang, Guiping Zhao, RanRan Liu, QingHe Li, MaiQing Zheng, ShouFeng Li, Zhong Liang, ZhenHua Zhao, and Jie Wen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b00714 • Publication Date (Web): 14 Oct 2015 Downloaded from http://pubs.acs.org on October 20, 2015
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Brain Natriuretic Peptide Stimulates Lipid Metabolism through Its Receptor NPR1 and the Glycerolipid Metabolism Pathway in Chicken Adipocytes FUNDING SOURCE STATEMENT: The research was supported by grants: 863 project (2013AA102501);
China
Agricultural
Science
and
Technology
Innovation
Project
(ASTIP-IAS04); the earmarked fund for modern agro-industry technology research system (CARS-42). Huang HY
1,2#
, Zhao GP
1,3#
1,3
, Liu RR , Li QH
1,3
1,3
2
2
2
, Zheng MQ , Li SF , Liang Z , Zhao ZH , Wen
J 1,3* 1
Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.
China; 2
Institute of Poultry Science, Chinese Academy of Agriculture Sciences, Jiangsu 225125, P. R.
China; 3
State Key Laboratory of Animal Nutrition, Beijing 100193, P. R. China;
Email: Huang HY - huanghuayun520@163; Zhao GP -
[email protected] Liu RR -
[email protected]; Li QH -
[email protected]; Zheng MQ –
[email protected]; Li SF -
[email protected] ; Liang Z -
[email protected]; Zhao ZH-
[email protected]; Wen J * -
[email protected] Huang HY and Zhao GP contributed equally to this work. *
Corresponding author: Wen J, Institute of Animal Sciences, Chinese Academy of Agricultural
Sciences, Beijing 100193, P.R. China. Tel: +86-10-62815856，Fax: +86-10-62816018.
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ABBREVIATIONS: ANP : Atrial natriuretic peptide; BNP: Brain natriuretic peptide; CNP: C-type natriuretic peptide; NPR1 : Natriuretic peptide receptor 1; NPR2: Natriuretic peptide receptor 2; NPPB : Natriuretic
peptide
precursor
B;
High-abdominal-fat
HAbF:
chicken
group;
LAbF
Low-abdominal-fat group; DEGs: Differentially expressed genes; KEGG: Kyoto Encyclopedia of Genes and Genomes; DGKG: Diacylglycerol kinase; LIPG: Lipase, endothelial; AGPAT1: 1-acylglycerol-3-phosphate O-acyltransferase 1;
AGPAT2: 1-acylglycerol-3-phosphate
O-acyltransferase 2; QPCR: quantitative real-time PCR; CCK8: Cell Counting Kit-8; SD: standard deviation; NPR-1/cGMP/PKG: natriuretic peptide receptor 1/ cyclic guanosine monophosphate/ protein kinase G; DAG: diacylglycerol.
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ABSTRACT Brain natriuretic peptide (BNP) is related to lipid metabolism in mammals, but its effect and the molecular mechanisms underlying it in chickens is incompletely understood. We found that natriuretic peptide precursor B (NPPB, which codes BNP) mRNA expression in high-abdominal-fat chicken groups was significantly higher than that of low-abdominal-fat groups. Partial correlations indicated that changes in the weight of abdominal fat were positively correlated with NPPB mRNA expression. In vitro, compared with the control group, preadipocytes with NPPB interference showed reduced proliferation, differentiation and glycerin concentration in media. Treatments of cells with BNP led to enhanced proliferation/differentiation of cells and glycerin concentration, and mRNA expression of its receptor natriuretic peptide receptor 1 (NPR1) was up-regulated significantly. In cells exposed to BNP, 482 differentially expressed genes were determined compared with controls without BNP. Four genes known to be related to lipid metabolism (diacylglycerol kinase; lipase, endothelial; 1-acylglycerol-3-phosphate O-acyltransferase 1; 1-acylglycerol-3-phosphate O-acyltransferase 2) were enriched in the glycerolipid metabolism pathway and expressed differentially. In conclusion, BNP stimulates the proliferation, differentiation and lipolysis of preadipocytes through up-regulation of the expressions of its receptor NPR1 and key genes enriched in glycerolipid metabolic pathway
Keywords: NPPB interference; BNP induction; Chicken; Differentially expressed genes; Lipid metabolism
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INTRODUCTION Natriuretic peptides are a group of hormones characterized by a conserved ring structure comprising 17 amino acids with N-terminal and C-terminal extensions1, 2. Main natriuretic 3
4
peptides in mammals are atrial natriuretic peptide (ANP) , brain natriuretic peptide (BNP) and c-type natriuretic peptide (CNP)
5
. Natriuretic peptides play important parts in multiple
biological processes though two receptors: natriuretic peptide receptor (NPR)1 and NPR2. NPR1 mRNA has been identified in the fat tissues of rats and humans
6-9
. BNP promotes
lipolysis of isolated fat cells in humans10 and its biological actions are mediated by NPR1 in mammals
11
. It has been shown that triglyceride levels in the liver are reduced significantly in
high fat-fed BNP knockout mice compared with those of wild-type mice, and that triglyceride levels in the liver are not significantly different between rats fed standard chow and wild-type 12
mice . Clinical investigations have demonstrated that BNP levels are inversely associated with several components of the metabolic syndrome (e.g., glucose intolerance, dyslipidemia, insulin resistance)
13
. Plasma levels of BNP have been shown to be inversely related with
obesity in community-based subjects in Japan14. These results have suggested a link between BNP and lipid metabolism in mammals. However, in birds (and different from mammals), ANP has not been isolated, and BNP (coded by natriuretic peptide precursor B (NPPB)) is the sole circulating hormone secreted in 15
the heart . In birds, the relationship between BNP and lipid metabolism is far from certain though chicken BNP has been shown to inhibit the ability of angiotensin II to stimulate release of aldosterone into the circulation 16. Studies from our research team have shown that two SNPs were associated with abdominal 4
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fat percentage by genome-wide association study in chicken. These SNPs are within 8.3 Kb of the natriuretic peptide B (NPPB) gene. Furthmore, NPPB expression in a high-abdominal-fat chicken (HAbF) group was significantly higher than that of a 17
low-abdominal-fat (LAbF) group . These findings suggest that BNP influences fat deposition in chickens. Thus, the present study was undertaken to clarify the effect of BNP on lipid metabolism in chickens and the molecular mechanisms underlying this phenomenon. MATERIAL AND METHODS ETHICAL APPROVAL OF THE STUDY PROTOCOL The study protocol was conducted in accordance with the Guidelines for the Experimental Animals established by the Ministry of Science and Technology (Beijing, China). All experimental protocols were approved by the Science Research Department (in charge of animal welfare) of the Institute of Animal Sciences, CAAS (Beijing, China). CULTURE OF PREADIPOCYTES Preadipocytes were isolated from the abdominal adipose tissue from female Beijing-You chickens (age, 2–4 weeks; Institute of Animal Science, CAAS) following methods described previously 18, 19. Cells were cultured in 96-well and six-well culture dishes. The culture medium was DMEM/F12 (1:1) containing 10% FBS and 1% penicillin/streptomycin solution at 37°C in a humidified atmosphere of 5% CO2 in air. Cells reached 70% confluence, then differentiation was induced with monocyte differentiation-inducing factors (3-isobutyl-1-methylxanthine, 0.5 mmol/L; dexamethasone, 1 µmol/L; insulin, 1 mg/L) for 2 days followed by another 2 days with only insulin (l mg/L) added to the basic culture medium. CONSTRUCTION AND TRANSFECTION OF NPPB INTERFERENCE VECTORS 5
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Vector construction. NPPB interference vector was synthesized by GenePharma (Shanghai GenePharma, Shanghai, China). According to the mRNA sequence of chicken NPPB (NM_204925.1), Four pairs of shRNA primers were designed by Oligo Designer v3.0 (Genepharma, shanghai, china) (Table1)
with TTCAAGAGA as the internal loop. The primer
NPPB-gga-201 was further validated in vitro. Annealed shRNA oligonucleotides were ligated into a pGPU6/GFP vector (Clontech Laboratories, Shiga, Japan) using BamHI and BpiI, and then transformed into Escherichia coli DH5-α. Positive clones were identified by sequencing. Along with these synthesized oligonucleotides, manufacturer (Clontech Laboratories)-supplied scrambled shRNA oligonucleotides were also ligated to the pGPU6/GFP vector and used as negative controls. Transfection. Preadipocytes were plated on six-well or 96-well plates in DMEM/F12 without antibiotics. Transfections were undertaken at 70–80% confluence using Fugene 6 (Promega, Madison, WI, USA). Adipocytes cultured in six-well plates were transfected with shNPPB (1.5 µg per well) to detect gene expression and cell differentiation using qPCR and staining with Oil Red O in three replicate wells, respectively. Cells of the same group in 96-well plates were transfected with shNPPB (200 ng per well) to monitor cell proliferation using Cell Counting Kit-8 (CCK-8; Dojindo, Fukuoka, Japan) in eight replicate wells. BNP TREATMENTS Starting on day-0, chicken BNP (Phoenix, AZ, USA) was included in media at 0.001, 0.01, and 1 nmol/L. Cell proliferation was detected using CCK-8 in eight replicate wells from each treatment. Chicken BNP was added with differentiation media at 0 and 1 nmol/L in three replicate wells from each treatment. Adipogenesis was monitored by morphologic examination 6
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of the cellular accumulation of lipid droplets by staining (Oil Red O). On days 2, 4 and 6, adipocytes were fixed with 10% formaldehyde, washed with PBS, and stained with Oil Red O (0.3% in 60% isopropanol) followed by extensive washes. Then, stained triglyceride droplets were visualized and photographed. LIPOLYSIS For differentiated adipocytes incubated for 6 days, chicken BNP (0 and 1 nmol/L) was added and adipocytes incubated for a further 24 h. The glycerol concentration in the medium was determined in three replicate wells from each treatment using a Glycerol kit (Jiancheng, Nanjing, China). The glycerol concentration in the medium was detected using this Glycerol kit in three replicate wells from each treatment 2 days and 4 days after shNPPB transfection. RNA EXTRACTION Total RNA was isolated from fat cells using a Total RNA kit (TIANGEN, Beijing, China) according to manufacturer instructions. Concentration and purity of RNA were determined at an absorbance of 260 nm (A260) and A280 as well as the ratio A260:280 (A260:280 ≥1.8 and ≤2.0) using a ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). RNA integrity (RIN ≥7) was assessed on an 2100 Bioanalyzer Lab-on-chip system (Agilent Technologies, Palo Alto, CA, USA). RNA samples were stored at −80°C until use. PROFILING OF GENE EXPRESSION Profiling of gene expression based on ultra-high throughput sequencing (HiSeq 2500; Illumina, San Diego, CA, USA) was undertaken by RiboBio (Guangdong, China) using total RNA from three replicate differentiated adipocytes treated with BNP (0 and 1 nmol/L) for 4 days, respectively. Raw data were converted to FASTQ files using bcl2fastq. “Clean” data 7
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were obtained by filtering and removing low-quality sequences, and mapped to the reference genome and genes of chickens (Gallus_gallus-4.0; available at www.ncbi.nlm.nih.gov/genome) using TopHat. Reads per kilo-base per million mapped reads was employed to quantify gene expression.Differentially expressed genes (DEGs) were identified by ≥2 fold-changes and P
