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Comparative transcriptome analysis reveals more complicated adipogenic process in intramuscular stem cells than that of subcutaneous vascular stem cells Luxi Chen, Yue Zhang, Hu Chen, Xumeng Zhang, Xiaohong Liu, Peiqing Cong, Zuyong He, Yaosheng Chen, and Delin Mo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00856 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 2, 2019
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Journal of Agricultural and Food Chemistry
Comparative transcriptome analysis reveals more complicated
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adipogenic process in intramuscular stem cells than that of
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subcutaneous vascular stem cells
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Luxi Chen#, Yue Zhang#, Hu Chen#, Xumeng Zhang, Xiaohong Liu, Zuyong He,
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Peiqing Cong, Yaosheng Chen, Delin Mo*
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State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University,
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Guangzhou, Guangdong 510006, P.R. China
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#
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* Corresponding author
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Tel: +86-020-39332991
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Fax: +86-020-39332940
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Email:
[email protected] These authors contributed equally to this work.
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Abstract
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Fat-related traits have great influences on pork quality. As different fat tissues have
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different biochemical profiles depending on its location, intramuscular fat contributes
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to the gustatory qualities, while subcutaneous fat is considered as a negative factor
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associated with growth performance. In this study, both primary intramuscular and
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subcutaneous vascular stem cells (IVSCs and SVSCs) could be differentiated into
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mature adipocytes, though the IVSCs differentiation efficiency was lower. By
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comparative analysis of transcriptomes, 2524 differentially expressed genes (DEGs)
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were found between two VSCs before differentiation, while only 551 DEGs were found
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and enriched in two pathways including biosynthesis of unsaturated fatty acids after
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differentiation. This result indicated that differentiated VSCs were more similar. During
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differentiation, more DEGs existed in IVSCs than that in SVSCs, suggesting that
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adipogenesis of IVSCs might be more complex. Additionally, the expression level of
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DEGs involved in adipogenic process helps to explain the difference of differentiation
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efficiency between IVSCs and SVSCs.
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Keywords: Pig; Adipogenesis; Adipocyte; Fatty acid content; PPARγ
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Introduction
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Intramuscular fat (IMF) has been regarded as a potential factor responsible for meat
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quality including meat tenderness, juiciness, and taste in livestock and poultry industry
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1-3
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fat has become hotspot in recent years. What’s more, pigs can be considered an ideal
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animal model in biomedical research, as its similarities with humans in physiological,
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pathological and genomic characteristics 4. It has been clearly demonstrated that
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adipose tissue can produce a variety of secretory factors in proportion to adiposity that
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exert multiple effects on a series of metabolic diseases including type 2 diabetes and
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insulin resistance 5-7.
. How to improve the content of IMF while reduce the accumulation of subcutaneous
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Adipogenesis is a complex process with a temporally regulated set of gene-
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expression event and a cellular morphology change. Adipocyte differentiation models
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in vitro such as 3T3-F442A, 3T3-L1 and C3H10T1/2 provide powerful tools to study
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signaling networks regulating adipocyte differentiation 8. Hence, isolation of Vascular
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Stem Cells (VSCs) may bring new remarkable breakthroughs in obesity and diabetes
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research 9-11. Moreover, the VSCs separated from the subcutaneous and intramuscular
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adipose tissue are not only just different in their distribution, but also in lipid
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metabolism, insulin sensitivities, secretory functions and adipogenic capacities
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The intramuscular adipocytes use glucose as the main carbon source of fatty acid
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synthesis, while subcutaneous adipocytes employ acetate 14. Intramuscular adipocyte is
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more sensitive to insulin compared with subcutaneous adipocytes 15. Mixed conjugated
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linoleic acid inhibits the differentiation of subcutaneous adipocytes while promotes
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intramuscular adipocytes differentiation
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adipocytes derived from subcutaneous adipose tissue, but has no significant effect on
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adipocytes from intramuscular adipose tissue
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12, 13
.
. Starvation suppresses lipid synthesis in
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. These differences between the two
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adipocytes might be due to intrinsic differences
. Increasing concentrations
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of oleic acid will increase free fatty acid receptor 2 (FFAR2) protein level and its
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mRNA expression in intramuscular adipocytes but not in subcutaneous adipocytes
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19
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genes has been identified in subcutaneous adipocytes using microarray
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findings suggest that adipocytes from different white fat depots originate from different
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progenitors maintain their unique developmental transcriptional profiles.
. 46 genes highly expressed in intramuscular adipocytes while 87 highly expressed 20
. These
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In this study, we focused on the differences between subcutaneous and
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intramuscular adipocytes pre-/post-differentiation, as well as the different mechanisms
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that regulate their differentiation. Firstly, we separated the 1-day-old Landrace
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intramuscular and subcutaneous vascular stem cells (IVSCs and SVSCs, respectively),
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and successfully induced them to differentiate into mature adipocytes. Then the two
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types of adipocytes pre-/post- differentiation were collected for Solexa/Illumina’s
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digital gene expression (DGE) profiles sequencing. Through the screening of
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differentially expressed genes (DEGs), especially the specifically expressed genes, as
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well as the Gene Ontology (GO) and pathway bioinformatics analysis, we conducted a
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comprehensive and comparative analysis of the two preadipocyte types and the
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regulatory mechanisms of their adipogenesis.
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Materials and Methods
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Ethics statement
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All animal procedures were performed according to the guidelines developed by
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the China Council on Animal Care and protocols were approved by the Animal Care
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and Use Committee of Guangdong Province, China. The approval ID or permit
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numbers are SCXK (Guangdong) 2004-0011 and SYXK (Guangdong) 2007-0081.
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Journal of Agricultural and Food Chemistry
Isolation and adipogenic induction of cells
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Three healthy Landrace boar piglets, no more than 24h old, were prepared for
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separating intramuscular vascular stem cells (IVSC) and subcutaneous vascular stem
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cells (SVSC). The longissimus dorsi muscle and subcutaneous fat tissue were dissected,
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primary IVSC and SVSC cells were isolated according to previous published protocol
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USA) at 37°C in the 5% CO2, humidified atmosphere.
, and cultured in growth medium that contained DMEM/F12 with 20% FBS (Gibco,
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The procedures of differentiation were modified from previous report 22. Primary
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cells grew to confluence, and two days later, these cells were stimulated for 3 days in
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differentiation medium: DMEM/F12 containing 20% FBS and MDI (0.5 mM 3-
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isobutyl-1-methylxanthine, 0.25 μM dexamethasone, and 5 mg/L insulin). Then the
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cells were maintained in DMEM/F12 containing 20% FBS and 5 mg/L insulin, and the
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medium was replaced every two days until the end of the checkpoint. Mature adipocytes
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were visualized by staining with Oil red O solution.
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RNA extraction, library construction and sequencing
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Cells derived from three individual piglets were pooled for IVSC, IVSC-MDI,
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SVSC and SVSC-MDI, respectively. Thus, a total of four libraries were utilized in the
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transcriptome analysis. Total RNA was extracted using MagZol Reagent (Magen,
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Guangzhou, China) according to the manufacturer’s instructions and then treated with
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DNaseⅠto remove potential genomic DNA contamination. Total RNA integrity and
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concentration were evaluated using Agilent 2100 Bioanalyzer (Agilent Technologies,
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USA). The samples used had an average RIN (RNA integrity number) value of 9.4 and
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a minimum RIN value of 9.1. Constructing library and sequencing was performed
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according to previous published paper 23.
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Computational analysis of sequencing data
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The constructed libraries underwent Illumina proprietary sequencing chip (flow
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cell) for cluster generation and were deep-sequenced using Illumina Genome Analyzer
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IIX. Image analysis, base calling, and quality calibration were performed using the
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Solexa Automated Pipeline, after which the raw data were produced.
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Clean tags were obtained by filtering raw data to remove adaptor tags, low quality
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tags and final tags with typical length and copy>=2 will be remained. The clean tags
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were classified according to their copy number in the library and the proportion of each
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category in relation to total clean tags was determined. Subsequently, we analyzed the
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capacity of each library.
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Tag mapping
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The clean tags were blasted against sequences in Sus scrofa UniGene database
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(version 10.2) with one mismatch allowed. Tags mapped to more than one transcript
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were included in our analysis. When multiple types of sequences were aligned to the
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different positions of the same gene, the gene expression levels were calculated with
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the summation of all.
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qPCR analysis
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The RNA samples of all qPCR analyses came from the rest of library construction.
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First-strand cDNA of mRNAs were obtained using StarScript II First-strand cDNA
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Synthesis Kit (Genstar). qPCR was applied using StarScript II Green Fast One-Step
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qRT-PCR Kit (Genstar) according to the manufacturer’s instructions on a
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LightCycler480 (Roche), relative quantification ( △ △ Ct) was perform with
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instrumental software. Endogenous GAPDH mRNA was used as reference for mRNA
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in cells as mentioned previously. All reactions were performed in triplicate, and data
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were presented as mean ± SE. The primers of mRNAs were all listed in Table S1.
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Western blotting
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Cells at different days after MDI stimulation were lysed in cell lysis buffer (Beyotime)
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containing 1 mM phenylmethylsulfonyl fluoride. Equal amounts of total cellular
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protein were fractionated by 12% (w/v) SDS/PAGE and electronically transferred to
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0.2 mm PVDF membrane (Bio-Rad). The membrane was rinsed with TBS-Tween20
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(TBST), blocked for 2 h in 5% (w/v) skimmed milk, and incubated with primary
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antibody for 1h. The membrane was washed with TBST and incubated for 1h with
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secondary antibody conjugated to HRP. Blots were visualized using an ECL detection
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kit (Thermo Scientific). Rabbit anti-C/EBPα (#2295, Cell Signaling Technology) for
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C/EBPα, rabbit anit-PPARγ (C26H12, Cell Signaling Technology) and mouse anti-
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GAPDH (sc-59540, Santa Cruz Biotechnology) for GAPDH were utilized in this study.
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Measurement of fatty acid composition
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After adipogenesis induction, IVSC-MDI and SVSC-MDI were collected for lipid
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extraction. Samples were added with 10 μL（10 mg/mL）C19:0 internal standard and
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analyzed by gas chromatography mass spectrometry (7890A-5975C, Agilent
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Technologies) on DB-5 capillary column (30m×0.25mm×0.25 µm). Fatty acid
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composition was calculated by comparison with internal standard for quantification and
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normalized by cell number for each sample. Result data expressed as µg/1×106 cells.
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Differentiation expression analysis
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Digital
gene
expression
analysis
was
carried
out
by
DeSeq
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(http://bioconductor.org/packages/release/bioc/html/DESeq.html) working without any
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replicates which based on the negative binomial distribution. We compared the
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differences between different types of cells at the same state and differences between
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pre-/post-differentiated cells of the same cell type.
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To compare the DEGs across samples, the number of raw clean tags in each library
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was normalized to Tags Per Million (TPM) to obtain normalized gene expression levels.
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Genes were deemed significantly differentially expressed with a P-value
