S100a10基因对小鼠前体脂肪细胞白色和棕色成脂分化的影响

doi:10.11843/j.issn.0366-6964.2019.06.007

摘要/Abstract

摘要： 旨在用小鼠模型探究全基因组关联分析（GWAS）筛选到的西门塔尔牛大理石花纹评分性状正相关基因S100钙结合蛋白A10（S100A10）对小鼠前体脂肪细胞成脂分化的影响。本研究以来源于C57BL/6品系小鼠腹股沟两侧白色脂肪组织的前体脂肪细胞为材料，体外进行白色/棕色成脂分化。通过siRNA干扰S100a10基因表达，通过油红O染色检测前体脂肪细胞分化效果，通过荧光定量PCR（qRT-PCR）、蛋白免疫印迹和细胞免疫荧光检测基因和蛋白表达变化。结果显示，敲低S100a10后，通过油红O染色发现脂滴明显变少；通过qRT-PCR检测白色成脂分化标志基因Pparg、C/ebpa、Fabp4、Glut4、Adiponectin、Leptin表达量显著下降（P<0.01）；棕色成脂分化基因Ucp1、Pgc1a、Fabp4、Elovl3、Cox7a表达量显著下降（P<0.01）。敲低S100a10后，通过蛋白免疫印迹检测发现，FABP4和PPARG在白色成脂分化中表达量显著下降（P<0.01），通过细胞免疫荧光检测发现FABP4在白色成脂分化中表达量下降；敲低S100a10后通过蛋白免疫印迹和细胞免疫荧光检测发现，UCP1和FABP4在棕色成脂分化中表达量显著下降（P<0.01）。综上表明，敲低S100a10基因后抑制小鼠前体脂肪细胞的白色/棕色成脂分化，本研究将为探究S100A10基因对于肉牛肌内脂肪沉积的影响提供科学依据。

关键词: 脂肪细胞, S100a10, 白色/棕色成脂分化, 肌内脂肪

Abstract: The objective of this study was to investigate the effects of the S100A10 gene, which was identified as a candidate gene regulating Simmental marbling score by genome-wide association study (GWAS), on adipogenic differentiation of mice preadipocytes. In this study, white preadipocytes were isolated from inguinal white adipose tissue of C57BL/6 mice and induced white/brown adipogenic differentiation in vitro. The expression of the S100a10 gene was interfered using siRNA. The differentiation of preadipocytes were detected by Oil Red O staining. Gene and protein expression levels were measured by qRT-PCR, Western blot and immunofluorescence. The results showed that lipid droplets decreased significantly after S100a10 knocking down. And qRT-PCR assay showed that the expression level of white adipocyte-specific genes Pparg, C/ebpa, Fabp4, Glut4, Adiponectin and Leptin were significantly down regulated in white adipogenic differentiation(P<0.01). The expression level of brown adipocyte-specific genes Ucp1, Pgc1a, Fabp4, Elovl3 and Cox7a were significantly decreased in brown adipogenic differentiation(P<0.01). Western blot result showed that the expression of FABP4 and PPARG were significantly decreased in white adipogenic differentiation(P<0.01), the immunofluorescence assay showed that the expression of FABP4 was decreased in white adipogenic differentiation. The protein expression of brown adipogenic differentiation specific genes FABP4 and UCP1 were obviously decreased in brown adipogenic differentiation(P<0.01). The results showed that knocking down S100A10 inhibited the white/brown adipogenic differentiation in mice preadipocytes. This study provides scientific basis for the further research of the effect of S100A10 on intramuscular fat deposition in beef cattle.

Key words: adipocyte, S100a10, white/brown adipogenic differentiation, intramuscular fat

中图分类号:

S823.99

任玲, 胡鑫, 邢义珅, 王亚慧, 李俊雅, 张路培. S100a10基因对小鼠前体脂肪细胞白色和棕色成脂分化的影响[J]. 畜牧兽医学报, 2019, 50(6): 1171-1178.

REN Ling, HU Xin, XING Yishen, WANG Yahui, LI Junya, ZHANG Lupei. Effects of S100a10 on White and Brown Adipogenic Differentiation of Mice Preadipocytes[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(6): 1171-1178.

参考文献

[1] DODSON M V,JIANG Z H,CHEN J,et al.Allied industry approaches to alter intramuscular fat content and composition in beef animals[J].J Food Sci,2010,75(1):R1-R8.
[2] LISTRAT A,LEBRET B,LOUVEAU I,et al.How muscle structure and composition influence meat and flesh quality[J].Sci World J,2016,2016:3182746.
[3] VISSCHER P M,BROWN M A,MCCARTHY M I,et al.Five years of GWAS discovery[J].Am J Hum Genet,2012,90(1):7-24.
[4] XIA J W,QI X,WU Y,et al.Genome-wide association study identifies loci and candidate genes for meat quality traits in Simmental beef cattle[J].Mamm Genome,2016,27(5-6):246-255.
[5] BHARADWAJ A,BYDOUN M,HOLLOWAY R,et al.Annexin A2 heterotetramer:structure and function[J].Int J Mol Sci,2013,14(3):6259-6305.
[6] RESCHER U,GERKE V.S100A10/p11:family,friends and functions[J].Pflügers Archiv - Eur J Phys,2008,455(4):575-582.
[7] REDDY T R K,LI C,FISCHER P M,et al.Three-dimensional pharmacophore design and biochemical screening identifies substituted 1,2,4-triazoles as inhibitors of the annexin A2-S100A10 protein interaction[J].ChemMedChem,2012,7(8):1435-1446.
[8] LIU M W,GE R,LIU W L,et al.Differential proteomics profiling identifies LDPs and biological functions in high-fat diet-induced fatty livers[J].J Lipid Res,2017,58(4):681-694.
[9] SALAMEH A,DAQUINAG A C,STAQUICINI D I,et al.Prohibitin/annexin 2 interaction regulates fatty acid transport in adipose tissue[J].JCI Insight,2016,1(10),doi:10.1172/jci.insight.86351.
[10] SARJEANT K,STEPHENS J M.Adipogenesis[J].Cold Spring Harb Perspect Biol,2012,4(9):a008417.
[11] LOWE C E,O'RAHILLY S,ROCHFORD1 J J.Adipogenesis at a glance[J].J Cell Sci,2011,124:2681-2686.
[12] BRENDLE C,STEFAN N,STEF I,et al.Impact of diverse chemotherapeutic agents and external factors on activation of brown adipose tissue in a large patient collective[J].Sci Rep,2019,9:1901.
[13] DA SILVAC P V,HERNÁNDEZ-SAAVEDRA D,WHITE J D,et al.Cold and exercise:therapeutic tools to activate brown adipose tissue and combat obesity[J].Biology,2019,8(1):9.
[14] BARBATELLI G,MURANO I,MADSEN L,et al.The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation[J].Am J Phys-Endocrinol Metab,2010, 298(6):E1244- E1253.
[15] PARK A,KIM W K,BAE K H.Distinction of white,beige and brown adipocytes derived from mesenchymal stem cells[J].World J Stem Cells,2014,6(1):33-42.
[16] FRONTINI A,VITALI A,PERUGINI J,et al.White-to-brown transdifferentiation of omental adipocytes in patients affected by pheochromocytoma[J].Biochim Biophys Acta-Mol Cell Biol Lipids,2013,1831(5):950-959.
[17] SCHULZ T J,HUANG P,HUANG T L,et al.Brown-fat paucity due to impaired BMP signalling induces compensatory browning of white fat[J].Nature,2013,495(7441):379-383.
[18] FISHER F M,KLEINER S,DOURIS N,et al.FGF21 regulates PGC-1α and browning of white adipose tissues in adaptive thermogenesis[J].Genes Dev,2012,26(3):271-281.
[19] SEALE P,CONROE H M,ESTALL J,et al.Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice[J].J Clin Invest,2011,121(1):96-105.
[20] 尹靖东,李德发.猪肉质形成的分子机制与营养调控[J].动物营养学报,2014,26(10):2979-2985.
YIN J D,LI D F.Molecular mechanism underlying meat quality formation and its nutritional regulation in pigs[J].Chinese Journal of Animal Nutrition,2014,26(10):2979-2985.(in Chinese)
[21] 李嫒,张秀秀,黄万龙,等.大白猪和莱芜猪肌内脂肪组织circRNAs的鉴定与分析[J].畜牧兽医学报,2018,49(7):1343-1353.
LI Y,ZHANG X X,HUANG W L,et al.Identification and analysis of circRNAs in intramuscular adipose tissues between Large White and Laiwu pigs[J].Acta Veterinaria et Zootechnica Sinica,2018,49(7):1343-1353.(in Chinese)
[22] YU Z Y,CAI Y Z,DENG M W,et al.Fat extract promotes angiogenesis in a murine model of limb ischemia:a novel cell-free therapeutic strategy[J].Stem Cell Res Ther,2018,9:294.
[23] WANG Y L,LIU X Y,HOU L M,et al.Fibroblast growth factor 21 suppresses adipogenesis in pig intramuscular fat cells[J].Int J Mol Sci,2015,17(1):E11.
[24] YAMADA T,KAWAKAMI S,NAKANISHI N.Fat depot-specific differences in angiogenic growth factor gene expression and its relation to adipocyte size in cattle[J].J Vet Med Sci,2010,72(8):991-997.
[25] YONEKURA S,TOKUTAKE Y,HIROTA S,et al.Proliferating bovine intramuscular preadipocyte cells synthesize leptin[J]. Domest Anim Endocrinol,2013,45(1):33-37.
[26] CARMELIET P.Angiogenesis in life,disease and medicine[J].Nature,2005,438(7070):932-936.
[27] BOSMA E K,VAN NOORDEN C J F,SCHLINGEMANN R O,et al.The role of plasmalemma vesicle-associated protein in pathological breakdown of blood-brain and blood-retinal barriers:potential novel therapeutic target for cerebral edema and diabetic macular edema[J].Fluids Barriers CNS,2018,15:24.
[28] LEFERE S,VAN DE VELDE F,HOORENS A,et al.Angiopoietin-2 promotes pathological angiogenesis and is a therapeutic target in murine nonalcoholic fatty liver disease[J].Hepatology,2019,69(3):1087-1104.
[29] SUN F,SHAIKH A S,WANG J,et al.Higher anti-angiogenesis activity,better cellular uptake and longer half-life of a novel glyco-modified endostatin by polysulfated heparin[J].Curr Pharm Biotechnol,2018,19(12):996-1004.
[30] PODKALICKA P,MUCHA O,DULAK J,et al.Targeting angiogenesis in Duchenne muscular dystrophy[J].Cell Mol Life Sci, 2019, doi:10.1007/s00018-019-03006-7.
[31] GAO Y F,ZHOU S,PANG L Z,et al.Celastrol suppresses nitric oxide synthases and the angiogenesis pathway in colorectal cancer[J].Free Radic Res,2019,doi:10.1080/10715762.2019.1575512.
[32] HOSSEINI S,BEHJATI F,RAHIMI M,et al.Relationship between PIK3CA amplification and P110α and CD34 tissue expression as angiogenesis markers in Iranian Women with sporadic breast cancer[J].Iran J Pathol,2018,13(4):447-453.
[33] ZHAO S H,HUANG L N,WU J H,et al.Vascular endothelial growth factor upregulates expression of annexin A2in vitro and in a mouse model of ischemic retinopathy[J].Mol Vis,2009,15:1231-1242.
[34] MADUREIRA P A,SURETTE A P,PHIPPS K D,et al.The role of the annexin A2 heterotetramer in vascular fibrinolysis[J]. Blood, 2011,118(18):4789-4797.
[35] SURETTE A P,MADUREIRA P A,PHIPPS K D,et al.Regulation of fibrinolysis by S100A10in vivo[J].Blood, 2011, 118(11):3172-3181.
[36] STEPANOVA V,JAYARAMAN P,ZAITSEV S V,et al.Urokinase-type plasminogen activator (uPA) promotes angiogenesis by attenuating proline-rich homeodomain protein (PRH) transcription factor activity and de-repressing vascular endothelial growth factor (VEGF) receptor expression[J].J Biol Chem,2016,291(29):15029-15045.