LPIN1基因对水牛乳腺上皮细胞甘油三酯和脂肪酸合成的影响

doi:10.16431/j.cnki.1671-7236.2020.10.012

Abstract

Abstract: The purpose of this study was to construct the adenovirus pDC316-LPIN1-eGFP vector and package the Ad-LPIN1 adenovirus particles,in order to explore the effect of LPIN1 gene on triglyceride and fatty acid synthesis in buffalo mammary epithelial cells.The expression of LPIN1 gene and lipin1 protein in buffalo mammary epithelial cell were detected by Real-time quantitative PCR and immunofluorescence (IF) after Ad-LPIN1 infected.The triglyceride was detected by oil red O staining and triglyceride measurement and the genes expression of triglyceride synthesis and fatty acid synthesis after overexpressing LPIN1 were detected by Real-time quantitative PCR.The results showed that high-quality Ad-LPIN1 adenovirus particles were packaged with the pDC316-LPIN1-eGFP vector.Ad-LPIN1 adenovirus particles infected the buffalo mammary epithelial cell (MOI=50),LPIN1 mRNA was relatively up-regulated more than 5 000-fold,which was extremely significant difference (P<0.01).lipin1 protein expression was up-regulated and mainly expressed in the nucleus.Oil red O staining results showed that lipid droplets in cells were significantly reduced after overexpressing LPIN1 gene.The results of triglyceride measurement showed that the triglyceride content in cells decreased after overexpressing LPIN1 gene,and the difference was extremely significant (P<0.01).The mRNA of peroxisome proliferator activated receptor gama (PPARγ),peroxisome proliferator activated receptor alpha (PPARα) and sterol regulatory element binding transcription factor 1 (SREBP1) after overexpressing LPIN1 gene were significantly or extremely significant increased (P<0.05;P<0.01).And the mRNA of stearoyl-CoA desaturase (SCD),fatty acid synthase (FASN),acyl-CoA synthetase long chain family member 1 (ACSL1) and acetyl-CoA carboxylase alpha (ACACA) were decreased to 83.17%,51.30%,44.24%,41.18% (P<0.01),respectively.The above-mentioned results showed that adenovirus efficiently mediated the expression of LPIN1 gene in buffalo mammary epithelial cell,and increased the expression of lipin1 protein in the nucleus.lipin1 protein,as a common transcription activation factor,inhibited the synthesis of triglycerides by down-regulating mRNA of ACACA,FASN,ACSL1 and SCD genes.

Key words: buffalo; mammary epithelial cell; LPIN1 gene; triglyceride; fatty acid

CLC Number:

S823.8⁺3

DUAN Anqin, LU Xingrong, MA Xiaoya, LIANG Shasha, DENG Tingxian. Effect of LPIN1 Gene on Triglyceride and Fatty Acid Synthesis in Buffalo Mammary Epithelial Cell[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(10): 3140-3148.

References 34

[1]	EATON J M,MULLINS G R,BRINDLEY D N,et al.Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association[J].Journal of Biological Chemistry,2013,288(14):9933-9945.
[2]	谢秉锵,瓦云超,伍云,等.水牛乳资源及其理化特性研究进展[J].中国乳品工业,2018,46(11):22-25. XIE B Q,WA Y C,WU Y,et al.Progress on distribution and physicochemical characteristics of buffalo milk[J].China Dairy Industry,2118,46(11):22-25.(in Chinese)
[3]	BIONAZ M,LOOR J J.ACSL1,AGPAT6,FABP3,LPIN1,and SLC27A6 are the most abundant isoforms in bovine mammary tissue and their expression is affected by stage of lactation[J].The Journal of Nutrition,2008,138(6):1019-1024.
[4]	YADAV P,MUKESH M,KATARIA R S,et al.Semi-quantitative RT-PCR analysis of fat metabolism genes in mammary tissue of lactating and non-lactating water buffalo (Bubalus bubalis)[J].Tropical Animal Health and Production,2012,44(4):693-696.
[5]	YADAV P,KUMAR P,MUKESH M,et al.Kinetics of lipogenic genes expression in milk purified mammary epithelial cells (MEC) across lactation and their correlation with milk and fat yield in buffalo[J].Research in Veterinary Science,2015,99:129-136.
[6]	HAN B,YUAN Y,LIANG R,et al.Genetic effects of LPIN1 polymorphisms on milk production traits in dairy cattle[J].Genes,2019,10(4):265.
[7]	WANG X,ZHOU H,HICKFORD J G H,et al.Variation in the yak lipin-1 gene and its association with milk traits[J].Journal of Dairy Research,87(2):166-169.
[8]	段安琴,陆杏蓉,马小娅,等.水牛原代体细胞慢病毒感染体系的建立[J].中国畜牧兽医,2019,46(10):3023-3031. DUAN A Q,LU X R,MA X Y,et al.Establishment of buffalo primary somatic lentivirus infection system[J].China Animal Husbandry & Veterinary Medicine,2019,46(10):3023-3031.(in Chinese)
[9]	段安琴,庞春英,朱鹏,等.水牛乳汁中乳腺上皮细胞的分离培养及鉴定[J].中国畜牧兽医,2017,44(11):3243-3249. DUAN A Q,PANG C Y,ZHU P,et al.Isolation,culture and identification of buffalo mammary epithelial cells from milk[J].China Animal Husbandry & Veterinary Medicine,2017,44(11):3243-3249.(in Chinese)
[10]	段安琴,庞春英,朱鹏,等.腺病毒载体转染水牛不同原代体细胞的效果比较[J].中国畜牧兽医,2016,43(10):2661-2665. DUAN A Q,PANG C Y,ZHU P,et al.Comparison of adenovirus vector mediated gene transfer into different buffalo cells[J].China Animal Husbandry & Veterinary Medicine,2016,43(10):2661-2665.(in Chinese)
[11]	王鉴.Lipinl参与的细胞代谢调控作用研究[D].广州:华东理工大学,2018. WANG J.Study on the roles of lipinl in the regulation of cell metabolism[D].Guangzhou:East China University of Science and Technology,2018.(in Chinese)
[12]	KHALIL M B,SUNDARAM M,ZHANG H,et al.The level and compartmentalization of phosphatidate phosphatase-1(lipin-1) control the assembly and secretion of hepatic VLDL[J].Journal of Lipid Research,2008,50(1):47-58.
[13]	PETERFY M,PHAN J,REUE K.Alternatively spliced lipin isoforms exhibit distinct expression pattern,subcellular localization,and role in adipogenesis[J].Journal of Biological Chemistry,2005,280(38):32883-32889.
[14]	YANG C,WANG X,WANG J,et al.Rewiring neuronal glycerolipid metabolism determines the extent of axon regeneration[J].Neuron,2020,105(2):276-292.
[15]	REN H,FEDERICO L,HUANG H,et al.A phosphatidic acid binding/nuclear localization motif determines lipin1 function in lipid metabolism and adipogenesis[J].Molecular Biology of the Cell,2010,21(18):3171-3181.
[16]	PÉTERFY M,HARRIS T E,FUJITA N,et al.Insulin-stimulated interaction with 14-3-3 promotes cytoplasmic localization of lipin-1 in adipocytes[J].Journal of Biological Chemistry,2010,285(6):3857-3864.
[17]	PETERSON T R,SENGUPTA S S,HARRIS T E,et al.mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway[J].Cell,2011,146(3):408-420.
[18]	段安琴,庞春英,邓廷贤,等.水牛LPIN1基因克隆及序列分析[J].中国畜牧兽医,2018,45(10):2653-2660. DUAN A Q,PANG C Y,DENG T X,et al.Cloning and sequence analysis of buffalo LPIN1 gene[J].China Animal Husbandry & Veterinary Medicine,2018,45(10):2653-2660.(in Chinese)
[19]	TAKEUCHI K,REUE K.Biochemistry,physiology,and genetics of GPAT,AGPAT,and lipin enzymes in triglyceride synthesis[J].American Journal of Physiology-Endocrinology and Metabolism,2009,296(6):E1195-E1209.
[20]	KUMAR M,VOHRA V,RATWAN P,et al.Influence of FASN gene polymorphism on milk production and its composition traits in Murrah buffaloes[J].Indian Journal of Animal Research,2015,640-643.
[21]	XU W,CHEN Q,JIA Y,et al.Isolation,characteriza-tion,and SREBP1 functional analysis of mammary epithelial cell inbuffalo[J].Journal of Food Bioche-mistry,2019,43:e12997.
[22]	LIAN S,GUO J R,NAN X M,et al.microRNA Bta-miR-181a regulates the biosynthesis of bovine milk fat by targeting ACSL1[J].Journal of Dairy Science,2016,99(5):3916-3924.
[23]	LI Z,LU S,CUI K,et al.Fatty acid biosynthesis and transcriptional regulation of Stearoyl-CoA Desaturase 1(SCD1) in buffalo milk[J].BioMed Central Genetics,2020,21(1):23-32.
[24]	陈秋明.SREBP1基因在水牛乳腺上皮细胞中的功能研究[D].南宁:广西大学,2016. CHEN Q M.Functional study of SREBP1 in buffalo mammary epithelial cell[D].Nanning:Guangxi University,2016.(in Chinese)
[25]	MA L,CORL B A.Transcriptional regulation of lipid synthesis in bovine mammary epithelial cells by sterol regulatory element binding protein-1[J].Journal of Dairy Science,2012,95(7):3743-3755.
[26]	XU H F,LUO J,ZHAO W S,et al.Overexpression of SREBP1(sterol regulatory element binding protein 1) promotes de novo fatty acid synthesis and triacylglycerol accumulation in goat mammary epithelial cells[J].Journal of Dairy Science,2016,99(1):783-795.
[27]	XU H,LUO J,TIAN H,et al.Lipid metabolic gene expression and triacylglycerol accumulation in goat mammary epithelial cells are decreased by inhibition of SREBP-1[J].Journal of Animal Science,2018,96(6):2399-2407.
[28]	LAKHIA R,YHESKEL M,FLATEN A,et al.PPARα agonist fenofibrate enhances fatty acid β-oxidation and attenuates polycystic kidney and liver disease in mice[J].American Journal of Physiology.Renal Physiology,2018,314(1):F122-F131.
[29]	HUANG D,ZHAO Q,LIU H,et al.PPAR-alpha agonist WY-14643 inhibits LPS-induced inflamma-tion in synovial fibroblasts via NF-κB pathway[J].Journal of Molecular Neuroscience,2016,59(4):544-553.
[30]	GESSNER D K,GRÖNE B,ROSENBAUM S,et al.Treatment of lactating sows with clofibrate as a synthetic agonist of PPARα does not influence milk fat content and gains of litters[J].BioMed Central Veterinary Research,2015,11(1):54-62.
[31]	TIAN H B,LUO J,SHI H B,et al.Role of peroxisome proliferator-activated receptor-α on the synthesis of monounsaturated fatty acids in goat mammary epithelial cells[J].Journal of Animal Science,2020,98(3):skaa062.
[32]	OSORIO J S,LOHAKARE J,BIONAZ M.Biosynthesis of milk fat,protein,and lactose:Roles of transcriptional and posttranscriptional regulation[J].Physiological Genomics,2016,48(4):231-256.
[33]	GUO Z,ZHAO K,FENG X,et al.mTORC2 regulates lipogenic gene expression through PPARγ to control lipid synthesis in bovine mammary epithelial cells[J].BioMed Research International,2019,2019:5196028.
[34]	VARGAS-BELLO-PÉREZ E,ZHAO W,BIONAZ M,et al.Nutrigenomic effect of saturated and unsaturated long chain fatty acids on lipid-related genes in goat mammary epithelial cells:What is the role of PPARγ?[J].Veterinary Sciences,2019,6(2):54-73.

Effect of LPIN1 Gene on Triglyceride and Fatty Acid Synthesis in Buffalo Mammary Epithelial Cell

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 34

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	YANG Guitao, MA Jideng, LI Xuewei, GE Liangpeng, ZHANG Jinwei. Research Advance on Regulation of Skeletal Muscle Physiology Function by Short Chain Fatty Acids [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2286-2295.
[2]	XU Yuanyuan, ZHENG Haiying, YANG Chunyan, DENG Tingxian, HUANG Chenqian, FENG Chao, LU Xingrong, DUAN Anqin, MO Xia, MA Xiaoya, SHANG Jianghua. Effects of Inhibition of BMP/Smad Signaling Pathway on the Growth and Steroid Hormone Synthesis of Buffalo Ovarian Granulosa Cells [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2354-2362.
[3]	XIAO Peng, SHANG Jianghua, YANG Chunyan, LI Mengqi, DUAN Anqin, MA Xiaoya, FENG Chao, HUANG Chenqian, ZHANG Bo, ZHOU Jinchen, WEI Kelong, ZHENG Wei, ZHENG Haiying. Effects of Alpha-linolenic Acid on Buffalo Ovarian Granulosa Cells Cultured in vitro [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2380-2387.
[4]	LI Xuting, WANG Bin, LI Sicong, LI Jinliang, LIANG Ge. Effect of Xiaochengqi Powder on Intestinal Microbial Community in Constipation Sow [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1675-1683.
[5]	ZHANG Tiantian, HUA Zaidong, REN Hongyan, GU Hao, ZHOU Bin, BI Yanzhen. Effect of FADS1 Gene on Unsaturated Fatty Acid Synthesis [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 451-459.
[6]	CHEN Jing, YU Shuyi, HUANG Hui, LI Jianzhe. Effect of Clostridium butyrate on the Composition of Plasma Free Fatty Acid in Mice Fed with High Choline Diet [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 519-530.
[7]	PAN Siyao, ZHANG Dandan, LIU Quanhui, WANG Guodong, LYU Danwei, HUANG Ben. Effects of Inhibition of miR-142-3p on Lactation Function of Chemical Induced Mammary Epithelial Cells in Dairy Goats [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 58-66.
[8]	YANG Min, GAO Yu, XU Zihao, ZHANG Jiulong, DELINUER·Baishanbieke, REN Wanping. Effects of Feeding Steam-flaked Corn on Weight Gain,Rumen Fermentation and Microbial Diversity of Xinjiang Brown cattle [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 2961-2970.
[9]	CAI Meng, ZHU Xiaoyan, WANG Mengling, LIU Zihao, XIONG Benhai, YANG Liang. Effect of Staphylococcus aureus on Exosome Characterization of Mammary Epithelial Cells in Dairy Cows [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3091-3098.
[10]	ZHANG Yuanxin, WANG Yanyu, YUN Yanhong, CHEN Junpu, SHI Huiyu, WANG Xuemei. Establishment of Oxidative Damage Model of Goat Mammary Epithelial Cells [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1917-1925.
[11]	SUN Qingyun, FAN Xiang, XING Ya, ZHAO Minmeng, LIU Long, GENG Tuoyu, GONG Daoqing. Study on the Relationship Between OTUD7A Gene and Goose Fatty Liver Formation [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1295-1303.
[12]	YUAN Chongshan, WANG Jun, LYU Wenfa. Research Progress on Effects of Polyunsaturated Fatty Acids on Animal Semen Quality [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1422-1429.
[13]	LUO Shiqi, ZHANG Xiuwen, SU Jianyu, CHEN Longhai, YANG Lin, WANG Wence. Advances on Intestinal Microorganisms in Waterfowl [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 845-856.
[14]	LIU Xinyong, TAN Zhengzhun, LI Hui, HUANG Jian, LUO Hua, ZENG Linghu, CHENG Juanru, ZHONG Huapei, YANG Chunyan, WANG Xiaoli, QIN Guangsheng. Effects of Heat Stress on Blood Biochemical Indexes of Murrah and Nili-Ravi Buffaloes [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 521-528.
[15]	ZHANG Qiang, LIU Zhenni, LIAO Qiang, LEI Xiaowen, KONG Zhiwei, XIE Hualiang, TAN Donghai, LI Jianjun, ZHONG Yunping. Effects of Fermented Andrographis Powder on Production Performance, Serum Biochemical Indexes, Immune Related Indexes, Antioxidant Indexes and Fatty Acid Contents of Muscle in Muscovy Ducks [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4614-4624.