Study on Expressions of CNR1 and FABP4 Genes in Ovine Intramuscular Preadipocytes

doi:10.13304/j.nykjdb.2021.0120

Abstract

Abstract:

To study the pattern and difference of expression of CNR1 and FABP4 during the period of proliferation and differentiation of adipocytes， the longissimus dorsi muscle tissue sourced from a 3-day-old Hu lambs was collected， and then digested by the type II collagenase to separate and observe the primary preadipocytes. The preadipocytes were cultured and the growth curve was drawn. The preadipocytes were induced by insulin， dexamethasone and 3-isobutyl-1-methylxanthine， and the morphology of mature adipocytes was also observed by oil red O staining. The expressions of CNR1 and FABP4 genes were detected using real-time PCR method at different time of the adipocytes differentiation. The results showed that the preadipocytes were successfully isolated from the longissimus dorsi muscle of lamb， and the adherent cells were shown to be spindle shape. The formation of lipid droplets were observed in the preadipocytes stained with oil red O， which showed the characteristics of adipocytes. The relative expression levels of CNR1 and FABP4 genes were lower at the early stage of differentiation， then gradually increased at the middle and late stage. There were no significant differences in relative expression level of CNR1 and FABP4 on the 2^nd， 4^th and 8^th day， but the relative expression level of FABP4 was significantly higher than that of CNR1 on the 12^th day. It showed that CNR1 and FABP4 genes showed different expression characteristics in ovine intramuscular fat cells， which provided reference data for further revealing the regulatory relationship between two genes of intramuscular fat deposition and improving ovine meat traits.

Key words: sheep, intramuscular preadipocytes, FABP4, CNR1, gene expression

摘要：

为研究绵羊脂肪细胞增殖分化过程中参与动物能量代谢调控的大麻素受体1（cannabinoid receptor 1）基因CNR1和调控脂肪沉积的脂肪酸结合蛋白4（fatty acid binding protein 4）基因FABP4的表达规律和差异，采集一只3日龄湖羊羔羊背最长肌组织，背肌组织经Ⅱ型胶原酶消化后分离观察原代前体脂肪细胞；开展前体脂肪细胞培养并绘制生长曲线；应用胰岛素、地塞米松和3-异丁基-1-甲基黄嘌呤诱导前体脂肪细胞后，采用油红O染色观察成熟脂肪细胞形态；收集不同分化时间点细胞，应用实时荧光定量PCR方法测定CNR1和FABP4基因表达量。结果表明，羔羊背最长肌组织成功分离出了前体脂肪细胞，贴壁细胞呈梭形；诱导前体脂肪细胞经油红O染色后观察到脂滴形成，具有脂肪细胞特征；CNR1和FABP4基因表达量在诱导分化早期均较低，但在诱导分化中后期表达量逐步升高；CNR1和FABP4基因表达量在诱导分化后的第2、第4和第8天均差异不显著，但FABP4基因在第12天的表达量显著高于CNR1基因。上述结果为进一步探究绵羊CNR1和FABP4基因调控关系及利用两基因改良绵羊肉品性状提供了参考数据。

关键词: 绵羊, 肌内前体脂肪细胞, FABP4, CNR1, 基因表达

CLC Number:

S826

Wei YAN, Yutao WANG, Yonghao ZHANG, Haixia LIU, Dayong HAN, Aiwen ZHU. Study on Expressions of CNR1 and FABP4 Genes in Ovine Intramuscular Preadipocytes[J]. Journal of Agricultural Science and Technology, 2022, 24(3): 95-102.

闫伟, 王玉涛, 张永浩, 刘海霞, 韩大勇, 朱爱文. 绵羊肌内前体脂肪细胞CNR1和FABP4基因表达研究[J]. 中国农业科技导报, 2022, 24(3): 95-102.

Figures/Tables 6

Table 1 Sequences of amplified primers

基因名Gene name	引物序列Primer sequence （5’-3’）	扩增片段长度 Length of amplifed fragment/bp
GAPDH	F： GCAAGTTCCACGGCACAG	249
	R： GGTTCACGCCCATCACAA
CNR1	F： GACCAAGAGCGGAGAACG	144
	R： TCAATGACGGAGAAGGGAC
FABP4	F： CATGAAAGAAGTGGGTGTGGG	150
	R： TCCTGGCCCAATTTGAAGGA

Fig. 1 Primary cultured cells of intramuscular preadipocyte in sheep （200 ×）

Fig. 2 Growth curve of intramuscular preadipocyte cells in sheep

Fig. 3 Induced differentiation of intramuscular preadipocyte in sheep （100 ×）

Fig. 4 CNR1 and FABP4 expression level of intramuscular preadipocyteNote： * and ** represent significant differences between groups at P<0.05 and P<0.01 levels， respectively.

Fig. 5 Difference of CNR1 and FABP4 ExpressionNote： * represents significant differences between groups at P<0.05 level.

References 50

1	VOLK M G. An examination of the evidence supporting the association of dietary cholesterol and saturated fats with serum cholesterol and development of coronary heart disease [J]. Altern. Med. Rev., 2007, 12:228-245.
2	CORLEY M M, WARD J. Expression of fat and cholesterol biomarkers in meat goats [J]. J. Mol. Biol. Res., 2013, 3:78-90.
3	JEREMIAH L E, DUGAN M E, AALHUS J L, et al.. Assessment of the relationship between chemical components and palatability of major beef muscles and muscle groups [J]. Meat Sci., 2003, 65(3):1013-1019.
4	WEBB E C, O'NEILL H A. The animal fat paradox and meat quality [J]. Meat Sci., 2008, 80(1):28-36.
5	HAUSMAN G J, BERGEN W G, ETHERTON T D, et al.. The history of adipocyte and adipose tissue research in meat animals [J]. J. Anim. Sci., 2018, 96(2):473-486.
6	MOODY W, CASSENS R A. Quantitative and morphological study of bovine longissimus fat cells [J]. J. Food Sci., 1968, 33(1):47-52.
7	UEZUMI A, FUKADA S, YAMAMOTO N, et al.. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle [J]. Nat. Cell Biol., 2010, 12(2):143-152.
8	RODRÍGUEZ DE FONSECA F, ARCO IDEL I, BERMUDEZ-SILVA F J, et al.. The endocannabinoid system: physiology and pharmacology [J]. Alcohol Alcoholism, 2005, 40:2-14.
9	CREPILLO A, SUÁREZ J, BERMUDEZ-SILVA F J, et al.. Expression of the cannabinoid system in muscle: effects of a high-fat diet and CB1 receptor blockade [J]. Biochem. J., 2010, 433:175-185.
10	WITKAMP R F. The role of fatty acids and their endocannabinoid-like derivatives in the molecular regulation of appetite [J]. Mol. Aspects Med., 2018, 64(12):45-67.
11	OSEI-HYIAMAN D, HARVEY-WHITE J, BATKAI S, et al.. The role of the endocannabinoid system in the control of energy homeostasis [J]. Int. J. Obesity, 2006, 30(S1):33-38.
12	OSEI-HYIAMAN D, DEPETRILLO M, PACHER P, et al.. Endocannabinoid activation at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to diet-induced obesity [J]. J. Clin. Invest., 2005, 115:1298-1305.
13	OSEI-HYIAMAN D, LIU J, ZHOU L, et al.. Hepatic CB1 receptor is required for development of diet-induced steatosis, dyslipidemia, and insulin and leptin resistance in mice [J]. J. Clin. Invest., 2008, 118:3160-3169.
14	FALOMIR-LOCKHART L J, LABORDE L, KAHN P C, et al.. Protein-membrane interaction and fatty acid transfer from intestinal fatty acid–binding protein to membranes: support for a multistep process [J]. J. Biol. Chem., 2006, 281:14232-14240.
15	WOLFRUM C. Cytoplasmic fatty acid binding protein sensing fatty acids for peroxisome proliferator activated receptor activation [J]. Cell. Mol. Life Sci., 2007, 64:2465-2476.
16	SYAMSUNARNOM R, TATSUYA I, HANAOKA H, et al.. A critical role of fatty acid bind protein 4 and 5(FABP4/5) in the systemic response to fasting [J/OL]. PLoS One, 2013, 8(11):e79386 [2020-12-28]. .
17	YAN W, ZHOU H T, LUO Y Z, et al.. Variation in the FABP4 gene affects carcass and growth traits in sheep [J]. Meat Sci., 2018, 145:334-339.
18	HUI X Y, LI H Y, ZHOU Z G, et al.. Adipocyte fatty acid-binding protein modulates inflammatory responses in macrophages through a positive feedback loop involving c-jun NH2-terminal kinases and activator protein-1 [J]. J. Biol. Chem., 2010, 285(14):10273-10280.
19	HOWLETT A C. Cannabinoid receptor signaling [J]. Handb. Exp. Pharmacol., 2005, 168:53-79.
20	杜琛,付绍印,韩志玲,等.绒山羊肌内前体脂肪细胞的基因表达分析[J].畜牧兽医学报,2013,44(10):1532-1538.
	DU S, FU S Y, HAN Z L, et al.. Gene expression analysis on intramuscular preadipocytes in the cashmere goats [J]. Acta Veterinaria Et Zootechnica Sin., 2013, 44(10):1532-1538.
21	杜琛,付绍印,高鸿雁,等.绒山羊肌内脂肪细胞成熟前后比较转录组分析[J].畜牧兽医学报,2014,45(5):714-721.
	DU S, FU S Y, GAO H Y, et al.. Transcriptome analysis of intramuscular preadipocytes and matureadipocyte in cashmere goats [J]. Acta Veterinaria Et Zootechnica Sin., 2014, 45(5):714-721.
22	郑竹清,杜琛,付绍印,等.山羊肌内脂肪细胞lncRNA鉴别及特征分析[J].畜牧兽医学报,2014,45(12):1924-1931.
	ZHENG Z Q, DU S, FU S Y, et al.. Identification and characterization analysis of long non-coding RNA from RNA-seq data of intramuscular adipocytes in goats [J]. Acta Veterinaria Et Zootechnica Sin., 2014, 45(12):1924-1931.
23	崔京京,富俊才,孙岩,等.绵羊前体脂肪细胞的分离培养及诱导分化[J].中国畜牧杂志,2012,48(1):22-26.
	CUI J J, FU J C, SUN Y, et al.. Culture and differentiation of ovine preadipocytes [J]. Chin. J. Anim. Sci., 2012, 48(1):22-26.
24	罗燕,赵宗胜,谷新利,等.绵羊肌内前体脂肪细胞的体外培养[J].石河子大学学报(自然科学版), 2011,29(3):291-295.
	LUO Y, ZHAO Z S, GU X L, et al.. Culture of ovine intramuscular preadipocyte in vitro [J]. J. Shihezi Univ. (Nat. Sci.), 2011, 29(3):291-295.
25	李戡,刘文忠,张瑞鑫,等.AMPK调控绵羊肌内前体脂肪细胞分化的研究[J].畜牧兽医学报,2018,49(8):1594-1604.
	LI K, LIU W Z, ZHANG R X, et al.. AMPK regulates sheep muscle derived preadipocytes differentiation [J]. Acta Veterinaria Et Zootechnica Sin., 2018, 49(8):1594-1604.
26	FRONTIN A, CINTI S. Distribution and development of brown adipocytes in the murine and human adipose organ [J]. Cell Metab., 2010, 11(4):253-256.
27	康丹菊.绵羊脂肪沉积关键基因的筛选及ALDH1As在脂肪细胞分化中的调控研究[D].陕西杨凌:西北农林科技大学,2018.
	KANG D J. Identification of key genes related to adipose deposition in sheep and regulatory study of ALDH1As in adipocyte differentiation [D]. Shaanxi Yangling: Northwest Agriculture and Forestry University, 2018.
28	KAWAI M, MUSHIAKE S, BESSHO K, et al.. Wnt/Lrp/β-catenin signaling suppresses adipogenesis by inhibiting mutual activation of PPARγ and C/EBPα [J]. Biochem. Biophys. Res. Commun., 2007, 363(2):276-282.
29	胡艳霞,曾勇庆,崔志峰,等.莱芜猪前体脂肪细胞的原代培养及诱导分化研究[J].畜牧兽医学报,2012,43(9):1346-1352.
	HU Y X, ZENG Y Q, CUI Z F, et al.. Primary culture and induced differentiation of laiwu black pig preadipocytes [J]. Acta Veterinaria Et Zootechnica Sin., 2012, 43(9):1346-1352.
30	孟金花,蔡刚志,肖红卫,等.猪肌内脂肪前体细胞分离培养和成脂诱导分化研究[J].中国畜牧杂志,2019,55(12):98-101.
	MENG J H, CAI Z G, XIAO H W, et al.. Culture and adipogenic differentiation of porcine preadipocytes [J]. Chin. J. Anim. Sci., 2019, 55(12):98-101.
31	吴森.牛原代肌肉卫星细胞和前体脂肪细胞不同混合比例共培养细胞活性的比较[J].青海畜牧兽医杂志,2019,49(2):14-20, 50.
	WU S. Comparison of cellular viability under different cell mix proportion co-culture system of bovine primary skeletal muscle satellite cells and preadipocytes [J]. Chin. Qinghai J. Anim. Vet. Sci., 2019, 49(2):14-20, 50.
32	陈涛,马立霞,李志鑫,等.新西兰兔肌内和皮下前体脂肪细胞原代培养与诱导分化的研究[J].中国畜牧杂志,2019,55(6):39-44.
	CHENG T, MA L X, LI Z X, et al.. Comparative study on primary culture and induced differentiation of intramuscular and subcutaneous preadipocytes of New Zealand rabbits [J]. Chin. J. Anim. Sci., 2019, 55(6):39-44.
33	张清月,王雪,刘树林,等.绒山羊羔羊和成年羊前体脂肪细胞的原代培养及传代方法[J].动物营养学报,2018,30(9):3551-3558.
	ZHANG Q Y, WANG X, LIU S L, et al.. Primary culture and passage method of preadipocytes of cashmere lambs and adult goats [J]. Chin. J. Anim. Nutr., 2018, 30(9):3551-3558.
34	叶承志, 张龙江, 王宏伟, 等.油酸诱导SW872前脂肪细胞分化的作用 [J]. 实用儿科临床杂志, 2006, 21(7):395-397.
	YE C Z, ZHAGN L J, WANG H W, et al.. Effect of oleic acid on differentiation of SW872 preadipocytes [J]. J. Appl. Clin. Pediatrics, 2006, 21(7):395-397.
35	卢慧玲,王宏伟,Cianfloned K,等.诱导SW872前脂肪细胞分化的最优方法探讨[J].基础医学与临床,2006,26(3):315-318.
	LU H L, WANG H W, CIANFLONED K, et al.. To optimize the method for inducing SW872 preadipocyte to differentiation [J]. Basic Clin. Med., 2006, 26(3):315-318.
36	张罕星,朱晓彤,束刚,等.猪脂肪前体细胞分化过程中聚脂相关基因的表达模式[J].动物学报,2007,53(1):143-150.
	ZHANG H X, ZHU X T, SHU G, et al.. Expression profile of the genes involved in adipogenesis during porcine preadipocyte differentiation [J]. Acta Zoolog. Sin., 2007, 53(1):143-150.
37	张艳芳,任阳,朱琳娜,等.猪肌内前体脂肪细胞的体外培养[J].农业生物技术学报,2011,19(1):116-121.
	ZHANG Y F, REN Y, ZHU L N, et al.. Culture of porcine intramuscular preadipocyte in vitro [J]. J. Agric. Biotechnol., 2011, 19(1):116-121.
38	林森,林亚秋,朱江江,等.Wnt10b对山羊前体脂肪细胞分化相关基因表达的影响[J].畜牧兽医学报,2018,49(4):685-692.
	LIN S, LIN Y Q, ZHU J J, et al.. Effect of Wnt10b on the expression of precursor adipocytes differentiation related genes in goat [J]. Acta Veterinaria Et Zootechnica Sin., 2018, 49(4):685-692.
39	HAUSMAN G J, BERGEN W G, ETHERTON T D, et al.. The history of adipocyte and adipose tissue research in meat animals [J]. J. Anim. Sci., 2018, 96(2):473-486.
40	STRABLE M S, NTAMBI J M. Genetic control of de novo lipogenesis: role in diet-induced obesity [J]. Crit. Rev. Biochem. Mol., 2010, 45:199-214.
41	CRESPILLO A,SUÁREZ J,BERMÚDEZ-SILVA F J, et al.. Expression of the cannabinoid system in muscle: effects of a high-fat diet and CB1 receptor blockade [J]. Biochem. J., 2010, 433:175-185.
42	RAVINET T C, DEIGORGE C, MENET C, et al.. CB1 cannabinoid receptor knockout in mice leads to leanness, resistance to diet-induced obesity and enhanced leptin sensitivity [J]. Int. J. Obesity Relat. Metab., 2004, 28:640-648.
43	COTA D, MARSICANO G, TSCHOP M, et al.. The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis [J]. J. Clin. Invest., 2003, 112:423-431.
44	闫伟,刘海霞,张力,等.中国和新西兰绵羊群体FABP4基因遗传特性比较分析[J].中国农业科技导报,2018,20(9):40-48.
	YAN W, LIU H X, ZHAGN L, et al.. Comparative analysis of FABP4 genetic characteristcs of ovine population from China and New Zealand [J]. J. Agric. Sci. Technol., 2018, 20(9):40-48.
45	徐蕾,闫伟,胡江,等.绵羊脂肪型脂肪酸结合蛋白基因(A-FABP)的变异研究[J].甘肃农业大学学报,2016,51(3):29-34.
	XU L, YAN W, HU J, et al.. Variations on ovine adipocyte fatty-acid binding protein (A-FABP) gene [J]. J. Gansu Agric.Univ., 2016, 51(3):29-34.
46	YAN W, ZHOU H T, LUO Y Z, et al.. Identification of more than two paternal haplotypes of the ovine fatty acid-binding protein 4 (FABP4) gene in half-sib families: evidence of intragenic meiotic recombination [J/OL]. PLoS One, 2014, 9(2):e88691 [2020-12-10]. .
47	YAN W, ZHOU H T, LUO Y Z, et al.. Allelic variation in ovine fatty acid-binding protein (FABP4) gene [J]. Mol. Biol. Rep., 2012, 39 (12):10621-10625.
48	JUDITH S, BETINA C. The emerging functions and mechanisms of mammalian fatty Acid-binding proteins [J]. Annu. Rev. Nutr., 2008, 28:73-95.
49	MASATO F, GÖKHAN S H. Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets [J]. Nat. Rev. Drug. Discovery, 2008, 7(6):489-503.
50	MARGARITA V, PATRICIA R, ANA L G, et al.. CB1 blockade potentiates down-regulation of lipogenic gene expression in perirenal adipose tissue in high carbohydrate diet-induced obesity [J/OL]. PloS One, 2014, 9(2):e90016 [2020-12-10]. .

[1]	Xiaowei CHEN, Yuzhu SHA, Xiu LIU, Pengyang SHAO, Fanxiong WANG, Zhuanhui XIE, Wenxin YANG, Qianling CHEN, Min GAO, Wei HUANG. Analysis of Gene Expression Characteristics Associated with Quality， Nutrient Composition， and Meat Quality of Tibetan Sheep Meat During Different Phenological Stages [J]. Journal of Agricultural Science and Technology, 2025, 27(7): 161-171.
[2]	Taotao MAO, Xiaoqiang ZHAO, Xiaodong BAI, Bin YU. Effect of Low Temperature Stress on Photosynthetic Performance， Antioxidant Enzyme System and Related Gene Expression in Maize Seedlings [J]. Journal of Agricultural Science and Technology, 2025, 27(5): 49-60.
[3]	Guoshun CHEN, Bin TIAN, Yancheng GAO, Yancui LI, Wenjin TIAN, Wenjing YANG, Wancheng WU, Lijun HUANG. Effects of Astragalus Polysaccharides on Production Performance， Carcass Performance and Meat Quality of Stellar-fed Tan Sheep [J]. Journal of Agricultural Science and Technology, 2025, 27(2): 150-157.
[4]	Fanxiong WANG, Ying XU, Yuzhu SHA, Pengyang SHAO, Zhuanhui XIE, Wenhao LI, Jiqing WANG, Shaobin LI, Xiaowei CHEN, Wenxin YANG, Xiu LIU. Analysis of Muscle Fatty Acid Characteristics of Tibetan Sheep of Different Ages [J]. Journal of Agricultural Science and Technology, 2024, 26(8): 74-83.
[5]	Longbin WANG, Mingna LI, Yuzhu LUO, Jiqing WANG, Zhiyun HAO, Jiyuan SHEN, Huimin ZHEN, Yuting ZHANG, Shutong YANG. Functional Analysis of SMAD2， SMAD3 and SMAD4 Genes in Ovarian Activity in Lesser Caecilian Sheep [J]. Journal of Agricultural Science and Technology, 2024, 26(7): 69-79.
[6]	Ying XU, Yue REN, Yuzhu SHA, Xiaoning PU, Xinyu GUO, Liangwei YAO, Pengyang SHAO, Jiqing WANG, Shaobin LI, Xiu LIU. Characteristics Analysis of Meat Fatty Acids of Tibetan Sheep at Different Altitudes Under Natural Grazing Conditions [J]. Journal of Agricultural Science and Technology, 2024, 26(4): 67-76.
[7]	Liangwei YAO, Yuzhu SHA, Xinyu GUO, Xiaoning PU, Ying XU, Jiqing WANG, Shaobin LI, Zhiyun HAO, Xiu LIU. Analysis of Meat Quality， Nutritional Components and Expression Characteristics of Meat Quality-related Genes in Tibetan Sheep at Different Altitudes [J]. Journal of Agricultural Science and Technology, 2024, 26(3): 66-75.
[8]	Guangjun YANG, Yuan LIAO, Zhichao ZHANG, Rongzhen ZHONG, Ziyuan DUAN. Characteristics of Temporal Expression of Key Genes During Differentiation from Sheep Preadipocyte [J]. Journal of Agricultural Science and Technology, 2024, 26(2): 100-108.
[9]	Huabing LIU, Wei DANG, Qi LI, Xiaobing ZHANG, Zhiqiang XU, Yongjian ZHONG, Zhiguang REN, Yonggang ZHANG, Kailong YUAN, Hao YANG, Hui WANG, Jutao SUN. Study on Differences in Nitrogen Absorption and Assimilation among Tobacco Varieties [J]. Journal of Agricultural Science and Technology, 2024, 26(11): 66-78.
[10]	Yiwei YAN, Jie TIAN. Identification and Expression Analysis of NAC Gene Family Under Low Temperature in Allium sativum L. [J]. Journal of Agricultural Science and Technology, 2023, 25(4): 67-76.
[11]	Lingwei SUN, Mengxian HE, Jianjun DAI, Caifeng WU, Defu ZHANG, Yuexia LIN. Metabolomics in Neonatal Lambs of Hu-sheep with Intrauterine Growth Retardation [J]. Journal of Agricultural Science and Technology, 2022, 24(7): 123-131.
[12]	Ming CHENG, Ying ZHU, Xiaonan WANG, Ping LUO, Yong CHEN, Zhuanfang HAO, Zhangying XI. Drought Resistance Regulated by Allelic Variations of ZmSNAC13 in Maize [J]. Journal of Agricultural Science and Technology, 2022, 24(5): 24-31.
[13]	Tao YANG, Xiaoqian MA, Quan ZHANG, Hongliang ZHANG. Research Progress of Histone Modification in Rice [J]. Journal of Agricultural Science and Technology, 2022, 24(4): 11-20.
[14]	Xiaochun SUN, Wenjing HUANG, Bo LI. Effects of Exogenous Salicylic Acid on Physiological and Biochemical Indexes and Related Gene Expression in Platycodongrandiflorus Under Drought Stress [J]. Journal of Agricultural Science and Technology, 2022, 24(1): 63-70.
[15]	BAI Hao, LI Xiaofan, ZHONG Li, SONG Qianqian, LIU Benshuai, ZHANG Xin, ZHANG Yang, WANG Zhixiu, JIANG Yong, XU Qi, CHANG Guobin, CHEN Guohong, . Mineral Element Depositions and Gene Expression Across Different Tissues of the Runzhou White Crested Ducks [J]. Journal of Agricultural Science and Technology, 2021, 23(8): 63-73.