Characteristics of Temporal Expression of Key Genes During Differentiation from Sheep Preadipocyte

doi:10.13304/j.nykjdb.2022.0930

Abstract

Abstract:

To analyze the expression pattern and characteristics of key genes in sheep preadipocytes during adipogenic differentiation， 3T3-L1 cell line was selected as reference， the preadipocytes were isolate from the tail adipose tissue of female lambs from Hulun Buir sheep 20-day after birth using tissue digestion method and prepared into primary cell line. The 3T3-L1 cell line and sheep preadipocyte cells were induced to differentiate into mature adipocyte by cocktail method， and the temporal expressions of key genes during differentiation were detected by real-time fluorescence quantitative PCR. The results showed that the adipogenic differentiation of sheep precursor adipocytes took longer， and some cells still had differentiation ability after 16 d of induction. The patterns of temporal expression indicate that the expressions of DLK1， SREBP1， ACC1 and FASN were basically consistence both in the sheep preadipocytes and 3T3-L1 cell lines， the expression trends of ZFP423， ADIPOQ and C/EBPα were highly similar， however， the genes of PPARγ， FABP4， LPL and HSL were different. Compared with 3T3-L1 cell line， the relative expression levels of ZFP423， ADIPOQ， C/EBPα and FABP4 in sheep preadipocytes still remained at a high level in the middle and late stages of induced differentiation， and the relative expressions of PPARγ， LPL and HSL showed a continuous increase trend， which illustrated that sheep preadipocytes had its own characteristics in adipogenic differentiation. It was speculated that the cells from the sheep adipocytes synthesized triglycerides and then deposited in mature adipocytes through ingesting fatty acids from the external environment after 16 d of induction according to the expression of related genes. It indicated that sheep adipose cells had strong ability to absorb external fatty acids， which might be one of reasons for the excessive deposition of subcutaneous fat in native sheep. So the sheep adipocyte was more suitable as a cell model in research of sheep fat metabolism， which provided reliable experimental model for research of sheep fat metabolism.

Key words: sheep, preadipocytes, adipogenic differentiation, gene temporal expression

摘要：

为分析绵羊前体脂肪细胞在成脂分化过程中关键基因的表达规律及特点，以3T3-L1细胞系为参照，选取出生20 d的呼伦贝尔羊母羔羊，利用组织块消化法从其尾部脂肪组织中分离前体脂肪细胞，建立原代细胞系。利用鸡尾酒法分别对3T3-L1细胞系和绵羊前体脂肪细胞系进行诱导分化，采用实时荧光定量PCR检测诱导分化过程中关键基因的时序表达。结果显示，绵羊前体脂肪细胞成脂分化用时更长，诱导分化16 d后依然有部分细胞具有分化能力。基因时序表达分析表明，DLK1、SREBP1、ACC1、FASN基因的时序表达趋势在2类细胞中基本一致；ZFP423、ADIPOQ、C/EBPα基因的表达时序在2类细胞系中高度相似；而PPARγ、FABP4、LPL、HSL基因在2类细胞系中的时序表达趋势差异较大。相比3T3-L1细胞系，绵羊前体脂肪细胞中ZFP423、ADIPOQ、C/EBPα、FABP4的相对表达量在诱导分化中、后期仍然处于较高水平，PPARγ、LPL、HSL的相对表达量呈现持续增长趋势，表明绵羊前体脂肪细胞的成脂分化具有自身特点。根据基因表达情况推测，绵羊前体脂肪细胞诱导分化16 d后通过外界环境摄取脂肪酸合成甘油三酯并沉积于细胞，这种较强的吸收外界环境脂肪酸的能力可能是本土绵羊皮下脂肪过度沉积的原因之一。综上所述，对绵羊脂肪代谢的相关研究以绵羊脂肪细胞为模型更加合理，为绵羊脂肪代谢研究提供了可靠的试验模型。

关键词: 绵羊, 前体脂肪细胞, 成脂分化, 基因时序表达

CLC Number:

S826

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.

杨广军, 廖媛, 张志超, 钟荣珍, 段子渊. 绵羊前体脂肪细胞分化过程中关键基因时序表达特征研究[J]. 中国农业科技导报, 2024, 26(2): 100-108.

Figures/Tables 4

References 46

1	MAO Y W, HOPKINS D L, ZHANG Y M, et al.. Consumption patterns and consumer attitudes to beef and sheep meat in China [J]. J. Food Nutr. Res., 2016, 4(42):30-39.
2	ROLLS B J, DREWNOWSKI A, LEDIKWE J H. Changing the energy density of the diet as a strategy for weight management [J]. J. Am. Diet. Assoc., 2005, 105(S5):98-103.
3	KANTONO K, HAMID N, MA Q, et al.. Consumers’ perception and purchase behaviour of meat in China [J/OL]. Meat Sci., 2021, 179:108548 [2022-09-12]. .
4	GHABEN A L, SCHERER P E. Adipogenesis and metabolic health [J]. Nat. Rev. Mol. Cell Biol., 2019, 20(4):242-258.
5	NUNN E R, SHINDE A B, ZAGANJOR E. Weighing in on adipogenesis [J/OL]. Front. Physiol., 2022, 13:821278 [2022-09-12]. .
6	SUL H S. Minireview: Pref-1: role in adipogenesis and mesenchymal cell fate [J]. Mol. Endocrinol., 2009, 23(11):1717-1725.
7	GUPTA R K, MEPANI R J, KLEINER S, et al.. Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells [J]. Cell Metab., 2012, 15(2):230-239.
8	ARSENIJEVIC T, GRÉGOIRE F, DELFORGE V, et al.. Murine 3t3-l1 adipocyte cell differentiation model: validated reference genes for qpcr gene expression analysis [J/OL]. PLoS One, 2012, 7(5):e37517 [2022-09-12]. .
9	LEE J H, CHO H D, JEONG J H, et al.. New vinegar produced by tomato suppresses adipocyte differentiation and fat accumulation in 3t3-l1 cells and obese rat model [J]. Food Chem., 2013, 141(3):3241-3249.
10	LEE J Y, KIM T Y, KANG H, et al.. Anti-obesity and anti-adipogenic effects of chitosan oligosaccharide (GO2KA1) in SD rats and in 3T3-L1 preadipocytes models [J/OL]. Molecules, 2021, 26(2): 0331 [2022-09-12]. .
11	PARK Y J, SEO D W, JU J Y, et al.. The antiobesity effects of buginawa in 3T3-L1 preadipocytes and in a mouse model of high-fat diet-induced obesity [J/OL]. Biomed. Res. Int., 2019, (3):3101987 [2022-09-12]. .
12	WONG M L, MEDRANO J F. Real-time PCR for mRNA quantitation [J]. Biotechniques, 2005, 39(1):75-85.
13	DUFAU J, SHEN J X, COUCHET M, et al.. In vitro and ex vivo models of adipocytes [J]. Am. J. Physiol. Cell Physiol., 2021, 320(5):822-841.
14	SCOTT M A, NGUYEN V T, LEVI B, et al.. Current methods of adipogenic differentiation of mesenchymal stem cells [J]. Stem Cell Dev., 2011, 20(10):1793-1804.
15	潘红梅,闫尊强,龙熙,等.3T3-L1前脂肪细胞诱导分化方法的建立[J].中国畜牧杂志,2016,52(15):94-97.
16	赵蕾,郑美丽,杨梅,等.3T3-L1前体脂肪细胞诱导分化方法优化的初步探讨[J].首都医科大学学报,2019,40(2):226-231.
	ZHAO L, ZHENG M L, YANG M, et al.. Optimization of induction and differentation of 3T3-L1 preadipocyte [J]. J. Captial Med. Univ., 2019, 40(2):226-231.
17	PU Y, VEIGA A. Pparγ agonist through the terminal differentiation phase is essential for adipogenic differentiation of fetal ovine preadipocytes [J/OL]. Cell. Mol. Biol. Lett., 2017, 22:6 [2022-09-12]. .
18	WANG Y, LI X, CAO Y, et al.. Effect of the acaa1 gene on preadipocyte differentiation in sheep [J/OL]. Front. Genet., 2021, 12:649140 [2022-09-12]. .
19	XIAO C, JIN H G, ZHANG L C, et al.. Effects of SPARCL1 on the proliferation and differentiation of sheep preadipocytes [J]. Adipocyte, 2021, 10(1):658-669.
20	DENG K, REN C, LIU Z, et al.. Characterization of RUNX1T1, an adipogenesis regulator in ovine preadipocyte differentiation [J]. Int. J. Mol. Sci., 2018, 19(5):1300 [2022-09-12]. .
21	ZENG J, ZHOU S W, ZHAO J, et al.. Role of OXCT1 in ovine adipose and preadipocyte differentiation [J]. Biochem. Biophys. Res. Commun., 2019, 512(4):779-785.
22	HAUSMAN G J, BASU U, WEI S, et al.. Preadipocyte and adipose tissue differentiation in meat animals: influence of species and anatomical location [J]. Annu. Rev. Anim. Biosci., 2014, 2:323-351.
23	BOHAN A E, PURVIS K N, BARTOSH J L, et al.. The proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°Celsius compared to euthermic conditions in pigs [J]. Adipocyte, 2014, 3(4):322-332.
24	PATEL N G, HOLDER J C, SMITH S A, et al.. Differential regulation of lipogenesis and leptin production by independent signaling pathways and rosiglitazone during human adipocyte differentiation [J]. Diabetes, 2003, 52(1):43-50.
25	LI B J, QIAO L Y, YAN X R, et al.. mRNA expression of genes related to fat deposition during in vitro ovine adipogenesis [J]. Can. J. Anim. Sci., 2019, 99(4):764-771.
26	WANG Y, KIM K A, KIM J H, et al.. Pref-1, a preadipocyte secreted factor that inhibits adipogenesis [J]. J. Nutr., 2006, 136(12):2953-2956.
27	ZHU Y, QI C, KORENBERG J R, et al.. Structural organization of mouse peroxisome proliferator-activated receptor gamma (mppar gamma) gene: alternative promoter use and different splicing yield two mppar gamma isoforms [J]. Proc. Natl. Acad. Sci. USA, 1995, 92(17):7921-7925.
28	WANG Q A, TAO C, JIANG L, et al.. Distinct regulatory mechanisms governing embryonic versus adult adipocyte maturation [J]. Nat. Cell Biol., 2015, 17(9):1099-1111.
29	JANANI C, RANJITHA-KUMARI B D. PPAR gamma gene: a review [J]. Diabetes Metab. Syndrome., 2015, 9(1):46-50.
30	KIM J B, WRIGHT H M, WRIGHT M, et al.. ADD1/SREBP1 activates PPAR gamma through the production of endogenous ligand [J]. Proc. Natl. Acad. Sci. USA, 1998, 95(8):4333-4337.
31	ZHAO X, FENG D, WANG Q, et al.. Regulation of lipogenesis by cyclin-dependent kinase 8-mediated control of SREBP-1 [J]. J. Clin. Invest., 2012, 122(7): 2417-2427.
32	HORTON J D. Sterol regulatory element-binding proteins: transcriptional activators of lipid synthesis [J]. Biochem. Soc. Trans., 2002, 30(6):1091-1095.
33	EBERLÉ D, HEGARTY B, BOSSARD P, et al.. SREBP transcription factors: master regulators of lipid homeostasis [J]. Biochimie, 2004, 86(11):839-848.
34	DOROTEA D, KOYA D, HA H. Recent insights into SREBP as a direct mediator of kidney fibrosis via lipid-independent pathways [J/OL]. Front. Pharmacol., 2020, 11:265 [2022-09-12]. .
35	ELSTNER E, MÜLLER C, KOSHIZUKA K, et al.. Ligands for peroxisome proliferator-activated receptor gamma and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice [J]. Proc. Natl. Acad. Sci. USA, 1998, 95(15):8806-8811.
36	OLZMANN J A, CARVALHO P. Dynamics and functions of lipid droplets [J]. Nat. Rev. Mol. Cell. Biol., 2019, 20(3):137-155.
37	SMITH S, WITKOWSKI A, JOSHI A K. Structural and functional organization of the animal fatty acid synthase [J]. Prog. Lipid Res., 2003, 42(4):289-317.
38	WANG H, ECKEL R H. Lipoprotein lipase: from gene to obesity [J]. Am. J. Physiol-Endocrinol. Metab., 2009, 297(2):271-288.
39	TSUBAKIO-YAMAMOTO K, SUGIMOTO T, NISHIDA M, et al.. Serum adiponectin level is correlated with the size of HDL and LDL particles determined by high performance liquid chromatography [J]. Metabolism, 2012, 61(12):1763-1770.
40	CHAN D C, WATTS G F, NG T W, et al.. Adiponectin and other adipocytokines as predictors of markers of triglyceride-rich lipoprotein metabolism [J]. Clin. Chem., 2005, 51(3):578-585.
41	YANAI H, YOSHIDA H. Beneficial effects of adiponectin on glucose and lipid metabolism and atherosclerotic progression: mechanisms and perspectives [J/OL]. Int. J. Mol. Sci., 2019, 20(5):1190 [2022-09-12]. .
42	FURUHASHI M, HOTAMISLIGIL G S. Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets [J]. Nat. Rev. Drug. Discov., 2008, 7(6):489-503.
43	ZIMMERMAN A W, VEERKAMP J H. New insights into the structure and function of fatty acid-binding proteins [J]. Cell. Mol. Life Sci., 2002, 59(7):1096-1116.
44	RODRÍGUEZ-CALVO R, GIRONA J, ALEGRET J M, et al.. Role of the fatty acid-binding protein 4 in heart failure and cardiovascular disease [J]. J. Endocrinol., 2017, 233(3):173-184.
45	OLZMANN J A, CARVALHO P. Dynamics and functions of lipid droplets [J]. Nat. Rev. Mol. Cell Biol., 2019, 20(3):137-155.
46	ROSAS-BALLINA M, GUAN X L, SCHMIDT A, et al.. Classical activation of macrophages leads to lipid droplet formation without de novo fatty acid synthesis [J/OL]. Front. Immunol., 2020, 11:131 [2022-09-12]. .

基因名称 Gene name	登录号 GenBank ID	引物序列 Primer sequence （5’-3’）	产物长度 Product size/bp
m-DLK1	NM_001190703.1	F： GCGTGGACCTGGAGAAAG R： ACAGAAGTTGCCTGAGAAGC	194
m-ZFP423	NM_001310520.1	F： CACCTGCGATCACTGTCAG R： GACGCAACATCCTTGCTGGA	141
m-SREBF1	NM_001313979.1	F： CTTTGGCCTCGCTTTTCGG R： TGGGTCCAATTAGAGCCATCTC	118
m-PPARG	NM_001127330.3	F： CTCCAAGAATACCAAAGTGCGA R： GCCTGATGCTTTATCCCCACA	150
m-CEBPA	NM_001287514.1	F： GCGGGAACGCAACAACATC R： GTCACTGGTCAACTCCAGCAC	97
m-FABP4	NM_001409513.1	F： TGGGAACCTGGAAGCTTGTCTC R： GAATTCCACGCCCAGTTTGA	197
m-ADIPOQ	NM_009605.5	F： TCACCTACGACCAGTATCAG R： GAGAAGAAAGCCAGTAAATGT	167
m-ACC1	NM_133360.3	F： AACATCCCCACGCTAAACAG R： CTGACAAGGTGGCGTGAAG	117
m-FASN	NM_007988.3	F： ATTGGCTCCACCAAATCCAAC R： CCCATGCTCCAGGGATAACAG	90
m-LPL	NM_008509.2	F： TCAGAGCCAAGAGAAGCAGCAA R： TTGTGTTGCTTGCCATCCTCA	118
m-HSL	NM_001039507.2	F： GATTTACGCACGATGACACAGT R： ACCTGCAAAGACATTAGACAGC	114
m-β-actin	NM_007393.5	F： TCTGGCACCACCTTCTACAATG R： AGCACAGCCTGGATAGCAACG	171
o-DLK1	XM_027957402.2	F： TCTGCGAGATCATGACCAAC R： GGCTTGCACAGACACTTGAA	214
o-ZFP423	XM_027977817.2	F： TTCCTGACCGAGTCCTCCCT R： TCTTGTGGTTCTCCTTGATGTGC	193
o-SREBF1	XM_027974786.2	F： GAGCTTCGTGGTTTCCAGAG R： CTCAGGCTACGGTCCAGAAG	158
o-PPARG	NM_001100921.1	F： CCCTGGCAAAGCATTTGTAT R： ACTGACACCCCTGGAAGATG	222
o-CEBPA	NM_001308574.1	F： CAAAGCCAAGAAGTCCGT R： CTCAGTTGTTCCACCCGC	180
o-FABP4	NM_001114667.1	F： AGTGGGTGTGGGCTTTGCTA R： TTTTCTCTTTATGGTGGTTG	256
o-ADIPOQ	XM_042243690.1	F： TCGTTGGTCCTAAGGGTGAC R： TTGGTAAAGCGAATGGGAAC	184

基因名称 Gene name	登录号 GenBank ID	引物序列 Primer sequence （5’-3’）	产物长度 Product size/bp
m-DLK1	NM_001190703.1	F： GCGTGGACCTGGAGAAAG R： ACAGAAGTTGCCTGAGAAGC	194
m-ZFP423	NM_001310520.1	F： CACCTGCGATCACTGTCAG R： GACGCAACATCCTTGCTGGA	141
m-SREBF1	NM_001313979.1	F： CTTTGGCCTCGCTTTTCGG R： TGGGTCCAATTAGAGCCATCTC	118
m-PPARG	NM_001127330.3	F： CTCCAAGAATACCAAAGTGCGA R： GCCTGATGCTTTATCCCCACA	150
m-CEBPA	NM_001287514.1	F： GCGGGAACGCAACAACATC R： GTCACTGGTCAACTCCAGCAC	97
m-FABP4	NM_001409513.1	F： TGGGAACCTGGAAGCTTGTCTC R： GAATTCCACGCCCAGTTTGA	197
m-ADIPOQ	NM_009605.5	F： TCACCTACGACCAGTATCAG R： GAGAAGAAAGCCAGTAAATGT	167
m-ACC1	NM_133360.3	F： AACATCCCCACGCTAAACAG R： CTGACAAGGTGGCGTGAAG	117
m-FASN	NM_007988.3	F： ATTGGCTCCACCAAATCCAAC R： CCCATGCTCCAGGGATAACAG	90
m-LPL	NM_008509.2	F： TCAGAGCCAAGAGAAGCAGCAA R： TTGTGTTGCTTGCCATCCTCA	118
m-HSL	NM_001039507.2	F： GATTTACGCACGATGACACAGT R： ACCTGCAAAGACATTAGACAGC	114
m-β-actin	NM_007393.5	F： TCTGGCACCACCTTCTACAATG R： AGCACAGCCTGGATAGCAACG	171
o-DLK1	XM_027957402.2	F： TCTGCGAGATCATGACCAAC R： GGCTTGCACAGACACTTGAA	214
o-ZFP423	XM_027977817.2	F： TTCCTGACCGAGTCCTCCCT R： TCTTGTGGTTCTCCTTGATGTGC	193
o-SREBF1	XM_027974786.2	F： GAGCTTCGTGGTTTCCAGAG R： CTCAGGCTACGGTCCAGAAG	158
o-PPARG	NM_001100921.1	F： CCCTGGCAAAGCATTTGTAT R： ACTGACACCCCTGGAAGATG	222
o-CEBPA	NM_001308574.1	F： CAAAGCCAAGAAGTCCGT R： CTCAGTTGTTCCACCCGC	180
o-FABP4	NM_001114667.1	F： AGTGGGTGTGGGCTTTGCTA R： TTTTCTCTTTATGGTGGTTG	256
o-ADIPOQ	XM_042243690.1	F： TCGTTGGTCCTAAGGGTGAC R： TTGGTAAAGCGAATGGGAAC	184

基因名称 Gene name	登录号 GenBank ID	引物序列 Primer sequence （5’-3’）	产物长度 Product size/bp
o-ACC1	NM_001009256.1	F： ACCACCAACGCGAAGGT R： GTCAATGGCGGACAGGA	114
o-FASN	XM_027974304.2	F： GCCATCCTCTCTGCCTACTG R： CTGCTTCACGAACTCCAACA	198
o-LPL	NM_001009394.1	F： CTTCAACCACAGCAGCAAAA R： AAACTTGGCCACATCCTGTC	211
o-HSL	NM_001128154.1	F： CTCCGACTCAGACCAGAAGG R： AGGGCTGCTTCAGACACACT	192
o-β-actin	NM_001009784.3	F： CGGCAATGAGCGGTTCC R： CCGTGTTGGCGTAGAGGT	143

基因名称 Gene name	登录号 GenBank ID	引物序列 Primer sequence （5’-3’）	产物长度 Product size/bp
o-ACC1	NM_001009256.1	F： ACCACCAACGCGAAGGT R： GTCAATGGCGGACAGGA	114
o-FASN	XM_027974304.2	F： GCCATCCTCTCTGCCTACTG R： CTGCTTCACGAACTCCAACA	198
o-LPL	NM_001009394.1	F： CTTCAACCACAGCAGCAAAA R： AAACTTGGCCACATCCTGTC	211
o-HSL	NM_001128154.1	F： CTCCGACTCAGACCAGAAGG R： AGGGCTGCTTCAGACACACT	192
o-β-actin	NM_001009784.3	F： CGGCAATGAGCGGTTCC R： CCGTGTTGGCGTAGAGGT	143

[1]	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.
[2]	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.
[3]	YAN Wei1, LIU Haixia1, ZHANG Li1, HAN Dayong1, ZHU Aiwen1, ZHAO Xuting1, LUO Yuzhu2. Comparative Analysis of FABP4 Genetic Characteristcs of Ovine Population from China and New Zealand [J]. Journal of Agricultural Science and Technology, 2018, 20(9): 40-48.
[4]	LIU Yu-feng, WANG Ming-li*, SHI Zi-zhong, WANG Hong-\|yu. Analysis of Technical Efficiency and Technological Progress Contribution in Mutton Sheep Production [J]. , 2014, 16(3): 156-161.
[5]	JIN Ling-yan\|GU Xin\|CAI Jin-hua\|LIU Ya-ni. Detection of Dovine and Sheep Derived Materials in Feed by Two PCR Methods [J]. , 2008, 10(S2): 76-80.
[6]	MA Yu-zhen, WANG Rui,YAN Zhen, WANG Li-min\| LIU Dong-jun\| XIA Guo-liang. Comparison Between Sheep Fetal Fibroblast Cells transfected with \|GFP by Lipofectamine^TM and by Fugene-6 [J]. , 2008, 10(1): 108-112.