绵羊Musclin基因真核表达载体的构建及其对肌细胞糖代谢和胰岛素作用的影响

doi:10.11843/j.issn.0366-6964.2021.03.008

Abstract

Abstract: To investigate the effects of sheep Musclin on glucose metabolism and insulin function, the structure properties of sheep Musclin protein were predicted by using various bioinformatics softwares and the eukaryotic expression vector of Musclin gene was constructed. The sheep fetal myoblasts were divided into 4 groups and treated as follows:mock-vector group (pcDNA3.1), Musclin-overexpressed group (pcDNA3.1-Musclin), mock-vector + insulin group (pcDNA3.1 + Insulin) and Musclin-overexpressed + insulin group (pcDNA3.1-Musclin + Insulin). Then the cells were collected at 48 and 72 h after differentiation induction, and the effects of Musclin on glucose metabolism in normal or insulin-treated differentiating myoblasts were analyzed and the changes of the related gene expression were detected by real-time PCR. The restriction endonuclease digestion and sequencing results indicated that the expression vector (pcDNA3.1-Musclin) was successfully constructed. The results of bioinformatics analysis showed that the sheep Musclin protein contained a signal peptide and had a restriction site at the 28th amino acid. The subcellular localization showed that it was in the extracellular space, and belonged to the secretory protein. Its secondary and tertiary structure mainly included alpha helixes and random coils. The cellular assays showed that the overexpression of sheep Musclin gene significantly reduced the glycogen content in the normal or insulin-treated sheep differentiating myoblasts and increased the glucose content in medium. The overexpression of Musclin inhibited the expressions of IRS1, AKT1, AKT2, GLUT1, GLUT4 mRNA, and promoted the expression of GSK3β mRNA in the Musclin-overexpressed group and Musclin-overexpressed + insulin group sheep myoblasts induced differentiation for 72 h, while only the expression of GLUT4 mRNA was inhibited after cells were induced differentiation for 48 h. In conclusion, the overexpression of the sheep Musclin gene affected the glucose metabolism and attenuated the functions of insulin in sheep differentiating myoblasts, and they coordinated with each other to maintain the homeostasis of glucose metabolism. The functional mechanisms of sheep Musclin were involved in the expression changes of the above genes and were related to the differentiation state of sheep myoblasts.

Key words: sheep, Musclin gene, bioinformatics, eukaryotic expression, glucose metabolism, insulin

CLC Number:

S826.2

ZHENG Tengfei, XIN Xiang, HAN Gaolian, LI Mengxin, LI Junling, QIN Jian, DU Rong. Construction of Eukaryotic Expression Vector of Sheep Musclin Gene and Its Effects on Glucose Metabolism and Insulin Function in Myocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(3): 641-652.

References 39

[1]	NISHIZAWA H,MATSUDA M,YAMADA Y,et al.Musclin,a novel skeletal muscle-derived secretory factor[J].J Biol Chem,2004,279(19):19391-19395.
[2]	LIU Y,HUO X,PANG X F,et al.Musclin inhibits insulin activation of akt/protein kinase B in rat skeletal muscle[J].J Int Med Res,2008,36(3):496-504.
[3]	BANZET S,KOULMANN N,SANCHEZ H,et al.Musclin gene expression is strongly related to fast-glycolytic phenotype[J].Biochem Biophys Res Commun,2007,353(3):713-718.
[4]	GUO Q,HU H L,LIU X H,et al.C/EBPβ mediates palmitate-induced musclin expression via the regulation of PERK/ATF4 pathways in myotubes[J].Am J Physiol Endocrinol Metab,2019,316(6):E1081-E1092.
[5]	ZHANG W Q,YANG D,YUAN Y M,et al.Muscular G9a regulates muscle-liver-fat axis by musclin under overnutrition in female mice[J]. Diabetes, 2020,69(12):2642-2654.
[6]	CHEN W J,LIU Y,SUI Y B,et al.Positive association between musclin and insulin resistance in obesity:evidence of a human study and an animal experiment[J].Nutr Metab (Lond),2017,14:46.
[7]	BEG M,ABDULLAH N,THOWFEIK F S,et al.Distinct Akt phosphorylation states are required for insulin regulated Glut4 and Glut1-mediated glucose uptake[J].eLife,2017,6:e26896.
[8]	ARAGÓN-HERRERA A,FEIJÓO-BANDÍN S,RODRÍGUEZ-PENAS D,et al.Relaxin activates AMPK-AKT signaling and increases glucose uptake by cultured cardiomyocytes[J].Endocrine,2018,60(1):103-111.
[9]	CUI X,QIAN D W,JIANG S,et al.Scutellariae radix and coptidis rhizoma improve glucose and lipid metabolism in T2DM rats via regulation of the metabolic profiling and MAPK/PI3K/Akt signaling pathway[J].Int J Mol Sci,2018,19(11):3634.
[10]	CHUKWUMA C I,MOPURI R,NAGIAH S,et al.Erythritol reduces small intestinal glucose absorption,increases muscle glucose uptake,improves glucose metabolic enzymes activities and increases expression of Glut-4 and IRS-1 in type 2 diabetic rats[J].Eur J Nutr,2018,57(7):2431-2444.
[11]	SUH H Y,CHOI H,PAENG J C,et al.Comprehensive gene expression analysis for exploring the association between glucose metabolism and differentiation of thyroid cancer[J].BMC Cancer,2019,19(1):1260.
[12]	JALDIN-FINCATI J R,PAVAROTTI M,FRENDO-CUMBO S,et al.Update on GLUT4 vesicle traffic:a cornerstone of insulin action[J].Trends Endocrinol Metab,2017,28(8):597-611.
[13]	SUBBOTINA E,SIERRA A,ZHU Z Y,et al.Musclin is an activity-stimulated myokine that enhances physical endurance[J].Proc Natl Acad Sci U S A,2015,112(52):16042-16047.
[14]	YU J,ZHENG J,LIU X F,et al.Exercise improved lipid metabolism and insulin sensitivity in rats fed a high-fat diet by regulating glucose transporter 4(GLUT4) and musclin expression[J].Braz J Med Biol Res,2016,49(5):e5129.
[15]	RE CECCONI A D,FORTI M,CHIAPPA M,et al.Musclin,a myokine induced by aerobic exercise,retards muscle atrophy during cancer cachexia in mice[J].Cancers (Basel),2019,11(10):1541.
[16]	MACHADO-NETO J A,FENERICH B A,ALVES A P N,et al.Insulin Substrate Receptor (IRS) proteins in normal and malignant hematopoiesis[J]. Clinics (Sao Paulo),2018,73(suppl 1):e566s.
[17]	SUN X J,LIU F.Phosphorylation of IRS proteins:Yin-Yang regulation of insulin signaling[J].Vitam Horm,2009,80:351-387.
[18]	ZHENG X,ZHAO M G,JIANG C H,et al.Triterpenic acids-enriched fraction from Cyclocarya paliurus attenuates insulin resistance and hepatic steatosis via PI3K/Akt/GSK3β pathway[J].Phytomedicine,2020,66:153130.
[19]	GUO Q,HU H L,ZHOU Y,et al.Glucosamine induces increased musclin gene expression through endoplasmic reticulum stress-induced unfolding protein response signaling pathways in mouse skeletal muscle cells[J].Food Chem Toxicol,2019,125:95-105.
[20]	CHEN W J,LIU Y,SUI Y B,et al.Increased circulating levels of musclin in newly diagnosed type 2 diabetic patients[J].Diab Vasc Dis Res,2017,14(2):116-121.
[21]	GALLO-VILLEGAS J,ARISTIZABAL J C,ESTRADA M,et al.Efficacy of high-intensity,low-volume interval training compared to continuous aerobic training on insulin resistance,skeletal muscle structure and function in adults with metabolic syndrome:study protocol for a randomized controlled clinical trial (Intraining-MET)[J].Trials,2018,19(1):144.
[22]	王伟杰,阮武营,李胜利,等.兔Musclin基因片段克隆与序列分析[J].农业开发与装备,2015(5):75,131.WANG W J,RUAN W Y,LI S L,et al.Molecular cloning and sequence analysis of rabbit musclin[J].Agricultural Development & Equipments, 2015(5):75,131.(in Chinese)
[23]	王伟杰,马超峰,李成山,等.牛Musclin基因片段克隆与序列分析[J].安徽农业科学,2013,41(27):10926-10927,10968.WANG W J,MA C F,LI C S,et al.Molecular cloning and sequence analysis of bovine musclin[J].Journal of Anhui Agricultural Sciences,2013,41(27):10926-10927,10968.(in Chinese)
[24]	姜东凤.猪Musclin基因cDNA的克隆、序列分析及表达研究[D].杨凌:西北农林科技大学,2008.JIANG D F.Cloning,sequence analysis and expression of musclin cDNA in pig[D].Yangling:Northwest A&F University,2008.(in Chinese)
[25]	LI W Z,ZHANG H Q,QI S,et al.Molecular cloning and motif identification of the sheep musclin gene promoter[J].DNA Cell Biol,2017,36(12):1093-1098.
[26]	亓帅.葡萄糖、胰岛素及地塞米松对绵羊Musclin基因启动子调控活性的影响[D].太谷:山西农业大学,2016.QI S.Effects of glucose,insulin and dexamethasone on the regulatory activity of the musclin promoter in sheep[D].Taigu:Shanxi Agricultural University,2016.(in Chinese)
[27]	宗贺.棕榈酸调控FoxO1基因表达及其机制的初步研究[D].哈尔滨:哈尔滨工业大学,2018.ZONG H.The preliminary study on regulation of foxo1 gene expression by palmitic acid and its mechanism[D].Harbin:Harbin Institute of Technology,2018.(in Chinese)
[28]	YASUI A,NISHIZAWA H,OKUNO Y,et al.Foxo1 represses expression of musclin,a skeletal muscle-derived secretory factor[J].Biochem Biophys Res Commun,2007,364(2):358-365.
[29]	陈文佳,隋宇玢,尹新华,等.Musclin与胰岛素抵抗[J].生理科学进展,2012,43(1):45-47.CHEN W J,SUI Y F,YIN X H,et al.Musclin and insulin resistance[J].Progress in Physiological Sciences,2012,43(1):45-47.(in Chinese)
[30]	WANG L T,ZHANG B,ZHENG W,et al.Exosomes derived from pancreatic cancer cells induce insulin resistance in C2C12 myotube cells through the PI3K/Akt/FoxO1 pathway[J].Sci Rep,2017,7(1):5384.
[31]	姚骏凯,高学敏,李伟,等.青钱柳叶对2型糖尿病胰岛素抵抗大鼠骨骼肌INsR/IRS1/PI3K/GLUT4通路的影响[J].中华中医药杂志,2019,34(1):86-90.YAO J K,GAO X M,LI W,et al.Effects of Cyclocarya paliurus leaves on INsR/IRS1/PI3K/GLUT4 pathway in skeletal muscle of type 2 diabetes mellitus insulin resistance rats[J].China Journal of Traditional Chinese Medicine and Pharmacy,2019,34(1):86-90.(in Chinese)
[32]	FERREIRA G D,GERMEYER A,DE BARROS MACHADO A,et al.Metformin modulates PI3K and GLUT4 expression and Akt/PKB phosphorylation in human endometrial stromal cells after stimulation with androgen and insulin[J].Eur J Obstet Gynecol Reprod Biol,2014,175:157-162.
[33]	龚又明,郑显辉,谢鸣坤,等.小檗碱对HepG2胰岛素抵抗细胞PI3K/AKT1/GLUT1信号通路的调控作用[J].新中医,2017,49(8):1-4.GONG Y M,ZHENG X H,XIE M K,et al.Berberine Can regulate PI3K,AKT1 and GLUT1 signal pathway in HepG2 insulin resistant cells[J].Journal of New Chinese Medicine,2017,49(8):1-4.(in Chinese)
[34]	黎宇,罗新新,严奉东,等.葛根上调肝胰岛素抵抗HepG2细胞OB-R,IRS2,GLUT1和GLUT2蛋白调节糖代谢的研究[J].中国中药杂志,2017,42(10):1939-1944.LI Y,LUO X X,YAN F D,et al.Puerariae Lobatae Radix elevated expression levels of OB-R,IRS2,GLUT1 and GLUT2 to regulate glucose metabolism in insulin-resistance HepG2 cells[J].China Journal of Chinese Materia Medica,2017,42(10):1939-1944.(in Chinese)
[35]	聂海涛,黄欣爱,刘浩,等.黄体期不同营养水平对湖羊卵泡发育及相关葡萄糖代谢途径的影响[J].畜牧兽医学报,2018,49(5):942-953.NIE H T,HUANG X A,LIU H,et al.Effect of different nutrient levels on follicle development and glucose metabolic pathway involved in folliculogenesis of hu sheep during luteal phase[J].Acta Veterinaria et Zootechnica Sinica,2018,49(5):942-953.(in Chinese)
[36]	张凯艺,朱文娟,谢宁,等.一种自发胰岛素抵抗的3T3-L1脂肪细胞模型构建[J].畜牧兽医学报,2019,50(7):1475-1485.ZHANG K Y,ZHU W J,XIE N,et al.Establishment of a spontaneous insulin resistant 3T3-LI adipocyte cell model[J].Acta Veterinaria et Zootechnica Sinica,2019,50(7):1475-1485.(in Chinese)
[37]	XU Z P,GAN G S,LIU Y M,et al.Adiponectin attenuates streptozotocin-induced tau hyperpho-sphorylation and cognitive deficits by rescuing PI3K/Akt/GSK-3β pathway[J].Neurochem Res,2018,43(2):316-323.
[38]	HERMIDA M A,KUMAR J D,LESLIE N R.GSK3 and its interactions with the PI3K/AKT/mTOR signalling network[J].Adv Biol Regul,2017, 65:5-15.
[39]	周欣茹.Musclin基因对成肌细胞增殖、葡萄糖摄取和氨基酸分解的影响及其分子机制[D].太谷:山西农业大学,2015.ZHOU X R.Influence of musclin gene on myoblast proliferation,glucose uptake and amino acid catabolism and its mechanism[D].Taigu:Shanxi Agricultural University,2015.(in Chinese)

Construction of Eukaryotic Expression Vector of Sheep Musclin Gene and Its Effects on Glucose Metabolism and Insulin Function in Myocytes

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 39

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	WANG Haibo, ZHAN Jinshun, GU Zhiyong, CHEN Xinfeng, PAN Yue, JIA Haobin, ZHONG Xiaojun, LI Kairong, ZHAO Shengguo, HUO Junhong. Comparative Study on Meat Quality Characteristics of Three-Way Hybrid Sheep Charolais×Duper×Hu and Charolais×Australian White×Hu and Hu Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 110-119.
[2]	DUAN Xiangru, KANG Jia, YANG Ruochen, SHAN Xinyu, LI Taichun, ZHAO Wen, ZHANG Yingjie, LIU Yueqin. Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 179-191.
[3]	ZHANG Hanyue, ZHAO Dan, LIANG Yu, ZHAO Bishi, FAN Mengdan, QIAO Liying, LIU Jianhua, YANG Kaijie, PAN Yangyang, LIU Wenzhong. miR-150 Regulates Ovine Preadipocyte Differentiation by Targeting AOC3 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3262-3274.
[4]	LIU Linli, PENG Xuelan, LI Bo, CHENG Lefan, CIREN Lamu, ZHANG Enping. Effect of Overexpression of UCP3 Gene on the Differentiation of Sahu Sheep Preadipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3275-3285.
[5]	HE Mingyang, MA Yujing, WANG Yong, YANG Ruochen, LIU Yueqin, ZHANG Yingjie, DUAN Chunhui. Effects of Melatonin on Proliferation, Apoptosis of Ovarian Granulosa Cells, and Its Secretion of Steroid Hormones of Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3313-3324.
[6]	SONG Meijun, HAO Kexing, HAI Siyu, CHEN Yan, WANG Jing, HU Guangdong. Effects of SRIF-14 on Proliferation and Apoptosis of Endometrial Epithelial Cells in Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3325-3334.
[7]	LI Yuexin, LIU Aiju, MA Xiaofei, ZHENG Zhong, HU Boxin, ZHI Yunxia, TIAN Shujun. Effect of TGFβR1 on the Function of Sheep Granulosa Cells Mediated by TGF-β/Smad Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3335-3347.
[8]	MA Youji, CHEN Pengfei, MA Qing, WU Yi. Effects of Different Exercise Amounts on Rumen Microflora Diversity of Tan Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3393-3405.
[9]	LIU Xinhuan, YUN Jialei, MAO Li, LI Jizong, HAO Fei, HE Miaofeng, YANG Leilei, ZHANG Wenwen, CHENG Zilong, SUN Min, LIU Maojun, WANG Shaohui, BAI Juan, LI Wenliang. Isolation, Identification, Virulence Genes and Drug Resistance Analysis of Escherichia coli Isolated from Diarrheal Goat and Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3445-3454.
[10]	ZHANG Ying, JIN Hua, HAN Yang, SUI Dan, XIAO Xin, HAO Xiujing, LI Min. Analysis of Microbial Diversity in Nasal Cavity of Sheep with Respiratory Disease and Their Environment [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3455-3465.
[11]	XIONG Chengkun, ZHANG Daoliang, YANG Yue, DING Hongyan, ZHAO Jie, LI Yu, WANG Xichun, FENG Shibin, ZHAO Chang, TANG Jishun, WU Jinjie. Effect of Rutin on Rumen Fermentation, Rumen Flora Structure and Antioxidant Properties in Perinatal Hu Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2898-2909.
[12]	DONG Yajie, HAO Xiaojing, WU Jinqiang, WANG Rong, ZHANG Pengxiang, WANG Haidong, HE Xiaoyan. Exploration of the Effect of SHH on Wool Bending through Krox20 Regulation of IGFBP5 Expression Based on Sheep Keratinocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2365-2375.
[13]	GENG Meimei, DOU Mengying, FU Dezhi, HE Qinghua, KONG Xiangfeng. Effects of Intrauterine Growth Retardation on Developmental Model of Insulin-Like Growth Factor in Suckling Piglets of Huanjiang Mini-Pig [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2414-2420.
[14]	DU Xiaodi, HOU Wei, SU Zhonghua, MA Qingmei, HE Xue, HUA Ruiqi, YANG Aiguo, YANG Guangyou. Bioinformatics and Expression Analysis of Ubiquitin-conjugating Enzyme Gene Family of Echinococcus granulosus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2605-2618.
[15]	ZHAO Donghao, YUAN Meng, MA Kaiteng, DUAN Zhuo, ZHU Yixin, TANG Fang, HAN Keguang, HUO Nairui. Chelating Role of Sheep Bone Collagen Peptide to Cadmium and Its Protection Role against Liver Injuries Induced by Cadmium in Chickens [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2641-2652.