Abstract：Myostatin (MSTN) is a member of the transforming growth factor β (TGF-β) superfamily, which plays a key role in muscle development. Numerous studies have shown that knockout of MSTN results in a widespread increase in skeletal muscle mass, and a significant reduction in fat accumulation. However, the mechanisms involved in fat deposition process yielded by MSTN gene suppression has not been understood. In the present study, we constructed 5 two-way expression antisense RNA vectors pMSTN-CMV-CAG-antiMSTN, then transfected bovine fetal fibroblast cells, and screened the best antisense RNA vector which significantly disturbed the MSTN expression in protein level with Western blot. The vectors were then confected to a bovine muscle satellite cells and the effect of MSTN knockdown to genes associated with adipogenesis such as fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase-1 (SCD-1), genes associated with lipolysis such as carnitine palmitoyltransferase 1b (CPT-1B), acyl-CoA oxidase(ACOX1) and enoyl-CoA hydratase 1 (ECHDC1), and fat transport protein (FATP) gene were investigated. The results showed that genes associated with adipogenesis and lipolysis were all up-regulated except the expression of ECHDC1 when MSTN knockdown in muscle satellite cells. The expression of gene associated with fat transport has no significant change. The results suggested that MSTN knockdown could enhance both adipogenesis and lipolysis, so the reduced fat mass resulted from MSTN knockdown was due to the increased lipolysis rather than inhibition of adipogenesis. And the expression of CPT-1B was up-regulated in the highest level among the genes investigated, this result suggested that the effect of MSTN to fat was mainly by the process of β oxidation, because CPT-1B was a main factor of β oxidation. This study provides a theoretical basis for the decreased fat content resulted from MSTN reduction, and makes a preliminary study for the effect of MSTN on fat deposition.
[1]McPherron A C, Lawler A M, Lee S J.1997. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member[J]. Nature, 387(6628):83-90.
[2]Harper G S, Pethick D W.2004. How might marbling begin? Australian Journal of Experimental Agriculture, 44(7):653-662 .
[3]Lee S H, Park E W, Cho Y M, et al.2007. Identification of differentially expressed genes related to intramuscular fat development in the early and late fattening stages of hanwoo steers. Journal of Biochemistry and Molecular Biology, 40(5): 757-764 .
[4]Goodpaster B H, Krishnaswami S, Resnick H, et al.2003. Association between regional adipose tissue distribution and both type 2 diabetes and impaired glucose tolerance in elderly men and women. Diabetes Care, 26(2):372-379.
[5]Albu J B, Kovera A J, Allen L, et al.2005. Independent association of insulin resistance with larger amounts of intermuscular adipose tissue and a greater acute insulin response to glucose in African American than in white nondiabetic women. American Journal of Clinical Nutrition, 82(6):1210-1217.
[6]Pethick D W, Harper G S, Oddy V H.2004. Growth, development and nutritional manipulation of marbling in cattle: a review. Australian Journal of Experimental Agriculture, 44(7):705-715 .
[7]Deng B, Wen J H, Ding Y, et al.2012. Different regulation role of myostatin in differentiating pig ADSCs and MSCs into adipocytes. Cell Biochemistry and Function, 30(2):145-150.
[8]Tomizawa J, Itoh T, Selzer G, et al.1981.Inhibition of ColE1 RNA primer formation by a plasmid-specified small RNA. Proceedings of the National Academy of Sciences of the United States of America, 78(3):1421-1425.
[9]赵云杰, 魏著英, 李欣欣, 等.2016.牛肌肉卫星细胞向胰岛素分泌细胞的诱导转分化研究. 农业生物技术学报，24(2): 224-232.
[10]Menendez J A, Vazquez-Martin F J A, Fernandez-Real J M, et al.2009. Fatty acid synthase: association with insulin resistance, type 2 diabetes, and cancer. Clinical Chemistry, 55(3):425-438.
[11]Jenni S, Leibundgut M, Boehringer D, et al.2007. Structure of fungal fatty acid synthase and implications for iterative substrate shuttling. Science, 316(5822):254-261.
[12]Chirala S S, HuangW Y, Jayakumar A, et al.1994. Animal fatty acid synthase: Functional mapping and cloning and expression of the domain I constituent activities. Proceedings of the National Academy of Sciences, 94(11):5588-5593.
[13]Livore V I, Tripodi K E J, Uttaro A D.2007. Elongation of polyunsaturated fatty acids in trypanosomatids. FEBS Journal, 274(1):264-274.
[14]Dobrzyn A, Dobrzyn P.2006. Stearoyl-CoA desaturase, a new player in skeletal muscle metabolism regulation [J]. Journal of Physiology and Pharmacology, 57(10): 31-42.
[15]Mololib, Contarini G, Avalli A, et al.2007. Short communication: Effect of stearoyl coenzyme A desaturase polymorphism on fatty acid composition of milk [J]. Journal of Dairy Science, 90(7): 3553-3558.
[16]王镜岩，朱圣庚，徐长法.2002. 生物化学[M]. 北京：高等教育出版社.
[17]Yamazaki N, Yamanaka Y, Hashimoto Y, et al.1997. Structural features of the gene encoding human muscle type carnitine palmitoyltransferase. FEBS Letters, 409(3): 401-406.
[18]Fournier B, Saudubray J M, Benichou B, et al.1994. Large deletion of the peroxisomal acyl-CoA oxidase gene in pseudoneonatal adrenoleukodystrophy [J]. Journal of Clinical Investigation, 94(2): 526-531.
[19]Varanasi U, Chu R, Chu S, et al.1994. Isolation of the human peroxisomal acyl-CoA oxidase gene: organization, promoter analysis, and chromosomal localization [J]. Proceedings of the National Academy of Sciences, 91(8): 3107-3111.
[20]Li Y, Tharappel J C, Cooper S, et al.2000. Expression of the hydrogen peroxide-generating enzyme fatty acyl CoA oxidase activates NF-kappa-B [J]. DNA Cell Biology, 19(2): 113-120.
[21]Sofia M, Anja K G, Michèle A, et al.2007.Conserved expression of alternative splicing variants of peroxisomal acyl-CoA oxidase 1 in vertebrates and developmental and nutritional regulation in fish [J]. Physiological Genomics, 28(3): 239-252.
[22]Zhang C, McFarlane C, Lokireddy S, et al.2012. Inhibition of myostatin protects against diet-induced obesity by enhancing fatty acid oxidation and promoting a brown adipose phenotype in mice. Diabetologia, 55(1):183-193.
[23]Anderson C M, Stahl A.2013. SLC27 fatty acid transport proteins. Molecular Aspects of Medicine, 34(2-3):516-528.
[24]Kazantzis M, Stahl A.2012[J].Fatty acid transport proteins, implications in physiology and disease. Biochimica et Biophysica Acta, 1821, (5):852-857
[25]Yuan H, Niu Y, Liu X, et al.2014. Exercise increases the binding of MEF2A to the Cpt1b promoter in mouse skeletal muscle. Acta Physiologica, 212(4):283-292.
[26]Li N, Yang Q, Walker R G, et al.2016. Myostatin attenuation in vivo reduces adiposity, but activates adipogenesis. Endocrinology, 157(1):282-291.
