Influence of Interfering CAT Gene by siRNA on Differentiation of Porcine (Sus scrofa) Subcutaneous Preadipocytes
SHAN Bao-Sen1, 2, LIU Xin1, LUO Wu1, SHAO Yong-Gang3, WEI Wei1, CHEN Jie1, ZHANG Li-Fan1, 2, *
1 College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China; 2 National Experimental Teaching Demonstration Center of Animal Science, Nanjing Agricultural University, Nanjing 210095, China; 3 College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China
Abstract As one of the major factors for affecting the growth and meat quality of pigs (Sus scrofa), subcutaneous fat deposition has always been a hot issue in pig production. Catalase (CAT) is a key antioxidant enzyme in the oxidative metabolism process and plays vital roles in the fat deposition of animals. To investigate the role of CAT gene in the porcine subcutaneous preadipocytes differentiation, the expression patterns of CAT during the preadipocyte differentiation process and CAT mRNA levels in various tissues of Erhualian pigs were detected using qRT-PCR. Moreover, CAT gene was knockdowned by small interfering RNA (siRNA) fragments, and then, the ability of subcutaneous preadipocytes differentiation was assessed via oil red O staining. The expression levels of important candidates in siCAT treated adipocytes, including the key adipocyte differentiation-related genes such as peroxisome proliferator activated receptor γ gene (PPARγ), CCAAT enhancer binding protein α gene (C/EBPα), fatty acid binding protein 4 gene (FABP4), apolipoprotein E gene (ApoE), adiponectin, C1Q and collagen domain containing gene (ADIPOQ), perilipin 1 gene (PLIN1), and hormone-sensitive lipase gene (HSL), and the antioxidant-related genes such as superoxide dismutase 2 gene (SOD2), glutathione synthetase gene (GSS), and peroxidase 3 gene (PRDX3) were detected by qRT-PCR. The results indicated that CAT gene was mainly expressed in subcutaneous adipose, kidney, and liver tissues of pigs and its expression levels in subcutaneous adipose tissues were significantly higher than that in intramuscular adipose tissues (P<0.01). During the differentiation process, the expression levels of CAT gene increased significantly at different differentiation stages, with the highest expression levels on the 4th day of the preadipocytes differentiation. Silencing of CAT increased the contents of hydrogen peroxide (H2O2) (P<0.01) and also decreased the triglyceride levels of subcutaneous preadipocytes (P<0.05). Furthermore, interfering CAT expression reduced the expression levels of ADIPOQ (P<0.05) and PLIN1 (P<0.01) and simultaneously increased the levels of HSL gene (P<0.05), with non-significant effect on the expression levels of PPARγ, C/EBPα, FABP4, ApoE, SOD2, GSS, and PRDX3 genes (P>0.05). Taken together, this study demonstrated CAT gene knockdown could inhibit the differentiation and lipid deposition of subcutaneous preadipocytes of Erhualian pigs, which might result from downregulating lipogenesis-related genes of ADIPOQ and PLIN1 and simultaneously upregulating the expressions of lipolysis-related HSL gene. These results could provide new evidence and ideas for further revealing the mechanism of CAT gene on adipogenesis and reducing the subcutaneous fat deposition of pigs.
SHAN Bao-Sen,LIU Xin,LUO Wu, et al. Influence of Interfering CAT Gene by siRNA on Differentiation of Porcine (Sus scrofa) Subcutaneous Preadipocytes[J]. 农业生物技术学报, 2019, 27(4): 666-676.
1 刘荣鑫. 2010. 氧化应激以不依赖Wnt/β-catenin通路的方式抑制猪前体脂肪细胞成脂分化[D]. 硕士学位论文, 西北农林科技大学, 导师: 杨公社, pp. 20-26. (Liu R X.2010. Oxidative stress inhibited porcine preadipocytes differentiation is independent of Wnt/β-catenin signaling[D]. Thesis for M.S., Northwest A&F University, Supervisor: Yang G S, pp. 20-26.) 2 屈长青, 张国华, 陈粉粉, 等. 2005. 猪前体脂肪细胞的原代培养[J]. 农业生物技术学报, 13(5): 104-108. (Qu C Q, Zhang G H, Chen F F, et al.2005. Primary culture of porcine preadipocyte[J]. Journal of Agricultural Biotechnology, 13(5): 104-108.) 3 孙成娟, 许厚强, 赵佳福, 等. 2017. 从江香猪肌内和皮下脂肪前体细胞分化过程中相关基因的表达[J]. 农业生物技术学报, 25(12): 1979-1988. (Sun C J, Xu H Q, Zhao J F, et al.2017. The expression of related genes on intramuscular and subcutaneous preadipocytes during differentiation in Congjiang Xiang pig (Sus scrofa)[J]. Journal of Agricultural Biotechnology, 25(12): 1979-1988.) 4 张秀娟, 王丽静, 刘小莺, 等. 2012. 氧化应激对3T3-L1脂肪细胞MCP-1、PAI-1、脂联素表达的影响[J]. 福建医科大学学报, 46(1): 1-6. (Zhang X J, Wang L J, Liu X Y, et al.2012. Effect of oxidative stress on the expression of MCP-1, PAI-1, adiponetin in 3T3-L1 adipocytes[J]. Journal of Fujian Medical University, 46(1): 1-6.) 5 赵志武. 2016. PLIN1基因沉默对3T3-L1脂肪细胞脂解的影响及机制探究[D]. 硕士学位论文, 山西医科大学, 导师: 燕炯, pp. 24-30. (Zhao Z W.2016. Effect and mechanism of PLIN1 gene silencing on lipolysis of 3T3-L1 adipocytes[D]. Thesis for M.S., Shanxi Medical University, Supervisor: Yan J, pp. 24-30.) 6 Amos D L, Robinson T, Massie M B, et al.2017. Catalase overexpression modulates metabolic parameters in a new 'stress-less' leptin-deficient mouse model[J]. Biochimica et Biophysica Acta-Molecular Basis of Disease, 1863(9): 2293-2306. 7 Bjørndal B, Burri L, Staalesen V, et al.2011. Different adipose depots: Their role in the development of metabolic syndrome and mitochondrial response to hypolipidemic agents[J]. Journal of Obesity, 2011: 490650. 8 Bouwman F G, Wang P, van Baak M, et al.2014. Increased beta-oxidation with improved glucose uptake capacity in adipose tissue from obese after weight loss and maintenance[J]. Obesity (Silver Spring), 22(3): 819-827. 9 Drehmer D L, de Aguiar A M, Brandt A P, et al.2016. Metabolic switches during the first steps of adipogenic stem cells differentiation[J]. Stem Cell Research, 17(2): 413-421. 10 Findeisen H M, Pearson K J, Gizard F, et al.2011. Oxidative stress accumulates in adipose tissue during aging and inhibits adipogenesis[J]. PLoS One, 6(4): e18532. 11 Furukawa S, Fujita T, Shimabukuro M, et al.2004. Increased oxidative stress in obesity and its impact on metabolic syndrome[J]. The Journal of Clinical Investigation, 114(12): 1752-1761. 12 Gao Y, Li F, Zhang Y, et al.2013. Silencing of ADIPOQ efficiently suppresses preadipocyte differentiation in porcine[J]. Cellular Physiology and Biochemistry, 31(2-3): 452-461. 13 Gondret F, Guitton N, Guillerm-Regost C, et al.2008. Regional differences in porcine adipocytes isolated from skeletal muscle and adipose tissues as identified by a proteomic approach[J]. Journal of Animal Science, 86(9): 2115-2125. 14 Heit C, Marshall S, Singh S, et al.2017. Catalase deletion promotes prediabetic phenotype in mice[J]. Free Radical Biology & Medicine, 103: 48-56. 15 Hernández-Guerrero C, Hernández-Chávez P, Romo-Palafox I, et al.2016. Genetic polymorphisms in SOD (rs2070424, rs7880) and CAT (rs7943316, rs1001179) enzymes are associated with increased body fat percentage and visceral fat in an obese population from central Mexico[J]. Archives of Medical Research, 47(5): 331-339. 16 Higuchi M, Dusting G J, Peshavariya H, et al.2013. Differentiation of human adipose-derived stem cells into fat involves reactive oxygen species and Forkhead box O1 mediated upregulation of antioxidant enzymes[J]. Stem Cells and Development, 22(6): 878-888. 17 Kraemer F B, Shen W J.2006. Hormone-sensitive lipase knockouts[J]. Nutrition & Metabolism, 3: 12. 18 Lee H, Lee Y J, Choi H, et al.2009. Reactive oxygen species facilitate adipocyte differentiation by accelerating mitotic clonal expansion[J]. The Journal of Biological Chemistry, 284(16): 10601-10609. 19 Loew O.1900. A new enzyme of general occurrence in organismis[J]. Science, 11(279): 701-702. 20 Lubos E, Loscalzo J, Handy D E.2011. Glutathione peroxidase-1 in health and disease: From molecular mechanisms to therapeutic opportunities[J]. Antioxidants & Redox Signaling, 15(7): 1957-1997. 21 Luo X, Li H X, Liu R X, et al.2009. Beta-catenin protein utilized by tumour necrosis factor-alpha in porcine preadipocytes to suppress differentiation[J]. BMB Reports, 42(6): 338-343. 22 Maurizi G, Petäistö T, Maurizi A, et al.2018. Key-genes regulating the liposecretion process of mature adipocytes[J]. Journal of Cellular Physiology, 233(5): 3784-3793. 23 Muraoka S, Nitta Y, Yamada T, et al.2017. Increase of anti-oxidative capacity during differentiation of 3T3-L1 preadipocytes into adipocytes[J]. Yakugaku Zasshi, 137(9): 1137-1145. 24 Okuno Y, Matsuda M, Kobayashi H, et al.2008. Adipose expression of catalase is regulated via a novel remote PPAR gamma-responsive region[J]. Biochemical and Biophysical Research Communications, 366(3): 698-704. 25 Park Y S, Uddin M J, Piao L, et al.2016. Novel role of endogenous catalase in macrophage polarization in adipose tissue[J]. Mediators of Inflammation, 2016: 8675905. 26 Ruiz-Ojeda F J, Gomez-Llorente C, Aguilera C M, et al.2016. Impact of 3-amino-1,2,4-triazole (3-AT)-derived increase in hydrogen peroxide levels on inflammation and metabolism in human differentiated adipocytes[J]. PLoS One, 11(3): e0152550. 27 Takahashi M, Arita Y, Yamagata K, et al.2000. Genomic structure and mutations in adipose-specific gene, adiponectin[J]. International Journal of Obesity and Related Metabolic Disorders, 24(7): 861-868. 28 Zhang J, Ma J, Long K, et al.2016. Dynamic gene expression profiles during postnatal development of porcine subcutaneous adipose[J]. PeerJ, 4: e1768.
