Abstract：Thioredoxin interacting protein (Txnip), a redox regulatory protein, binds to thioredoxin (Trx) and inhibits its activity, regulating the redox state in various physiological processes of cells, but its role in differentiation of porcine fat cells regulated by nutrient is unclear. In this study, the cDNA sequence of Sus scrofa Txnip gene was synthesized and the recombinant lentivirus (LV) overexpressing Txnip (LV5-Txnip) was constructed. The isolated porcine preadipocytes were cultured and transfected with LV5-Txnip and the transfection efficiency was around 90%. The Txnip mRNA level was raised by about 80 folds. The porcine preadipocytes transfected with LV5-Txnip were induced by adipogenic differentiation medium and treated with 5 or 15 mmol/L concentration of glucose, respectively. The differentiation and mRNA expressions of adipogenic-related genes for the cells were detected every other day using oil red O staining extraction assay and qRT-PCR, respectively. The results showed that differentiation of the cells transfected with LV5-Txnip was significantly inhibited (P<0.05), compared to the cells transfected with negative control virus and to the non-transfected cells, the expression level of peroxisome proliferator-activated receptor γ (PPARγ) and fatty acid synthase (FAS) was also reduced significantly (P<0.05). The differentiation and mRNA expression of PPARγ, carbohydrate response element binding protein (ChREBP) and glucose transporter 4 (Glut4) of control preadipocytes were induced significantly by high concentration of glucose, but this promotion was reduced by Txnip-overexpression (P<0.05). Txnip-overexpression suppressed differentiation of porcine preadipocytes through down-regulating PPARγ expression, which indicating Txnip could be an inhibitor of porcine adipocyte differentiation. Txnip impaired the promotion of glucose on the cell differentiation via decreased Glut4 expression leading to lower glucose transport and expression of ChREBP and PPARγ. The present study provides a reference for the investigation of the adipogenesis regulated by nutrient.
[1] 邓文珍,李杨,贾彦军,等.2017.过表达硫氧还蛋白相互作用蛋白(TXNIP)通过激活p38MAPK通路促进MIN6细胞凋亡[J].细胞与分子免疫学杂志,33(10):1323-1327.
(Deng W Z,Li Y,Jia Y J,et al.2017.Over-expression of thioredoxin-interacting protein promotes apoptosis of MIN6 cells via activating p38MAPK pathway[J].Chinese Journal of Cellular and Molecular Immunology,33(10):1323-1327.)
[2] 张国华,卢建雄,杨具田,等.2014.葡萄糖和含腺苷分子通过碳水化合物反应元件结合蛋白(ChREBP)诱导猪脂肪细胞硫氧还蛋白互作蛋白(Txnip)的表达[J].农业生物技术学报,22(03):280-288.
(Zhang G H,Lu J X,Yang J T,et al.2014.Glucose and adenosine-containing molecule stimulate expression of thioredoxin interacting protein (Txnip) mRNA through carbohydrate response element binding protein (ChREBP) in primary cultured porcine adipocytes[J].Journal of Agricultural Biotechnology,22(3):280-288.)
[3] 岳小婧,于淇,张晶,等.2019.Txnip基因siRNA慢病毒表达质粒构建及促进猪前体脂肪细胞分化[J].中国生物化学与分子生物学报,35(4):457-464.
(Yue X J,Yu Q,Zhang J,et al.2019.siRNA of Txnip gene constructed in lentiviral plasmids promotes porcine preadipocytes differentiation[J].Chinese Journal of Biochemistry and Molecular Biology,35(4):457-464.)
[4] Aprile M,Cataldi S,Ambrosio M R,et al.2018.PPARgammaDelta5,a naturally occurring dominant-negative splice isoform,impairs ppargamma function and adipocyte differentiation[J].Cell Reports,25(6):1577-1592 e1576.
[5] Bharti V,Tan H,Chow D,et al.2018.Glucocorticoid upregulates thioredoxin-interacting protein in cultured neuronal cells[J].Neuroscience,384:375-383.
[6] Boztepe T,Gulec S.2018.Investigation of the influence of high glucose on molecular and genetic responses:An in vitro study using a human intestine model[J].Genes Nutrition,13:11.
[7] Chutkow W A,Birkenfeld A L,Brown J D,et al.2010.Deletion of the alpha-arrestin protein Txnip in mice promotes adiposity and adipogenesis while preserving insulin sensitivity[J].Diabetes,59(6):1424-1434.
[8] Chutkow W A,Lee R T.2011.Thioredoxin regulates adipogenesis through thioredoxin-interacting protein (Txnip) protein stability[J].Journal of Biological Chemistry,286(33):29139-29145.
[9] Ehebauer F,Ghavampour S,Kraus D.2020.Glucose availability regulates nicotinamide N-methyltransferase expression in adipocytes[J].Science,248:117474.
[10] Gasiorek J J,Mikhael M,Garcia-Santos D,et al.2015.Thioredoxin-interacting protein regulates the differentiation of murine erythroid precursors[J].Experimental Hematology,43(5):393-403 e392.
[11] Hwang J,Suh H W,Jeon Y H,et al.2014.The structural basis for the negative regulation of thioredoxin by thioredoxin-interacting protein[J].Nature Communications,5:10-23.
[12] Huy H,Song H Y,Kim M J,et al.2018.TXNIP regulates AKT-mediated cellular senescence by direct interaction under glucose-mediated metabolic stress[J].Aging Cell,17(6):e12836.
[13] Ji S,Doumit M E,Hill R A.2015.Regulation of adipogenesis and key adipogenic gene expression by 1,25-dihydroxyvitamin D in 3T3-L1 cells[J].PLOS ONE,10(6):e0126142.
[14] Jung H,Kim D O,Byun J E,et al.2016.Thioredoxin-interacting protein regulates haematopoietic stem cell ageing and rejuvenation by inhibiting p38 kinase activity[J].Nature Communications,7:13674.
[15] Jung Y H,Bu S Y.2020.Suppression of long chain acyl-CoA synthetase blocks intracellular fatty acid flux and glucose uptake in skeletal myotubes[J].Biochimica Biophysica Acta-molecular and Cell Biology Lipids,1865(7):158678.
[16] Kim S Y,Suh H W,Chung J W,et al.2007.Diverse functions of VDUP1 in cell proliferation,differentiation,and diseases[J].Cellular & Molecular Immunology,4(5):345-351.
[17] Kim D O,Byun J E,Seong H A,et al.2018.Thioredoxin-interacting protein-derived peptide (TN13) inhibits LPS-induced inflammation by inhibiting p38 MAPK signaling[J].Biochemical and Biophysical Research Communications,507(1-4):489-495.
[18] Kwon E B,Kang M J,Ryu H W,et al.2020.Acacetin enhances glucose uptake through insulin-independent GLUT4 translocation in L6 myotubes[J].Phytomedicine,68:153178.
[19] Lowe C E,O'Rahilly S,Rochford J J.2011.Adipogenesis at a glance[J].Journal of Cell Science,124(Pt16):2681-2686.
[20] Lee J E,Ge K.2014.Transcriptional and epigenetic regulation of PPARgamma expression during adipogenesis[J].Cell and Bioscience,4:29.
[21] Lefterova M I,Haakonsson A K,Lazar M A,et al.2014.PPARgamma and the global map of adipogenesis and beyond[J].Trends in Endocrinology and Metabolism,25(6):293-302.
[22] Lu J,Holmgren A.2014.The thioredoxin antioxidant system[J].Free Radical Biology and Medicine,66:75-87.
[23] Mortuza R,Chen S,Feng B,et al.2013.High glucose induced alteration of SIRTs in endothelial cells causes rapid aging in a p300 and FOXO regulated pathway[J].PLOS ONE,8(1):e54514.
[24] Morrison J A,Pike L A,Same S B,et al.2014.Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer[J].Molecular Cancer,13:62.
[25] Mueller E.2014.Understanding the variegation of fat:Novel regulators of adipocyte differentiation and fat tissue biology[J].Biochimica et Biophysica Acta,1842(3):352-357.
[26] Panse M,Kluth O,Lorza-Gil E,et al.2018.Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms[J].PLOS ONE,13(5):e0198016.
[27] Park J W,Lee S H,Woo G H,et al.2018.Downregulation of TXNIP leads to high proliferative activity and estrogen-dependent cell growth in breast cancer[J].Biochemical and Biophysical Research Communications,498(3):566-572.
[28] Patwari P,Higgins L J,Chutkow W A,et al.2006.The interaction of thioredoxin with Txnip.Evidence for formation of a mixed disulfide by disulfide exchange[J].Journal of Biological Chemistry,281(31):21884-21891.
[29] Poulsen L,Siersbaek M,Mandrup S.2012.PPARs:Fatty acid sensors controlling metabolism[J].Seminars in Cell Development Biology,23(6):631-639.
[30] Parikh H,Carlsson E,Chutkow W A,et al.2007.TXNIP regulates peripheral glucose metabolism in humans[J].PLoS Medicine,4(5):e158.
[31] Ramirez-Zacarias J L,Castro-Munozledo F,Kuri-Harcuch W.1992.Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with oil red O[J].Histochemistry,97(6):493-497.
[32] Robinson K A,Brock J W,Buse M G.2013.Posttranslational regulation of thioredoxin-interacting protein[J].Journal of Molecular Endocrinology,50(1):59-71.
[33] Rajalin A M,Micoogullari M,Sies H,et al.2014.Upregulation of the thioredoxin-dependent redox system during differentiation of 3T3-L1 cells to adipocytes[J].Biological Chemistry,395(6):667-677.
[34] Ratneswaran A,Sun M M,Dupuis H,et al.2017.Nuclear receptors regulate lipid metabolism and oxidative stress markers in chondrocytes[J].Journal of Molecular Medicine (Berlin),95(4):431-444.
[35] Schulze P C,De Keulenaer G W,Yoshioka J,et al.2002.Vitamin D3-upregulated protein-1 (VDUP-1) regulates redox-dependent vascular smooth muscle cell proliferation through interaction with thioredoxin[J].Circulation Research,91(8):689-695.
[36] Sugii S,Olson P,Sears D D,et al.2009.PPARgamma activation in adipocytes is sufficient for systemic insulin sensitization[J].Proceedings of the National Academy of Sciences of USA,106(52):22504-22509.
[37] Siersbaek R,Nielsen R,Mandrup S.2010.PPARgamma in adipocyte differentiation and metabolism-novel insights from genome-wide studies[J].FEBS Letters,584(15):3242-3249.
[38] Schuster S,Johnson C D,Hennebelle M,et al.2018.Oxidized linoleic acid metabolites induce liver mitochondrial dysfunction,apoptosis,and NLRP3 activation in mice[J].Journal of Lipid Research,59(9):1597-1609.
[39] Stachecka J,Nowacka-Woszuk J,Kolodziejski P A,et al.2019.The importance of the nuclear positioning of the PPARG gene for its expression during porcine in vitro adipogenesis[J].Chromosome Research,27:271-284.
[40] Wu N,Zheng B,Shaywitz A,et al.2013.AMPK-dependent degradation of TXNIP upon energy stress leads to enhanced glucose uptake via GLUT1[J].Molecular Cell,49(6):1167-1175.
[41] Witte N,Muenzner M,Rietscher J,et al.2015.The glucose sensor ChREBP links de novo lipogenesis to ppargamma activity and adipocyte differentiation[J].Endocrinology,156(11):4008-4019.
[42] Xu X,So J S,Park J G,et al.2013.Transcriptional control of hepatic lipid metabolism by SREBP and ChREBP[J].Seminars in Liver Disease,33(4):301-311.
[43] Yoshioka J,Schulze P C,Cupesi M,et al.2004.Thioredoxin-interacting protein controls cardiac hypertrophy through regulation of thioredoxin activity[J].Circulation,109(21):2581-2586.
[44] Yoshihara E,Masaki S,Matsuo Y,et al.2014.Thioredoxin/Txnip:Redoxisome,as a redox switch for the pathogenesis of diseases[J].Frontiers in Immunology,4:514.
[45] Zhang G H,Lu J X,Chen Y,et al.2014.Comparison of the adipogenesis in intramuscular and subcutaneous adipocytes from Bamei and Landrace pigs[J].Biochemistry and Cell Biology,92(4):259-267.
[46] Zhang G H,Lu J X,Chen Y,et al.2016.Glucose and insulin stimulate lipogenesis in porcine adipocytes:Dissimilar and identical regulation pathway for key transcription factors[J].Molecules and Cells,39(11):797-806.
