摘要
牛肉作为一种高蛋白、低胆固醇的肉类食品倍受人们青睐,其嫩度和风味可随着肌内脂肪含量的增加而得到改善。而动物脂肪的形成与沉积是受多种转录因子调控的复杂生理过程,与脂肪细胞的增殖分化密切相关。CCAAT增强子结合蛋白家族(CCAAT/enhancer-binding protein family, C/EBPs)是一类非常重要的转录因子,在脂肪细胞的增殖分化过程中起关键调控作用。目前,关于C/EBPs对牛成纤维细胞成脂转分化的影响及其调控机理仍不清楚。为了探索牛C/EBPs基因的功能,本研究以C/EBPs家族中与脂肪细胞增殖分化有着紧密联系的3个成员——C/EBPα、C/EBPβ和C/EBP8为目标基因,以牛成纤维细胞为实验对象,以腺病毒介导的基因过量表达技术为手段,直接发掘3个目标基因诱导牛成纤维细胞转分化为脂肪细胞的潜在能力。同时,在转录水平上初步探索目标基因之间的相互调控作用,及其对一些重要的脂肪细胞分化相关基因的影响。
主要研究成果如下：
(1)成功克隆得到秦川牛C/EBPα、C/EBPβ和C/EBPδ基因的CDS区序列,其长度依次为1062bp、1047bp和771bp,分别编码353、348和256个氨基酸残基。序列信息分析结果显示,牛C/EBPs与人相应基因的氨基酸序列同源性分别达95%、97%和87%；牛C/EBPα和C/EBPβ分别在第233-252位,第190-208位氨基酸区域存在由外向内的显著跨膜结构,而C/EBPδ则在第125-141位氨基酸区域存在反方向的跨膜结构；牛C/EBPα、C/EBPβ和C/EBPδ都含一个有完全由α-螺旋构成的亮氨酸拉链,符合C/EBPs家族的一般结构特征。
(2)构建了牛C/EBPα、C/EBPβ和C/EBPδ基因的腺病毒过表达载体,并包装获得了高滴度重组腺病毒,建立了适合于牛成纤维细胞的腺病毒过表达载体系统。
(3)成功诱导了牛成纤维细胞向脂肪细胞的转分化。油红O染色结果显示,分别单一过量表达牛C/EBPα、C/EBPβ或C/EBPδ基因,可诱导成纤维细胞转分化为脂肪细胞或脂肪样细胞。同时过量表达2个或3个目标基因,也可诱导成纤维细胞转分化为脂肪细胞,且细胞的表型变化比单一表达时更明显。
(4) Real-time PCR分析结果显示,过量表达C/EBPα或C/EBPβ基因可互相激活对方的转录表达,而都对C/EBPδ基因的表达无明显影响；过量表达C/EBPδ基因可在诱导细胞分化早期上调C/EBPα基因的mRNA表达量,而对C/EBPβ基因的表达无明显影响。同时过量表达C/EBPβ和C/EBPδ基因可在诱导细胞分化中期上调C/EBPβ基因的表达量。
(5) Real-time PCR分析结果显示,过量表达C/EBPα或C/EBPβ基因可在诱导细胞分化后期,上调过氧化物酶体增殖激活受体-gamma (PPARγ)基因的mRNA表达量,而过量表达C/EBPδ基因对PPARγ基因的表达无明显影响；同时过量表达C/EBPβ和C/EBPα、C/EBPβ和C/EBPδ基因,也可在细胞分化后期上调PPARγ基因的表达量。单一或同时过量表达目标基因均对固醇调节元件结合蛋白-1(SREBP1)基因的表达无明显影响。
(6) Real-time PCR分析结果显示,过量表达C/EBPα或C/EBPβ基因可上调脂肪细胞分化相关基因的mRNA表达量,如脂蛋白脂酶(LPL)、乙酰辅酶A羧化酶-α(ACCα)、脂肪酸合成酶(FAS)、硬脂酰辅酶A去饱和酶(SCD)、脂肪酸转运蛋白-1(FATP1)和补体D (CFD)基因；而过量表达C/EBP8基因则仅可上调LPL、CFD和脂肪酸结合蛋白-4(FABP4)基因的表达量。同时过量表达2个或3个目标基因,均可上调除FATP1和FABP4以外的其他上述基因的表达量。
(7)FABP4基因的mRNA表达量仅在单一过量表达C/EBPδ基因的细胞组中显著上升,而在过量表达C/EBPβ基因的细胞组中还有一定程度的下降,表明过量表达牛C/EBPβ或C/EBPδ基因,对FABP4基因在转录水平上的调控作用可能有所差异。
综上所述,本研究利用腺病毒过表达技术介导牛C/EBPα、C/EBPβ和C/EBPδ基因过量表达,首次成功诱导牛成纤维细胞转分化为脂肪细胞或脂肪样细胞。同时,在转录水平上初步探索了这3个目标基因之间的相互作用关系,及其对一些重要的脂肪细胞分化相关基因的调控作用,为进一步研究牛体内脂肪的形成与沉积提供一定的理论依据,为下一步培育转基因牛的相关科研工作奠定基础。
Beef is considered as one of the healthy foods for human because of its abundant nutrient. Its flavor and tenderness can be improved by inducing intramuscular fat composition. Previous studies in vitro and in vivo on various kinds of animals elucidated that adipocytes proliferation and differentiation at cellular level were regulated by some important transcription factors. CCAAT-enhancer-binding protein family (C/EBPs) as one of these transcription factor families played pivotal role in the control of fat formation as well as fatty acids metabolism. However, there is little information concerning the function of bovine C/EBPα, C/EBPβ and C/EBPδ genes. Herein, we detected the potential specific effects of the three genes and their possible interactions during bovine fibroblaststrans-differentiation process. Furthermore, we also investigated their effects on several other fat cell differentiation related genes. The main results were as follows:
(1) The complete CDS sequences of bovine C/EBPα, C/EBPβ and C/EBPδ genes were successfully obtained. The CDS areas of bovine C/EBPα, C/EBPβ and C/EBPδ genes were1062bp,1047bp and771bp in length, coding353,348,256amino acid residues respectively. The putative amino acid sequences showed bovine C/EBPα, C/EBPβ and C/EBPδ shared higher similarities of95%,97%and87%with their homologue respectively. Bovine C/EBPα and C/EBPβ occupied one outside to inside transmembrane domain, locating within amino acid from233to252and from190to208respectively, while C/EBPδ occupied one inside to outside transmembrane domain, locating within amino acid from125to141. Besides, all the three putative proteins had one Basic leucine zipper domain (bZIP) consisted of a helixes, consistent with the predominant characteristic of C/EBPs.
(2) Recombinant adenovirus carrying bovine C/EBPα, C/EBPβ and C/EBPδ complete CDS fragments were successfully constructed, and further packaged and amplified in HEK293A cell lines.
(3) Oil O staining results demonstrated that over-expression of C/EBPα, C/EBPβ or C/EBPδ converted bovine fibroblasts into adipocytes or adipocytes-like cells. Over-expression of the combination of the two or the three isoforms converted fibroblasts into fat-laden adipocytes.
(4) Real-time PCR results indicated over-expression of C/EBPα or C/EBPβ enhanced the mRNA expression of each other during bovine fibroblasts trans-differentiation, while none of them affected C/EBPδ mRNA expression. Meanwhile, simultaneous up-regulation of C/EBPβ and C/EBP5enhanced C/EBPa mRNA expression at some level.
(5) Real-time PCR results indicated over-expression of C/EBPα and/or C/EBPβ upregulated PPARy mRNA expression level in the late phase of cell differentiation, while C/EBP8did not. All the three genes had no significant influence on SREBP1gene.
(6) Real-time PCR results indicated over-expression C/EBPα or C/EBPβ upregulated the mRNA expression level of genes related to adipocytes such as Lipoprotein lipase (LPL), Acetyl-CoA carboxylase alpha (ACC-a), Fatty acid synthase (FAS), Stearoyl-CoA desaturase (SCD), Fatty acid transporter member1(FATP1) and Complement Factor D (Adipsin/CFD) genes. Over-expression of C/EBPδ increased the mRNA expression level of LPL, CFD and Fatty acid binding protein4(FABP4) genes. Combination of C/EBPα, C/EBPβ and C/EBPδ over-expression enhanced the expression of the above mentioned genes except FABP4and FATP1.
(7) The mRNA level of FABP4gene significantly increased only in cells in which C/EBP8was upregulated, on the contrary, it decreased when C/EBPβ was upregulated to some extent, implying the two C/EBP isoforms may have dissimilar effects on FABP4gene at cellular level in bovine.
In conclusion, we successfully converted bovine fibroblasts into adipocytes or adipocytes-like cells by over-expressing bovine transcription factors namely C/EBPα, C/EBPβ and C/EBPδ, either individually or by combination. Also a simple sketch map between the three transcription factors interaction, and their effects on other related genes was obtained based on the present results.
引文
陈盼盼,张丽华,董为人,刘杰明,张易,乔伟光,陈英华,赵姝,郭家松.2011.脂肪组织源性干细胞分步诱导分化为神经元样细胞.南方医科大学学报,31(3)：512-517
杜纪坤,黄青阳.2007.脂蛋白脂酶基因的研究进展.遗传,29(1)：8-16
高运臻,潘玉春.2011.转录因子CCAAT增强子结合蛋白β (C/EBPβ)的研究进展.遗传.33(3)：198-206
韩冬,李建军,孙鸿斌,李春,王悦书.2007.骨形态发生蛋白一2腺病毒表达载体在成骨过程中与血管内皮细胞生长因子关系.15(9)：698-701
江千里,王健民,丽敏,江汕,周虹.2002.批量快速测定法测定标志基因为GFP的重组病毒滴度.第二军医大学学报,23(9)：1034-1035
李姣,袁峥嵘,高雪,许尚忠.2011.浅谈我国高档肉牛产业发展思路.《第六届中国牛业发展大会》论文集.
黎明,阴津华,张葵,吴从愿,邓洁英.2010.原代培养的人网膜前脂肪细胞分化过程中基因表达和脂肪因子分泌的特征.中华内分泌代谢杂志,26(1)：56-59
瞿礼嘉,顾红雅,胡苹,陈章良.1998.现代生物技术导论.高等教育出版社,施普林格出版社.
孙丽新,汤家铭,成国祥.2004.转分化研究进展.中国生物工程杂志,1(24)：27-31
宋彬,杨静.2007.曲格列酮对人前脂肪细胞增殖和分化的影响.山西医科大学学报.39(10)：890-892
单志新,林秋雄,李晓红,邓春玉,周志凌,黄薇,黄晓忠,余细勇.2008.腺病毒表达载体对骨髓间充质干细胞分化能力的影响.南方医科大学学报.28(7)：1132-1135
王宏伟,卢慧玲,温宇,Katherine Cianflone.2005.促酰化蛋白诱导前脂肪细胞分化过程中DGAT、 LPL、adipsin mRNA.表达华中科技大学学报,34(6)：707-710
王伟,罗军,赵旺生,建华,张晓,王龙坛.2010.两农萨能羊FAS基因shRNA序列筛选及其腺病毒载体的构建.西北农业学报.19(3)：6-12
赵晓蓉,曹利民,彭吉林,王敏,赵晓平,吴砂,李文涵,叶庆,沈关心.2006.稳定表达EGFR-GFP融合蛋白细胞系的建立.郧阳医学院学报,25(3)：129
咎林森,赵春平,刘扬,刘永峰.2009.中国肉牛产业现状、热点透析与发展趋势及对策.中国农业科技导报,11(5)：1-5
咎林森.2011.加强科技创新,促进我国肉牛种业建设.中国牛业科学,37(1)：3-5
Albalat A, Saera-Vila A, Capilla E, Gutierrez J, Perez-Sanchez J.Navarro I.2007. Insulin regulation of lipoprotein lipase (LPL) activity and expression in gilthead sea bream (Sparus aurata). Comp Biochem Physiol Biochem Mol Biol,148:151-159
Ana G C, Mitchell A L.2011. Forming functional fat:a growing understanding ofadipocyte differentiation. Mol Cel Bio,12:72-2734
Armengol J, Villena J A, Hondares E, Carmona M C, Sul H S, Iglesias R, Giralt M, Villarroya F.2012. Pref-1 in brown adipose tissue:specific involvement in brown adipocyte differentiation and regulatory role of C/EBP8. Biochem J,443 (3):799-810
Arner P.2005. Resistin:yet another adipokine tells us that men are not mice. Diabetologia,48:2203-2205
Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A,StruhI K.1994. Current Protocols in Molecular Biology. New York:Greene Publishing Associates and Wiley-Interscience.
Balazs Z, Panzenboeck U, Hammer A, Sovic A, Quehenberger O, Malle E, Sattler W.2004. Uptake and transport of high-density lipoprotein (HDL) and HDL-associated alpha-tocopherol by an in vitro blood-brainbarrier model. J Neurochem,89:939-950
Bedi R, Du J, Sharma A K, Gomes I, Ackerman S J.2009. Human C/EBP-epsilon activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation. Blood,113 (2): 317-327
Bennett C N, Hodge C L, MacDougald O A, Schwartz J.2003. Role of Wnt10b and C/EBPα in spontaneous adipogenesis of 243 cells. Biochem Bioph Res,302:12-16
Bichsel C, Neeld D, Hamazaki T, Chang LJ, Yang LJ, Terada N, Jin S.2013. Direct Reprogramming of Fibroblasts to Myocytes via Bacterial Injection of MyoD Protein. Cell Reprogram [Epub ahead of print].
Brownsey R W, Boone A N, Elliot J E, Kulpa J E, Lee W M.2006. Regulation of acetyl-CoA carboxylase. Biochem SocTrans,34:223-227
Burke Z D, Shen C N, Ralphs K L, Tosh D.2006. Characterization of liver function in transdifferentiated hepatocytes. J Cell Physiol,206 (1):147-159
Carrie A M, Jason D H, David S S, Eric C C, Ilya R B,Danielle A G, Stephen F P, Colleen MC.2007. Mice With a Deletion in the Gene for CCAAT/Enhancer-BindingProtein β are Protected Against Diet-Induced Obesity. Diabetes,56 (1):161-167
Cappellari O, Benedetti S, Innocenzi A, Tedesco FS, Moreno-Fortuny A, Ugarte G, Lampugnani MG, Messina G, Cossu G.2013. D114 and PDGF-BB Convert Committed Skeletal Myoblasts to Pericytes without Erasing Their Myogenic Memory. Dev Cell,13:S1534-5807
Christy R J, Yang V W, Ntambi J M, Geiman D E, Landschulz W H, Friedman A D, Nakabeppu Y, Kelly T J, Lane M D.1989. Differentiationinducedgene expression in 3T3-L1 preadipocytes: CCAAT/enhancerbinding protein interacts with and activates the promotersof two adipocyte-specific genes. Genes Dev,3:1323-1335
Du K, Ding J.2009. Insulin regulates TRB3 and other stress-responsive gene expression through induction of C/EBP beta. Mol Endocrinol,23 (4):475-485
Duttaroy A K.2009. Transport of fatty acids across the human placenta. Prog Lipid Res,48:52-61
Eberle D, Hegarty B, Bossard P, Ferre P, Foufelle F.2004. SREBP transcription factors:masterregulators of lipid homeostasis. Biochimie,86:839-848
Ebert A D, Yu J Y, RoseJr F F, Mattis V B, Lorson C L, Thomson J A, Svendsen C N.2009. Induced pluripotent stem cells from a spinal muscular atrophypatient. Nature,457:277-280
Elmasri H, Karaaslan C, Teper Y, Ghelfi E, Weng M Q, Ince T A, Kozakewich H, Bischoff J, Cataltepe S. 2009. Fatty acid bindingprotein 4 is a target of VEGF and a regulator of cell proliferation inendothelial cells. FASEB J,23:3865-3873
Evan D R, Sarraf P, Troy A E, Bradwin G, Moore K, Milstone, D S, Spiegelman B M, Mortensen R M. 1999. PPARy is required forthe differentiation of adipose tissue in vivo and in vitro. Mol Cell,4: 611-617
Evan D R, Chung-Hsin H, Wang X Z, Shuichi S, Mason W F, Frank J G, Bruce M S.2002. C/EBPa induces adipogenesis through PPARy:Aunified pathway. Genes & Dev,16:22-26
Evan D R.2005. The transcriptional basis of adipocyte development Prostaglandins. Prostag Leukotr Ess, 73:31-34
Evan D R, Ormond A M.2006. Adipocyte differentiation from the insideout. Nat Rev Mol Cell Biol,7 (12): 885-896
Fajas L, Schoonjans K, Gelman K, Kim J B, Najib J, Martin G, Fruchart J, Briggs M, Spiegelman B M Auwerx J.1999. Regulation of peroxisome proliferator-activated receptor y expression by adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1:implications for adipocyte differentiation and metabolism. Mol Cell Biol,19 (8):5495-5503
Farmer S R.2006. Transcriptional control of adipocyte formation. Cell Metab,4 (4):263-273
Farmer S R.2008. Molecular determinants of brown adipocyte formationand function. Genes Dev,22: 1269-1275
Frid M G, Kale V A, Stenmark K R.2002. Mature vascular endothelium can give rise to smooth muscle cells via endothelial-mesenchymal transdifferentiation:in vitro analysis. Circ Res,90 (11):1189-1196
Fujimoto M, Masuzaki H, Yamamoto Y, Norisada N, Imori M, Yoshimoto M, Tomita T, Tanaka T, Okazawa K, Fujikura J, Chusho H, Ebihara K, Hayashi T, Hosoda K, Inoue G, Nakao K.2005. CCAAT/enhancer binding protein alpha maintains the ability of insulin-stimulated GLUT4 translocation in 3T3-C2 fibroblastic cells. Biochim Biophys Acta,1745 (1):38-47
Fukuhara A, Otsuki M, Shimomura I.2011. Adiponectin, leptin, adipsin]. Nihon Rinsho, S1:221-221
Gao H, Parkin S, Johnson P F, Schwartz R C.2002. C/EBP gamma has a stimulatory role on the IL-6 and IL-8 promoters. JBiol Chem,277 (41):38827-38837
Goldberg I J, Merkel M.2001. Lipoprotein lipase:physiology, biochemistry, and molecular biology. Front Biosci,6:388-405
Gregory T R, Daotai N.2012. PPAR gamma, bioactive lipids, and cancer progression. Front Biosci.17: 1816-1834
Hakelien A M, Landsverk H B, Robl J M, Skalhegg B S, Collas P.2002. Reprogramming fibroblasts to express T-cell functions using cell extracts. Nat Biotechnol,20 (5):460-466
Hassan M, Latif N, Yacoub M.2012. Adipose tissue:friend or foe? Nat Rev Cardiol,9(12):689-702 Ieda M, Fu J D, Delgado-Olguin P, VedanthamV, Hayashi Y, Bruneau B G, Srivastava D.2010. Direct Reprogramming of Fibroblastsinto Functional Cardiomyocytesby Defined Factors. Cell,142: 375-386
Hausman G J, Poulos S P, Pringle T D, Azain M J.2008. The influence of thiazolidinediones on adipogenesis in vitro and in vivo:Potentialmodifiers of intramuscular adipose tissue deposition in meat animals. JAnim Sci,86:236-E243
Hishida T, Nishizuka M, Osada S, Imagawa M.2009. The role of C/EBP delta in the early stages of adipogenesis. Biochimie,91 (5):654-657
Hideyuki M.2011. Identification and utilization of genes associated with beef qualities. Anim Sci J,82: 1-7
Hotamisligil G S, Johnson R S, Distel R J, Ellis R, Papaioannou V E, Spiegelman B M.1996. Uncoupling of obesity from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid binding protein. Science,274(5291):1377-379
Huang P, He Z, Ji S, Sun H, Xiang D, Liu C, Hu Y, Wang X, Hui L.2011. Induction of functional hepatocyte-like cells from mouse fibroblasts bydefined factors. Nature,475:386-389
Jacob E F, Stephen O, Justin J R.2010. C/EBP transcription factors regulate SREBPlc gene expression during adipogenesis. Biochem J,425:215-223
Jensen-Urstad A P, Semenkovich C F.2012. Fatty acid synthase and liver triglyceride metabolism: housekeeper or messenger? Biochim Biophys Acta,1821 (5):747-753
Jiang Z H, Michal J J, Tobey D J, Daniels T F, Rule D C, MacNeil M D.2008 Significant associations of stearoyl-CoA desaturase (SCD1) gene with fatdeposition and composition in skeletal muscle. Int J Biol Sci,4 (6):345-351
Jinyong L, ZhongLiang D, Xiaoji L, Ni T, WenXin S, Jin C, Katie A S, Hue H L, Rex C H, Kenneth, W K, Bert V, TongChuan H.2007. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nature Protocols,2:1236-1247
Jonathan K H, Bae H P, Stephen R F.2001. A Role For C/EBPβ In Regulating Peroxisome Proliferator-Activated Receptor G Activity During Adipogenesis In 3T3-L1 Preadipocytes. J Biol Chem,276 (21):18464-18471
Jonathan D B, Jorge P.2007. Peroxisome Proliferator-Activated Receptors as Transcriptional Nodal Points and Therapeutic Targets. Circulation,115:518-533
Jopling C, Boue S, Belmonte J C I.2011. Dedifferentiation, transdifferentiationand reprogramming:three routes to regeneration. Nature Reviews:Molecular cell Biology,12:79-89
Julie L, Kirstin E, Nick A S.2009. Real-Time PCR:Current Technology and Applications, Caister Academic Press, Norfolk.
Karamanlidis G, Karamitri A, Docherty K, Hazlerigg D G, Lomax M A.2007. C/EBP beta reprograms white 3T3-L1 preadipocytes to a Brown adipocyte pattern of gene expression. Biol Chem,282 (34): 24660-24669
Kelesha S, Jacqueline M S.2012. Adipogenesis. Cold Spring Harb Perspect Biol, doi: 10.1101/cshperspect.a008417
Kim J B and Spiegelman B M.1996. ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. Genes Dev,10:1096-1107
Kimihiko M, Jeffrey M P, Frank J G.2004. PPARβ/δ potentiates PPARy-stimulated adipocytedifferentiation. The FASEB Journal,10.1096/fj.04-1944fje
Kralisch S, Fasshauer M.2013. Adipocyte fatty acid binding protein:a novel adipokineinvolved in the pathogenesis of metabolic and vasculardisease? Diabetologia,56:10-21
Kudo M, Sugawara A, Uruno A, Takeuchi K, Ito S.2004. Transcription suppression of peroxisome proliferator-activated receptor gamma2 gene expression by tumor necrosis factor alpha via an inhibition of CCAAT/enhancer-binding protein delta during the early stage of adipocyte differentiation. Endocrinology,145 (11):4948-4956
Kurian L, Sancho-Martinez I, Nivet E, Aguirre A, Moon K, Pendaries C, Volle-Challier C, Bono F, Herbert JM, Pulecio J, Xia Y, Li M, Montserrat N, Ruiz S, Dubova I, Rodriguez C, Denli A M, Boscolo F S, Thiagarajan R D, Gage F H, Loring J F, Laurent L C, Izpisua Belmonte J C.2013. Conversion of human fibroblasts to angioblast-like progenitor cells. Nat Methods,10(1):77-83
Landschulz W H, Johnson P F, McKnight S L.1988. The Leucine Zipper:A Hypothetical Structure Common to a New Class of DNA BindingProteins. Science,240 (4860):1759-1764
Lane M D, Qi-Qun T, Man-Shiow J.1999. Role of the CCAAT Enhancer Binding Proteins (C/EBPs) in Adipocyte Differentiation. Biochemical and Biophysical Research Communications,266:677-683
Lai P H, Wang W L, Ko C Y, Lee Y C, Yang W M, Shen T W, Chang W C, Wang J M.2008. HDAC1/HDAC3 modulates PPARG2 transcription through the sumoylated CEBPD in hepatic lipogenesis. Biochim Biophys Acta,1783(10):1803-1814
Lay L S, Lefrere I, Trautwein C, Dugail I, Krief S.2002. Insulin and sterol-regulatory element-binding protein-lc (SREBP-lc) regulation of gene expression in 3T3-L1 adipocytes. Identification of CCAAT/enhancer-binding protein beta as an SREBP-lc target. JBiol Chem,277 (38):35625-35634
Lefterova M I, Lazar M A.2009. New developments in adipogenesis. Trends Endocrin Met,20 (3): 107-114
Li L O, Klett E L, aColeman R A.2010. Acyl-CoA synthesis, lipid metabolism and lipotoxicity. Biochim Biophys Acta,1801 (3):246-251
Lin F T, Lane M D.1994. CCAAT/enhancer binding protein a is sufficient to initiate the 3T3-L1 adipocyte differentiation program. Biochemistry,91:8757-8761
Liu B C, Zhang J D, Zhang X L, Wu G Q, Li M X.2006. Role of connective tissue growth factor (CTGF) module 4 in regulating epithelial mesenchymal transition (EMT) in HK-2 cells. Clin Chim Acta,373 (122):144-150
Liu E Q, Kitajima S, Higaki Y, Morimoto M, Sun H J, Watanabe T, Yamada N, Fan J L.2005. High lipoprotein lipase activity increases insulin sensitivityin transgenic rabbits. Metabolism,54:132-138
Liu S, Wang Y X, Wang L, Wang N, Li Y, Li H.2010. Transdifferentiation of fibroblasts into adipocyte-like cells by chicken adipogenictranscription factors. Comparative Biochemistry and Physiology,156:502-508
Lobo S, Wiczer B M, Smith A J, Hall A M, Bernlohr D A.2007. Fatty acid metabolism in adipocytes: functional analysis of fatty acid transport proteins 1 and 4. J Lipid Res,48 (3):609-620
Mandard S, Muller M, Kersten S.2004. Peroxisome proliferator-activated receptor a target genes. Cell Mol Life Sci,61:393-416
Martina I L, Yong Z, David J S, Michael S, Jonathan S, Ana C, Dan F, David Z, Christian J S, Shirley L, Mitchell A L.2008. PPARy and C/EBP factors orchestrateadipocyte biology via adjacent bindingon a genome-wide scale. Gene Dev,22:2941-2952
Margawati E.2012. A Global Strategy of Using Molecular Genetic Information to Improve Genetics in Livestock. Reprod Domest Anim, 47(S1):7-9
Meivar-Levy I, Sapir T, Gefen-Halevi S, Aviv V, Barshack I, Onaca N, Mor E, Ferber S.2007. Pancreatic and duodenalhomeobox gene 1 induces hepatic dedifferentiation bysuppressing the expression of CCAAT/enhancer binding protein β. Hepatology,46:898-905
8Merkel M, Heeren J, Dudeck W, Rinninger F, Radner H, BreslowJ L, Goldberg I J, Zechner R, Greten H. 2002. Inactive lipoprotein lipase (LPL) alone increases selective cholesterol ester uptake in vivo, whereasin the presence of active LPL it also increases triglyceride hydrolysis andwhole particle lipoprotein uptake. JBiol Chem,277:7405-411
bMerkel M, Eckel R H, Goldberg I J.2002. Lipoprotein lipase:genetics, lipiduptake, and regulation. J Lipid Res,43:1997-2006
Miller M, Shuman J D, Sebastian T, Dauter Z, Johnson, P F.2003. Structural basis for DNA recognition by the basic region leucine zipper transcription factor CCAAT/enhancer-binding protein alpha. J Biol Chem,278:15178-15184
Miyazaki M, Ntambi J M.2003. Role of stearoyl-coenzyme A desaturasein lipid metabolism. Prostag Leukotr Ess,68:113-121
Moreno-Navarrete J M, Fernandez-Real J M.2012. Adipose Tissue Biology Chapter 2 Adipocyte Differentiation.
Nathan E W, Benjamin K O, James R S, Anatoly T, Changwon P, Kyunghee C, Perry E B.2006. OP9 mouse stromal cells rapidly differentiate intoadipocytes:characterization of a useful newmodel of adipogenesis. J Lipid Res,47:450-460
Ntambi J M, Miyazaki M, Stoehr J P, Lan H, Kendziorski C M, Yandell B S, Song Y, Cohen P, Friedman J M, Attie A D.2002. Loss of stearoyl-CoAdesaturase-1 function protects mice against adiposity. P Natl Acad Sci USA,99:11482-11486
Pessler-Cohen D, Pekala P H, Kovsan J, Bloch-Damti A, Rudich A, Bashan N.2006. GLUT4 repression in response to oxidative stress is associated with reciprocal alterations in C/EBP alpha and delta isoforms in 3T3-L1 adipocytes. Arch Physiol Biochem,112(1):3-12
Pfisterer U, Kirkeby A, Torper O, Wood J, Nelander J, Dufour A, Bjorklund A, Lindval O, Jakobsson J, Parmar M.2011. Direct conversion of human fibroblaststo dopaminergic neurons. P Natl Acad Sci USA,108(25):10343-10348
Raghunath C, Paramita B, Oksana G, Kimberly, Jaideep M, Max M, Stephanie P, William J, LioneF, Michael E, Charles V.2011. Suppression of the C/EBP family of transcription factorsin adipose tissue causes lipodystrophy. Journal of Molecular Endocrinology,46:175-192
Ramji D P, Foka P.2002. CCAAT/enhancer-binding proteins:structure, function and regulation. Biochem J, 365:561-575
Rogers P M, Fusinski K A, Rathod M A, Loiler S A, Pasarica M, Shaw M K, Kilroy G, Sutton G M, McAllister E J, Mashtalir N, Gimble J M, Holland T C, Dhurandhar N V.2008. Human adenovirus Ad-36 induces adipogenesisvia its E4 orf-1 gene. Int J Obes (Lond),32:397-406
Salma N, Xiao H, Mueller E, Imbalzano A N.2004. Temporal recruitment of transcription factors and SWI/SNF chromatin-remodeling enzymes during adipogenicinduction of the PPARy nuclear hormone receptor. MoI Cell Biol,24 (11):4651-4663
Sandeep T, Paras G, Arminder S S, Chaitnya K, Saurabh S.2011. The peroxisome proliferator-activated receptor:A family of nuclear receptors role in various diseases. Adv Pharm Technol Res,2 (4): 236-240
Schwenk R W, Holloway G P, Luikena J, Bonen A, Glatz J.2010. Fatty acid transport across the cell membrane:Regulation by fatty acid transporters. Prostag Leukotr Ess,4 (86):149-154
Sekiya S, Suzuki A.2011. Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Nature,475:390-393
Shigeki S, Peter O, Dorothy DSd, Maziyar S, Annette R A, Grant D B, Suk-Hyun H, Glenda L C, Yun-Qiang Y, Michael C N, Gene H, David R G, Michael D, Ruth T Y, Jerrold M O, Ronald M E. 2009. PPARgamma activation in adipocytes is sufficient for systemicinsulin sensitization. P Natl Acad Sci USA,106:22504-22509
Sidney M K, Lydia M F, Alfredo G, Ivoneda S D, Isabel A N S, Acta C B.2004. Experimental model for fibroblast culture. Special Edition 19:11-16
Siepel A, Bejerano G, Pedersen J S, Hinrichs A S, Hou M, Rosenbloom K, Clawson H, Spieth J, Hillier LW, Richards S, Weinstock G M.Wilson R K, Gibbs R A, Kent W J, Miller W, Haussler D.2005. Evolutionarily conserved elements in vertebrate, insect, worm, andyeast genomes. Genome Res,15(8): 1034-1050
Soren F S, Mette J, Yun C, Ronni N, Albin S, Susanne M.2011. Cross species comparison of C/EBPa and PPARy profiles in mouse and human adipocytes revealsinterdependent retention of binding sites. BMC Genomics,12:152-168
Song L, Liu M, Ono N, Bringhurst F R, Kronenberg H M, Guo J.2012. Loss of wnt/β-catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes. J Bone Miner Res,27 (11): 2344-2358
Spalding K L, Arner E, Westermark P O, Bernard S, Buchholz B A, Bergmann O, Blomqvist L, Hoffstedt J, Naslund E, Britton T, Concha H, Hassan M, Ryddn M, Frisen J, Arner P.2008. Dynamics of fat cell turnover in humans. Nature,453:783-787
Swierczynski J, Sledzinski T.2012. Metabolic and regulatory function of fatty acid synthase. Postepy Biochem,58(2):175-185
Tang Q Q, Otto T C, Lane M D.2003. Mitotic clonal expansion:a synchronous process required for adipogenesis. Proc Natl Acad Sci USA,100:44-49
Tanaka T, Yoshida N, Kishimoto T, Akira S.1997. Defective adipocyte differentiation in mice lacking the C/EBPβand/or C/EBP δ gene. EMBO Journal,16:7432-7443
Taniguchi Y, Sasaki Y.1996. Rapid communication:nucleotide sequence of bovine C/EBP alpha gene. J Anim Sci,74:2554
Taniguchi Y, Sasaki Y.1997. Rapid communication:nucleotide sequence of bovine C/EBP delta gene. J Anim Sci,75:586
Tchoukalova Y D, Hausman D B, Dean R G, Hausman G J.2000. Enhancing Effect of Troglitazone on PorcineAdipocyte Differentiation in Primary Culture:A Comparison with Dexamethasone. Obes Res, 8 (9):664-672
Tong L, Harwood H J.2006. Acetyl-Coenzyme A Carboxylases:Versatile Targets for Drug Discovery. J Cell Biochem,99:1476-1488
Tontonoz P, Hu E, Spiegelman B M.1994.Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell,79:1147-1156
Tomizawa M, Garfield S, Factor V, Xanthopulos K G. 1998. Hepatocytesdeficient in CCAAT/enhancer binding protein a (C/EBP α) exhibit both hepatocyteand biliary epithelial cell character. Biochem Biophys Res Commun,249:1-5
Tsukada J, Yoshida Y, Kominato Y, Auron, P E.2011. The CCAAT/enhancer (C/EBP) family of basic-leucine zipper (bZIP) transcription factors is a multifaceted highly-regulated system for gene regulation. Cytokine,54:6-19
Uysal K T, Scheja L, Wiesbrock S M, Bonner-Weir S, Hotamisligil G S.2000. Improved glucose and lipid metabolism in genetically obese mice lacking aP2. Endocrinology,141 (9):3388-3396
Van Herpen N A, Schrauwen-Hinderling V B.2008. Lipid accumulation in non-adipose tissue and lipotoxicity. Physiol Behav,94 (2):231-241
Victoria A P, Wo-Shing A U, Christopher E L, Shaikh M R, Jacob E F, Stephen O, Justin J R.2009. C/EBP transcription factors regulate SREBPlc gene expression during adipogenesis. Biochem J,425: 215-223
Vierbuchen T, Ostermeier A, Pang Z P, Kokubu Y, Sudhof T C, Wernig M.2010. Direct conversion of fibroblasts to functional neurons bydefined factors. Nature,463:1035-1041
Voshol P J, Jong M C, Dahlmans V E, Kratky D, Levak-Frank S, Zechner R, Romijn J A, Havekes L M. 2001. In muscle-specificlipoprotein lipase-overexpressing mice, muscle triglyceride contentis increased without inhibition of insulin-stimulated whole-body andmuscle-specific glucose uptake. Diabetes,50:2585-2590
Wang M, Fan J M, LiuX Y.2004. Effect of tutal saponins of Panax notonseng on transdiferentiation of rats tubular epithelial cell induced by IL-1 alpha. Zhongguo ZhongXi YiJie He Za Zhi,24(8):722-725
Wang X Z, Masahiko K, John S, Nikoleta B, Robin K, Peter C, Helene Z, David R.1998. Identification of novel stress-induced genes downstream of chop. The EMBO Journal,17(13):3619-3630
Wei S, Zan L S, Wang H B, Cheng G, Du M, Jiang Z, Hausman G J, McFarland D C, Dodson M V.2012. Adenovirus-mediated interference of FABP4 regulates mRNA expression of ADIPOQ, LEP and LEPR in bovine adipocytes. Genet Mol Res,12(1):494-505
Weinstock P H, Bisgaier C L, Aalto-Seta la K, Radner H, Ramakrishnan R, Levak-Frank S, Essenburg A D, Zechner R, Breslow J L.1995. Severe hypertriglyceridemia, reduced high density lipoprotein, and neonataldeath in lipoprotein lipase knockout mice. Mild hypertriglyceridemiawith impaired very low density lipoprotein clearance in heterozygotes. JClin Invest,96:2555-2568
Wu Z, Xie Y, Bucher N L, Farmer S R.1995. Conditional ectopic expression of C/EBP beta in NIH-3T3 mice with cells induces PPAR gamma and stimulates adipogenesis. Genes Dev,9(19):2350-2363
Wu Z Z, Bucher N L, Farmer S R.1996. Induction of peroxisome proliferator-activated receptor gamma during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBP beta, C/EBP delta, and glucocorticoids. Mol Cell Biol,16:4128-4136
Wu Z Z, Evan D R, Regina B, Stefanie H, Guillaume A, Amy E T, Catherine M.1999. Cross-regulation of C/EBP alpha and PPAR gamma controls the transcriptional pathway of adipogenesis and insulin sensitivity. Mol Cell,3:151-158
Wu Y V, Okada T, DeCarolis P, Socci N, O'Connor R, Geha R C, Joy S C, Antonescu C, Singer S.2012. Restoration of C/EBPa in dedifferentiated liposarcoma induces G2/M cell cycle arrest and apoptosis. Gene Chromosome Cane,51 (4):313-327
Xue Y C, Ouyang K, Huang J, Zhou Y, Ouyang H, Li H R, Wang G, Wu Q J, Wei C L, Bi Y Z, Jiang L, Cai Z Q, Sun H, Zhang K, ZhangY, Chen J, Fu X F.2013. Direct conversion of fibroblaststo neurons by reprogramming ptb-regulated microrna circuits. Cell,152:82-96
Yamamoto H, Kurebayashi S, Hirose T, Kouhara H, Kasayama S.2002. Reduced IRS-2 and GLUT4 expression in PPAR gamma2-induced adipocytes derived from C/EBP beta and C/EBP delta-deficient mouse embryonic fibroblasts. J Cell Sci,115(18):3601-3607
Yamanouchi K, Ban A, Shibata S,Hosoyama T, Murakami Y, Mishihara M.2007. Both PPAR gamma and C/EBPα are Sufficient to Induce Transdifferentiation of Goat Fetal Myoblasts into Adipocytes. J Reprod Dev,53 (3):563-572
Yamaoka I, Taniguchi Y, Sasaki Y.1997. Rapid communication:nucleotide sequence of bovine C/EBP beta gene. JAnim Sci,75 (2):587
Yang T T, Cheng L, Kain S R.1996. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res,24:4592-4593
Yourka D T, Dorothy B H, Roger G D, Gary J H.2000. Enhancing effect of troglitazone on porcine adipocyte differentiation in primary culture:a comparison with dexamethasone. Obes Res,8 (9): 664-672
Z Elizabeth F, Jacqueline M S.2012. Controlling a master switch of adipocyte development and insulin sensitivity:Covalent modifications of PPARy. Bba-Biomembranes,1822:1090-1095
Zhang J W, Tang Q Q, Vinson C, Lane M D.2004. Dominant-negative C/EBP disrupts mitotic clonal expansion and differentiation of 3T3-L1 preadipocytes. Proc Natl Acad Sci USA,101:43-47
Zhang G F, Budker VG, Ludtke J J, Wolff J A.2004. Naked DNA gene transfer in mammalian cells. Gene Delivery to Mammalian Cells,245:251-264
Zhang Y Y, Li X, Qian S W, Guo L, Huang H Y, He Q, Liu Y, Ma C G, Tang Q Q.2011.Transcriptional activation of histone H4 by C/EBPβ during the mitotic clonal expansionof 3T3-L1 adipocyte differentiation. Mol Biol Cell,22:2165-2174
Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton D A.2008. In vivo reprogramming of adultpancreatic exocrine cells to β-cells. Nature,455:627-632
Zhu J, Pang D, Zhou Y, Tang X, Huang Y, Xie W, Gao F, Lai L, Zhang M, Ouyang H.2012. Direct conversion of porcine embryonic fibroblasts into adipocytes by chemical molecules. Cell Reprogram, 14 (2):99-105
Zuo Y, Qiang L, Farmer S R.2006. Activation of CCAAT/enhancer-binding protein (C/EBP) alpha expression by C/EBP beta during adipogenesis requires a peroxisome proliferator-activated receptor-gamma-associated repression of HDAC1 at the C/EBP alpha gene promoter. J Biol Chem, 281(12):7960-7967