臺灣博碩士論文加值系統

SREBP (Sterol Regulatory Element Binding Protein)是bHLH-LZ (baic Helix-Loop-Helix Leucine Zipper) 家族成員之一，有1a、1c及2三種不同的isoform，SREBP蛋白質是以約125KD的precursor形式鑲嵌於核膜及endoplamic reticulum ( ER)上，再經過兩個蛋白脢作用後會釋放出有活性68KD的mature form進入核內調控標的基因的轉錄活性。分子生物學的研究顯示，SREBP-1主要調控參與脂肪酸代謝的相關基因表現，SREBP-2主要調控參與膽固醇代謝相關基因的轉錄作用。SREBP在in vivo及in vitro的研究顯示會受到營養狀況及荷爾蒙等的調控，但是關於這方面的研究多以肝臟作為研究重點，大多是研究脂肪酸對肝臟細胞及飲食中的PUFA對小鼠肝臟中SREBP-1的影響。本篇試圖觀察富含三酸甘油酯(triglyceride, TG)的乳糜微粒 (chylomicron, CM)對脂肪細胞及脂肪組織中SREBP-1c的影響，並建立偵測SREBP-1蛋白質的方法。
在偵測SREBP-1蛋白質方面，需注意轉印時間、轉印方式及抗體濃度，其中一級抗體以IgG2A4 hybridoma最佳，本實驗採用3T3L1脂肪細胞，在分化第十天時先給予low glucose含0.5% BSA的無血清DMEM培養16小時，在分別給予含或不含CM ( 200μgTG/mL )的 low glucose的無血清DMEM培養液培養6小時，脂肪細胞中的SREBP-1 mRNA沒有明顯的變化( p>0.05)，而SREBP-1蛋白質的表現量在precursor form有增加的情形，在mature form有下降的趨勢。
Wistar大鼠管餵soybean oil一到三小時間，血漿中TG濃度有明顯上升的情形( p<0.05)，soybean oil組在三小時血漿及CM中TG濃度明顯較corn starch組及PBS為高( p<0.05)。Wistar大鼠管餵corn starch及soybean oil一小時後，大鼠副睪脂中SREBP-1 mRNA有增加的情形，但是並無統計上差異( p>0.05)。而管餵三小時及六小時三組間均無顯著差異( p>0.05)。經統計分析，大鼠體內血漿或CM中TG濃度對於脂肪組脂中SREBP-1 mRNA的表現量並無相關性( p>0.05)。
關鍵詞：SREBP、脂肪細胞、chylomicron

Sterol Regulatory Element Binding Proteins ( SREBPs) are members of baic Helix-Loop-Helix Leucine Zipper ( bHLH-LZ) family of transcription factors. To date, three isoforms have been identified, 1a, 1c and 2, encoded by two separate genes. SREBPs are synthesized as 125 kDa precursor proteins that located to the endoplasmic reticulum. The precursor is cleaved by two proteases to release the mature, transcriptionally active 68 kDa amino terminal domain. SREBP-1 preferentially activate genes involved in fatty acid synthesis; SREBP-2 preferentially activate genes involved in cholesterol metabolism. Both in vitro and in vivo studies show SREBP-1 are regulated by nutritional status and hormones. A variety of reports are focused on hepatocytes and liver. These reports are about the effects of fatty acid and dietary PUFA to SREBP-1 in hepatocytes or liver. The regulation of SREBP in liver and in adipose tissue is probably not the same. So in this study , we are trying to observe the effect of chylomicron which is rich in exogenous TG on the expression of SREBP-1 mRNA and protein in adipocyte and adipose tissue. Moreover, establishment for detecting SREBP-1 protein.
In Western blot analysis, three antibodies against SREBP-1 we tried the monoclonal antibody IgG2A4 was found to be the best. At the same time, it’s necessary to notice transfer time, transfer method and concentration of antibody. On day 10, fully differentiated 3T3L1 adipocyte were serum-starvated for 16hrs and then transferred to low glucose serum-free DMEM containing 0.5% BSA with or without CM( 200μg TG/mL ) for 6 hrs. There’s no significant difference in the SREBP-1 mRNA level when cells incubated with or without CM( 200μg TG/mL ) for 6hrs (Fig 3-5). However, in both treatment, there’s a substantial increase in the amount of precursor SREBP-1, while a decrease in the amount of mature SREBP-1 (Fig.3-1).
Wistar rats were tube fed PBS、corn starch(12Kcal/rat)、soybean oil (12Kcal/rat). Plasma TG is significantly increasing during the first to the third hours in soybean oil group (p<0.05). After tube fed 3hrs, plasma TG and CM-TG are significantly higher in the soybean oil group than the other two groups (p<0.05). After tube fed 1hrs, SREBP-1 mRNA of epididymal fat pad is slightly increasing in corn starch and soybean oil group, but there is no significant diffenence among the corn starch、PBS and soybean oil groups (p>0.05). These data suggests there is no correlation between the concentration of plasma TG or CM-TG and SREBP-1 mRNA of epididymal fat pad in rats( p>0.05). The mechisms between these changes in both adipocyte and adipose tissue is not clear, deserve more investigation.

中文摘要 I
英文摘要 III
縮寫對照表 VI
第一章 前言
第一節 脂肪組織與脂肪細胞的介紹 1
第二節 SREBP的功能與介紹 2
一 、總論 2
二 、SREBP特性與其標的基因 4
三 、SREBP的調節因子 6
1. Sterols
2. Insulin
3. PUFA
四 、在脂肪組織及脂肪細胞中的SREBP 9
第三節 脂蛋白的背景介紹 11
Chylomicron的功能與介紹 12
第四節 實驗目的 13
第二章 以西方點墨法偵測SREBP-1蛋白質之方法建立
第一節 材料與方法 15
一 、樣品製備 15
二 、SDS PAGE 20
第二節 轉印方式 23
一 、半濕式蛋白質轉印法 23
二 、濕式蛋白質轉印法 24
第三節 一級抗體 25
一 、免疫染色 25
二 、一級抗體 26
三 、二級抗體 28
四 、一級抗體純化 28
第四節 實驗結果 29
第五節 討論 32
一 、轉印方法及時間 32
二 、一級抗體測試 34
三 、Cell fractionation測試 35
第三章 乳糜微粒對脂肪細胞及脂肪組織中SREBP-1的影響
第一節 動物實驗 42
第二節 細胞實驗 43
第三節 材料與方法 44
一 、西方點墨分析 44
二 、北方點墨分析 45
三 、脂蛋白分離 51
四 、血漿及脂蛋白中脂質分析 52
第四節 實驗結果 54
一 、脂肪細胞中SREBP-1的蛋白質表現量 54
二 、脂肪細胞中SREBP-1 mRNA表現量 55
三 、大鼠副睪脂中SREBP-1 mRNA之表現 55
四 、大鼠血漿及脂蛋白中脂質含量 56
第五節 討論 57
一 、脂肪細胞中SREBP-1蛋白質及mRNA表現量 57
二 、脂肪組織中SREBP-1 mRNA表現量及血漿與脂蛋白中脂質分析 60
第四章 結論 63
參考文獻 84
圖目錄
圖2-1、脂肪細胞以100gTG/mL CM培養3或6小時，loading不同量蛋白質，以K-10一級抗體偵測SREBP-1的表現量
圖2-2、脂肪細胞以100gTG/mL CM培養3或6小時，收集不同fraction，以K-10一級抗體偵測SREBP-1的表現量
圖2-3、脂肪細胞以100gTG/mL CM培養6小時，以濕式轉印法轉印蛋白質，以H-160一級抗體偵測SREBP-1的表現量
圖2-4、收集脂肪細胞的cell lysate，以濕式轉印法轉印蛋白質，分別以H-160及K-10一級抗體偵測SREBP-1的表現圖
圖2-5、脂肪細胞serum starvation 16小時, 以200gTG/mL CM培養6小時，以不同時間的濕式轉印法轉印蛋白質，IgG2A4偵測SREBP-1的表現圖
圖2-6、脂肪細胞serum starvation 16小時, 100nM Insulin的low glucose培養六小時，IgG2A4偵測SREBP-1的表現圖
圖3-1、脂肪細胞serum starvation 16小時, 200gTG/mL CM的low glucose培養六小時，以modified cell fractionation收集蛋白質，以IgG2A4偵測SREBP-1的表現圖
圖3-2、脂肪細胞serum starvation16小時, 200gTG/mL CM的low glucose培養不同時間，收集total cell lyaste，以IgG2A4偵測SREBP-1
圖3-3、脂肪細胞serum starvation 16小時, 100nM Insulin的low glucose培養六小時，收集total cell lyaste，IgG2A4偵測SREBP-1的表現圖(A)、CBR染色圖(B)
圖3-4、脂肪細胞serum starvation16小時後, 200gTG/mL CM的low glucose對SREBP-1 mRNA影響
圖3-5、脂肪細胞serum starvation16小時後, 200gTG/mL CM的low glucose對SREBP-1 mRNA影響影像量化
圖3-6、管餵corn starch 0到6小時對Wistar大鼠副睪脂中SREBP-1 mRNA表現量之影響
圖3-7、管餵corn starch 0∼6小時對Wistar大鼠副睪脂中SREBP-1 mRNA的影像量化結果
圖3-8、管餵soybean oil 0到6小時對Wistar大鼠副睪脂中SREBP-1 mRNA表現量之影響
圖3-9、管餵soybean oil 0∼6小時對Wistar大鼠副睪脂中SREBP-1 mRNA的影像量化結果
圖3-10、管餵PBS、corn starch或soybean oil一小時後，Wistar大鼠副睪脂中SREBP-1 mRNA表現量之放射顯影圖
圖3-11、管餵PBS、corn starch或soybean oil一小時後，Wistar大鼠副睪脂中SREBP-1 mRNA影像量化結果
圖3-12、管餵PBS、corn starch或soybean oil三小時後，Wistar大鼠副睪脂中SREBP-1 mRNA表現量之放射顯影圖
圖3-13、管餵PBS、corn starch或soybean oil三小時後，Wistar大鼠副睪脂中SREBP-1 mRNA影像量化結果
圖3-14、管餵PBS、corn starch或soybean oil六小時後，Wistar大鼠副睪脂中SREBP-1 mRNA表現量之放射顯影圖
圖3-15、管餵PBS、corn starch或soybean oil六小時後，Wistar大鼠副睪脂中SREBP-1 mRNA影像量化結果
圖3-16、管餵PBS、corn starch或soybean oil 0∼6小時，Wistar大鼠血漿中TG濃度變化
圖3-17、管餵PBS、corn starch或soybean oil 0∼6小時，Wistar大鼠CM中TG濃度變化
圖3-18、管餵PBS、corn starch或soybean oil 0∼6小時，Wistar大鼠VLDL中TG濃度變化
圖3-19、管餵PBS、corn starch或soybean oil 0∼6小時，Wistar大鼠VLDL中cholesterol濃度變化

參考文獻
張鈞堯（1998）膳食油脂之質與量對大鼠肝臟中PPAR-alpha和ACO mRNA表現之影響。台大農化所碩士論文。
吳雅玲（1999）膳食油脂對大鼠脂肪組脂中PPAR-r與相關基因mRNA及化學組成之影響。
Briggs, M.R., Yokoyama, C., Wang, X., Brown, M.S., and Goldstein, J.L. (1993). Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter I. Identification of the protein and delineation of its target nucleotide sequence. J. Biol. Chem. 268：14490—14496.
Cianflone, K., Rodriguez, M. A., Yasruel, Z., Sniderman, A. D., Rosenbloom, M., and Walsh, M. (1991) Effect of acylation stimulating protein on triacylglycerol synthetic pathway of human adipose tissue. Lipids 26：495-499.
DeBose, R. A., Brown, M.S ., Li, W.P., Nohturfft, A., Goldestein, J.L., and Espenshade, P.J. (1999) Transport-dependent proteolysis of SREBP: Relocation of Site-1 protease from Golgi to ER obviates the need for SREBP transport to Golgi. Cell. 99：703-712.
Duncan, E.A., Brown, M.S., Goldstein, J.L., and Sakai, J. (1997). Cleavage site for sterol-regulated protease localized to a Leu—Ser bond in lumenal loop of sterol regulatory element binding protein-2. J. Biol. Chem. 272：12778—12785.
Fajas, L., Fruchart, J. C. and Auwerx, J (1998) Transcriptional control of adipogenesis. Curr.Opin. Cell Biol. 10：165-173.
Foretz, M., Pacot, C., Dugail, I., Lemarchand, P., Guichard, C., Liepvre, X. Le., Berthelier-Lubrano, C.,Spiegelman, B., Kim, J.B., Ferre, P., and Foufelle,F. (1999) ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose. Mol. Cell. Biol. 19：3760-3768.
Foretz, F., Guichard, C., Ferre, P. and Foufelle,F. (1999) Proc. Natl. Acad. Sci. Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes. Proc. Natl. Acad. Sci. USA 96：12737-12742.
Goodridge, A.G. (1987) Dietary Regulation Of Gene Expression: Enzymes Involved In Carbohydrate And Lipid Metabolism. Annu. Rev. Nutr. 7：157-185.
Goldstein, J.L. and Brown, M.S. (1990). Regulation of the mevalonate pathway. Nature 343：425—430.
Gregoire, F. M., Smas, C.M. and Sul, H. S.(1998) Understanding adipocyte differentiation. Physiol. Rev. 78：783-809.
Guan, G., Jiang, G., Koch, R.L., and Shechter, I. (1995). Molecular cloning and functional analysis of the promoter of the human squalene synthase gene. J. Biol. Chem. 270：21958—21965.
Horton, J. D., Shimomura, I., Brown, M. S., Hammer, R. E., Goldstein, J. L., and Shimano, H. (1998a) Activation of Cholesterol Synthesis in Preference to Fatty Acid Synthesis in Liver and Adipose Tissue of Transgenic Mice Overproducing Sterol Regulatory Element-binding Protein-2. J. Clin. Invest. 101：2331-2339.
Horton, J.D., Pai, J., Guryev, O., Brown, M. S., and Goldstein, J. L. (1998b) Differential stimulation of cholesterol and unsaturated fatty acid biosynthesis in cells expressing individual nuclear sterol regulatory element-binding proteins. J. Biol. Chem. 273：26138-26148.
Horton, J.D., Bashmakov, Y., Shimomura, I., Shimano, H. (1998c) Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice. Proc. Natl. Acad. Sci. USA 95：5987-5992.
Hua, X., Sakai, J., Ho, Y.K., Goldstein, J.L., and Brown, M.S. (1995a). Hairpin orientation of sterol regulatory element binding protein-2 in cell membranes as determined by protease protection. J. Biol. Chem. 270：29422—29427.
Hua, X., Wu, J., Goldstein, J.L., Brown, M.S., and Hobbs, H.H. (1995b). Structure of human gene encoding sterol regulatory element binding protein-1 (SREBF1) and localization of SREBF1 and SREBF2 to chromosomes 17p11.2 and 22q13. Genomics 25：667—673.
Hua, X., Yokoyama, C., Wu, J., Briggs, M.R., Brown, M.S., Goldstein, J.L., and Wang, X. (1993). SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element. Proc. Natl. Acad. Sci. USA 90：11603—11607.
Inoue, J., Kumagai, H., Terada, T., Maeda, M., Shimieu, M., and Sato, R. (2001) Proteolytic activation of SREBPs during adipocyte differentiation. Biochem. Biophy. Res. Comm (283)：1157-1161.
Flier, J.S., and Hollenberg, A.N. (1999) ADD-1 provides major new insight into the mechanism of insulin action. Proc. Natl. Acad. Sci. USA 96：14191-14192.
Kern, P.A.,Saghizadeh, M., Ong, J.M., Bosch, R. J., Deem,R., Simsolo, R. B. (1995) The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase. J. Clin.Invest. 95：2111-2119.
Kim, J.B., Spotts, G.D., Halvorsen, Y.-D., Shih, H.-M., Ellenberger, T., Towle, H.C., and Spiegelman, B.M. (1995). Dual DNA binding specificity of ADD1/SREBP1 controlled by a single amino acid in the basic helix-loop-helix domain. Mol. Cell. Biol. 15：2582—2588.
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.
Loftus, T. M. and Lane, M. D. (1997) Modulating the transcriptional control of adipogenesis. Curr. Opin. Genet. Dev. 7：603-608.
Lopez, J.M., Bennett, M.K., Sanchez, H.B., Rosenfeld, J.M., and Osborne, T.F. (1996). Sterol regulation of acetyl CoA carboxylase：a mechanism for coordinate control of cellular lipid. Proc. Natl. Acad. Sci. USA 93：1049—1053.
Lowell, B.B., Solanes, G., Mueller, E., Yao, K.M., Wright, M., Sarraf, P., Kim, J.B., and Spiegelman, B.M. (1998) Nutrition and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1. J. Clin. Invest. 101：1-9.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. T. (1951) Protein measurement with Follin phenol reagent. J. Biol. Chem. 193：265-275.
Magana, M.M., and Osborne, T.F. (1996). Two tandem binding sites for sterol regulatory element binding proteins are required for sterol regulation of fatty-acid synthase promoter. J. Biol. Chem. 271：32689—32694.
Maslowska, M., Sniderman, A. D., Germinario, R., and Cianflone, K. (1997). ASP stimulates glucose transport in cultured human adipocytes. Int. J. Obes. 21：261-266.
Miserez, A.R., Cao, G., Probst, L., and Hobbs, H.H. (1997). Structure of the human gene encoding sterol regulatory element binding protein 2 (SREBF2). Genomics 40：31—40.
Murre, C., and Baltimore, D. (1992). The helix-loop-helix motif: structure and function. In Transcriptional Regulation. S.L. McKnight and K.R. Yamamoto, eds. (New York: Cold Spring Harbor Laboratory Press), pp. 861—879.
Osborne, T.F. (1995). Transcriptional control mechanisms in the regulation of cholesterol balance. Crit. Rev. Eukaryot. Gene Expr. 5, 317—335.
Osborne, T. (2000) Sterol regulatory element-binding protein：Key regulators of nutritional hemostasis and insulin action. J. Biol. Chem. 275：32379-32382.
Pelleymounter, M. A., Cullen, M. J., Baker, M. B., Hecht, R., Winters, D., Boone, T., and Collins, F. (1995) Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269：540-543.
Ricsson, J., Jackson, S.M., Lee, B.C., and Edwards, P.A. (1996). Sterol regulatory element binding protein binds to a cis element in the promoter of the farnesyl diphosphate synthase gene. Proc. Natl. Acad. Sci. USA 93：945—950.
Sato, R., Yang, J., Wang, X., Evans, M.J., Ho, Y.K., Goldstein, J.L., and Brown, M.S. (1994). Assignment of the membrane attachment, DNA binding, and transcriptional activation domains of sterol regulatory element binding protein-1 (SREBP-1). J. Biol. Chem. 269：17267—17273.
Scantlebury,T., Maslowska, M., and Cianflone, K. (1998). Chylomicron specific enhancement of acylation stimulating protein (ASP) and precursor protein C3 production in differentiated human adipocytes. J. Biol. Chem. 273：22903-22909.
Shimano, H., Horton, J.D., Shimomura, I., Hammer, R.E., Brown, M.S., and Goldstein, J.L. (1997). Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells. J. Clin. Invest. 99：846—854.
Shimano, H., Horton, J.D., Hammer, R.E., Shimomura, I., Brown, M.S., and Goldstein, J.L. (1996). Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a. J. Clin. Invest. 98：1575—1584.
Shimomura, I., Shimano, H., Horton, J.D., Goldstein, J.L., and Brown, M.S. (1997). Differential expression of exons 1a and 1c in mRNAs for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells. J. Clin. Invest. 99：838—845.
Shimomura, L., Bashmakov, Y., Ikemoto, S., Horton, J.D., Brown, M.S., and Goldstein, J.L. (1999) Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes. Proc. Natl. Acad. Sci. USA 96：13656-13661.
Tontonoz, P., Kim, J.B., Graves, R.A., and Spiegelman, B.M. (1993). ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation. Mol. Cell. Biol. 13：4753—4759.
Wang, X., Briggs, M.R., Hua, X., Yokoyama, C., Goldstein, J.L., and Brown, M.S. (1993). Nuclear protein that binds sterol regulatory element of LDL receptor promoter: II. Purification and characterization. J. Biol. Chem. 268：14497—14504.
Worgall, T.S., Sturley, S.L., Seo, T., Osborne, T.F., and Deckelbaum,R.J. (1998) Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing levels of mature sterol regulatory element-binding protein. J. Biol. Chem 273：25537-25540.
Yahagi, N., Shimano, H., Hasty, A.H., Amemiya-Kudo, M., Osuga, J., Harada, K., Gotoda, T., Nagai, R., Ishibashi, S., and Yamada,N. (1999) A crucial role of sterol regulatory element-binding protein-1 in the regulation of lipogenic gene expression by polyunsaturated fatty acids. J. Biol. Chem 274：35840-35844.
Yokoyama, C., Wang, X., Briggs, M.R., Admon, A., Wu, J., Hua, X., Goldstein, J.L., and Brown, M.S. (1993). SREBP-1, a basic helix-loop-helix leucine zipper protein that controls transcription of the LDL receptor gene. Cell 75：187—197.