飲食習慣不良是肥胖與代謝相關疾病發生的原因之一。相關文獻中，已瞭解高果糖飲食、高蔗糖飲食及含蔗糖飲水能誘導嚙齒類產生代謝異常，故進一步探究是否對於peroxisome proliferator-activated receptors (PPARs)與sterol regulatory element-binding proteins (SREBPs)等脂代謝相關基因有所影響。將雄性C57BL/6J小鼠依飲食條件區分為四組：正常飼料 (normal chow, N)、飼料含50%果糖 (high fructose, F)、飼料蔗糖組 (high sucrose, S)之飼料、正常飼料但飲用30%蔗糖水 (sucrose water, W)，進行9週餵養後，分析目標基因相對表現量。結果顯示，F組小鼠肝臟中，PPARα及所調控參與脂肪酸β oxidation之基因CPT1與ACO mRNA表現量相較於N組顯著上升，SREBP1c雖顯著上升，但其所調控參與脂質合成之基因ACC與FAS無顯著差異；肌肉中，CPT1與ACO表現量上升；脂肪組織中，ACC與FAS顯著下降。S組小鼠肝臟中，PPARα與SREBP1c表現相較N組無顯著差異；脂肪組織中，ACC相對表現量下降。W組小鼠中，PPARα雖無顯著差異，但其所調控之基因mRAN表現顯著上升，SREBP1c及其調控基因則皆顯著下降；脂肪組織中，SREBP1c其調控基因mRNA表現亦顯著下降。整體而言，高果糖飲食、高蔗糖飲食及含蔗糖飲水能促進肝臟及肌肉中脂質分解相關基因表現，且降低脂肪組織中參與脂質合成基因表現。此外，蔗糖飲水亦能降低小鼠肝臟中脂質合成相關基因表現。因此，此三種高糖飲食對小鼠脂代謝基因有不同之影響。

Diet represents one environmental factor that influences the onset of metabolic disorder. Numerous studies showed that high fructose diet, high sucrose diet or sucrose containing water induces metabolic derangements in rodents. In this study, we examined the effect of those diets on the genes expressions in mice, including peroxisome proliferator-activated receptors (PPARs), sterol regulatory element-binding proteins (SREBPs), and other genes related to lipid metabolism. C57BL/6J mice were fed with normal chow (N group), high fructose (F group), high sucrose (S group) or sucrose containing water (W group) for 9 weeks. Compared with N group, the genes expressions of hepatic PPARα and fatty acid oxidation enzymes, CPT1 and ACO, were significantly increased in F group. SREBP1c mRNA was significantly increased, but lipogenic related genes, ACC and FAS, were not significantly different. In F group, myotic CPT1 and ACO mRNA were increased, while ACC and FAS mRNA were reduced in adipose tissue. In S group, hepatic PPARα and SREBP1c mRNA were not significantly different compared with N group; adipotic ACC mRNA was reduced. In W group, hepatic PPARα mRNA was not significantly different compared with N group, but CPT1 and ACO were significantly increased. SREBP1c, ACC and FAS were reduced in muscle, and they were also reduced in adipose tissue. On the whole, high fructose diet, high sucrose diet or sucrose containing water increased the expression of lipolysis genes in liver and muscle, and reduced the expression of lipogenesis genes in adipose tissue. Moreover, sucrose containing water also reduced the expression of lipogenesis genes in liver. Therefore, those three different diets have different effects on the expression of genes
involved in lipid metabolism.

壹、緒論 1
1.1 前言 1
1.2 文獻探討 2
1.2.1 PPARs 2
1.2.2 SREBPs 4
1.2.3 FABPs 5
1.2.4 G6Pase 5
1.2.5脂肪酸主要代謝途徑 6
1.2.6脂質合成 7
1.2.7 高糖飲食對嚙齒類之影響 7
1.3 實驗設計概述 8
貳、材料 11
2.1 實驗動物 11
2.2 飲食條件 11
2.3 引子 11
2.3.1 Semi- quantitative RT-PCR 所使用之引子 11
2.3.2 Real-time RT-PCR 所使用之引子 13
2.4 藥品 14
2.5 試劑組 15
2.6 溶劑、緩衝溶液及培養基 15
2.7 儀器設備 16
參、方法 17
3.1 組織均質化 17
3.2 小鼠肝臟、肌肉、脂肪組織RNA萃取 17
3.3 RNA變性電泳分析 17
3.4 1.2 %甲醛變性電泳膠 18
3.5 互補去氧核醣核酸 (cDNA)之製備 18
3.6 Semi-quantitative RT-PCR 18
3.7 Real-time PCR 19
3.8 統計 19
肆、結果 20
4.1 cDNA之製備 20
4.2 以semi-quantitative RT-PCR檢測目標基因mRNA表現量 20
4.2.1 PPARs mRNA表現情況 20
4.2.2 ACO、 CPT1、 FABPs mRNA表現情況 21
4.2.3 G6Pase mRNA表現情況 22
4.2.4 SREBP1c mRNA表現情況 23
4.2.5 FAS、ACC mRNA表現情況 23
4.3以Real-time PCR檢測目標基因mRNA表現量 24
4.3.1 PPARs mRNA表現情況 24
4.3.2 ACO或CPT1 mRNA表現情況 25
4.3.3 SREBP1c mRNA表現情況 25
4.3.4 FAS、ACC mRNA表現情況 26
4.4 Semi-quantitative RT-PCR與Real-time PCR檢測結果之比較 26
伍、討論 28
5.1 高果糖飲食對C57BL/6J小鼠之影響 (F組) 28
5.2 高蔗糖飲食對C57BL/6J小鼠之影響 (S組) 30
5.3 含30%蔗糖飲水對C57BL/6J小鼠之影響 (W組) 31
5.4 含蔗糖飼料與含蔗糖飲水對C57BL/6J小鼠之影響比較 32
5.5 Semi-quantitative RT-PCR與Real-time PCR結果不一致之可能原因 33
陸、參考文獻 35

許珊菁 (2006).鼠模式中高脂飲食、肥胖與脂質調控基因之表現。台灣大學博士論文。
Aguilera AA, Diaz GH, Barcelata ML, Guerrero OA, Ros RM. (2004). Effects of fish oil on hypertension, plasma lipids, and tumor necrosis factor-alpha in rats with sucrose-induced metabolic syndrome. J Nutr Biochem. 15(6): 350–357.
Aoyama Y, Yoshida A, Ashida K. (1974). Effect of dietary fats and fatty acids on the liver lipid accumulation induced by feeding a protein-repletion diet containing fructose to protein-depleted rats. J Nutr. 104:741–746
Busserolles J, Gueux E, Rock E, Mazur A, Rayssiguier Y. (2002).Substituting honey for refined carbohydrates protects rats from hypertriglyceridemic and prooxidative effects of fructose. J Nutr. 132(33):79–82.
Burant CF, Saxena M. (1994).Rapid reversible substrate regulation of fructose transporter expression in rat small intestine and kidney. Am J Physiol. 267(1 Pt 1):G71-9.
Burant CF, Takeda J, Brot-Laroche E, Bell GI, Davidson NO. (1994). Fructose transporter in human spermatozoa and small intestine is GLUT5. J Biol Chem. 267(21):14523-6.
Chou YC, Wang SY, Chen GC, Lin YS, Chao PM (2009). The functional assessment of Alpinia pricei on metabolic syndrome induced by sucrose-containing drinking water in mice. Phytother Res. 23(4):558-63.
Clarke DC, Miskovic D, Han X-X, Calles-Escandon J, Glatz JFC, Luiken JJFP (2004). Overexpression associated fatty acid binding protein (FABPpm) in vivo increases fatty acid sarcolemmal transport and metabolism. Physiol Genomics. 17: 31–37
Desvergne, B., and W. Wahli (1999). Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Reviews. 20(5): 649-688.
Edvardsson U, von Lowenhielm HB, Panfilov O, Nystrom AC, Nilsson F & Dahllof B (2003). Hepatic protein expression of lean mice and obese diabetic mice treated with peroxisome proliferator-activated receptor activators. Proteomics. 3(4):468-478.
El Hafidi M, Cuellar A, Ramirez J, Banos G. (2001). Effect of sucrose addition to drinking water, that induces hypertension in the rats, on liver microsomal Delta9 and Delta5-desaturase activities. J Nutr Biochem. 12(7): 396–403.
Escher, P., and W. Wahli (2000). Peroxisome proliferator-activated receptors: insight into multiple cellular functions. Mutation Research. 448: 121-138.
Finck N. Brian, Bernal-Mizrachi Carlos, Han Dong Ho, Coleman Trey, Sambandam Nandakumar, LaRiviere L. Lori, Holloszy O. John, Semenkovich F. Clay, and Kelly P. Daniel (2005). A potential link between muscle peroxisome proliferatoractivated receptor-signaling and obesity-related diabetes. Cell Metab. 1(2):133-44.
Frayn KN and Kingman SM. (1995).Dietary sugars and lipid metabolism in humans. Am J Clin Nutr. 62(2):50– 63.
Hancock CR, Han DH, Chen M, Terada S, Yasuda T, Wright DC, Holloszy JO (2008). High-fat diets cause insulin resistance despite an increase in muscle mitochondria. Proc Natl Acad Sci U S A. 105(22):7815-20.
Horton J.D., Bashmakov Y., Shimomura I., Shimano H (1998). Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice. Proc Natl Acad Sci U S A. 95(11):5987-5992.
Horton JD, Shah NA, Warrington JA, Anderson NN, Park SW, Brown MS & Goldstein JL (2003). Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes. Proc Natl Acad Sci U S A. 100(21):12027-12032.
Ishikawa M., Iwasaki Y., Yatoh S., Kato T., Kumadaki S., Inoue N., Yamamoto T. , T. Matsuzaka, Nakagawa Y., Yahagi N., KobayashK. i, Takahashi A., Yamada N., Shimano H (2008). Overexpression of nuclear SREBP-2 in pancreatic beta-cells results in cholesterol accumulation leading to insulin secretion defects and diabetes: a new model for beta-cell lipotoxicity. J. Lipid Res. 49: 2524–2534.
Johnson J Richard, Segal S Mark, Sautin Yuri, Nakagawa Takahiko, Feig I Daniel, Kang Duk-Hee, Gersch S Michael, Benner Steven, and Laura G Sa´nchez-Lozada (2007). Potential role of sugar (fructose) in the epidemic of hypertension, obesity and the metabolic syndrome, diabetes, kidney disease, and cardiovascular disease. Am J Clin Nutr. 86(4): 899–906
Jordal, A. E., I. Hordvik, M. Pelsers, D. A. Bernlohr, and B. E. Torstensen (2006). FABP3 and FABP10 in Atlantic salmon (Salmo salar L.)--general effects of dietary fatty acid composition and life cycle variations. Comp Biochem Physiol B Biochem Mol Biol. 145(2):147-158.
Jürgens H, Haass W, Castañeda TR, Schürmann A, Koebnick C, Dombrowski F, Otto B, Nawrocki AR, Scherer PE, Spranger J, Ristow M, Joost HG, Havel PJ, Tschöp MH (2005). Consuming fructose-sweetened beverages increases body adiposity in mice. Obes Res. 13(7):1146-1156.
Kim, K.H., Lopez-Casillas, F., Bai, D.H., Luo, X., and Pape, M.E (1989). Role of reversible phosphorylation of acetyl-CoA carboxylase in long-chain fatty acid synthesis. FASEB J. 3:2250–2256.
Kimber L. Stanhopea, and Peter J. Havela. (2008). Fructose consumption: potential mechanisms for its effects toincrease visceral adiposity and induce dyslipidemia and insulin resistance. Current Opinion in Lipidology. 19:16–24
Kondo, H., R. Misaki, L. Gelman, and S. Watabe (2007). Ligand-dependent transcriptional activities of four torafugu pufferfish Takifugu rubripes peroxisome proliferator-activated receptors. Gen Comp Endocrinol. 154(1-3):120-127.
Lee CH, Olson P & Evans RM (2003). Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology. 144:2201-2207.
Lee SS, Pineau T, Drago J, Lee EJ, Owens JW, Kroetz DL, Fernandez-Salguero PM, Westphal H & Gonzalez FJ (1995). Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol Cell Biol. 15(6):3012-3022.
Leibowitz MD, Fievet C, Hennuyer N, Peinado-Onsurbe J, Duez H, Bergera J, Cullinan CA, Sparrow CP, Baffic J, Berger GD, Santini C, Marquis RW, Tolman RL, Smith RG, Moller DE & Auwerx J (2000). Activation of PPARdelta alters lipid metabolism in db/db mice. FEBS Lett. 473(3):333-336.
Litherland GJ, Hajduch E, Gould GW, Hundal HS. (2004). Fructose transport and metabolism in adipose tissue of Zucker rats: diminished GLUT5 activity during obesity and insulin resistance. Mol Cell Biochem. 261(1-2):23-33.
McArthur MJ, Atshaves BP, Frolov A, Foxworth WD, Kier AB, Schroeder F. (1999). Cellular uptake and intracellular trafficking of long chain fatty acids. J Lipid Res 40(8): 1371–1383.
Minassian C., Zitoun C. and Mithieux G. (1996). Differential time course of liver and kidney glucose-6-phosphatase activity during long-term fasting in rat correlates with differential time course of messenger RNA level. Mol. Cell. Biochem. 155(1):37-41.
Nagai Y, Nishio Y, Nakamura T, Maegawa H, Kikkawa R, Kashiwagi A (2002). Amelioration of high fructose-induced metabolic derangements by activation of PPARα. Am J Physiol Endocrinol Metab. 282(5):E1180-1190.
Najjar SM, Yang Y, Fernström MA, Lee SJ, Deangelis AM, Rjaily GA, Al-Share QY, Dai T, Miller TA, Ratnam S, Ruch RJ, Smith S, Lin SH, Beauchemin N, Oyarce AM (2005). Insulin acutely decreases hepatic fatty acid synthase activity. Cell Metab. 2(1):43-53.
O'Hea EK, Leveille GA (1969). Significance of adipose tissue and liver as sites of fatty acid synthesis in the pig and the efficiency of utilization of various substrates for lipogenesis. J Nutr. 99(3):338-344.
Peters JM, Lee SS, Li W, Ward JM, Gavrilova O, Everett C, Reitman ML, Hudson LD and Gonzalez FJ (2000). Growth, adipose, brain, and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor beta(delta). Mol Cell Biol. 20(14):5119-5128
Riby JE, Fujisawa T, Kretchmer N. (1993). Fructose absorption . Am J Clin Nutr. 58(7):48–53.
Roglans N, Vilà L, Farré M, Alegret M, Sánchez RM, Vázquez-Carrera M, Laguna JC (2007). Impairment of hepatic Stat-3 activation and reduction of PPARalpha activity in fructose-fed rats. Hepatology. 45(3):778-88.
Romsos DR, Belo PS, Miller ER, Leveille GA (1975). Influence of dietary 1,3-butanediol on wieght gain, blood, and liver metabolites and lipogenesis in the pig and chick. J Nutr. 105(2):161-70.
Sacchettini JC, Gordon JI, Banaszak LJ (1988). Thestructure of crystalline Escherichia coli- derived rat intestinal fatty acid-binding protein at 2.5-A resolution. J Biol Chem. 263(12): 5815–5819.
Schaap FG, van der Vusse GJ, Glatz JF (2002). Evolution of the family of intracellular lipid binding proteins in vertebrates. Mol Cell Biochem. 239(1-2): 69–77.
Schoonjans, K., B. Staels, and J. Auwerx (1996). The peroxisome proliferator activated receptors (PPARs) and their effects on lipid metabolism and adipocyte differentiation. Biochimica et Biophysica Acta. 1302(2): 93-109.
Shimomura I, Matsuda M, Hammer RE, Bashmakov Y, Brown MS, Goldstein JL (2000). Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice. Mol Cell. 6(1):77–86.
Shimano H, Horton JD, Shimomura I, Hammer RE, Brown MS, Goldstein JL (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(5):846-54.
Shimano H, Horton JD, Hammer RE, Shimomura I, Brown MS & Goldstein JL (1996). Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a. J Clin Invest. 98(7): 1575-1584.
Storch, J., and A. E. A. Thumser (2000). The fatty acid transport function of fatty acid-binding proteins. Biochimica et Biophysica Acta. 1486(1): 28-44.
Sumiyoshi M, Sakanaka M, Kimura Y. (2006). Chronic intake of high-fat and high-sucrose diets differentially affects glucose intolerance in mice. J Nutr. 136(3):582-7.
Thresher JS, Podolin DA, Wei Y, Mazzeo RS, Pagliassotti MJ. (2000). Comparison of the effects of sucrose and fructose on insulin action and glucose tolerance. Am J Physiol Regul Int Comp Physiol. 279(4):R1334–440.
Trinh, K. Y., O'Doherty, R. M., Anderson, P., Lange, A. J. and Newgard, C. B. (1998). Perturbation of fuel homeostasis caused by overexpression of the glucose-6-phosphatase catalytic subunit in liver of normal rats. J. Biol. Chem. 273(47):31615-31620.
Veerkamp JH, van Moerkerk HT (1993). Fatty acid-binding protein and its relation to fatty acid oxidation. Mol Cell Biochem. 123(1-2):101-106.
Wakil, S.J. (1989). Fatty acid synthase, a proficient multifunctional enzyme. Biochemistry. 28(11):4523–4530.
Yahagi N, Shimano H, Hasty AH, Amemiya-Kudo M, Okazaki H, Tamura Y, Iizuka Y, Shionoiri F, Ohashi K, Osuga J, Harada K, Gotoda T, Nagai R, Ishibashi S, 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 (50):35840–35844.