臺灣博碩士論文加值系統

果糖常用於大多數飲料和加工食品中。已知的研究顯示，攝取過多果糖會引起由果糖誘發的代謝症候群、腸道相關症狀和腸道屏障功能障礙，例如輕度的發炎細胞浸潤於黏膜層和腸道細菌過度生長。腸道屏障的破壞也與許多病理和發炎性疾病有關。本研究的目的是探討高果糖對代謝和腸道屏障功能的影響，特別是腸道通透性以及細胞與細胞間的緊密連接(Tight junction)。首先，我們測量培養的人類腸上皮細胞Caco-2中跨細胞膜上皮電阻值(TEER)的變化，以確保緊密連接的形成，之後處理不同濃度梯度(1%, 2%, 3 %, 4%, 5%)的果糖24小時以確定果糖對TEER變化的影響，接著透過異硫氰酸螢光素葡聚醣在體外測量腸道的通透性；同時，我們也利用細胞免疫螢光染色觀察這些不同濃度梯度果糖的處理對緊密連接結構型態的影響，並以西方墨點法確認處理組緊密連接蛋白的表現量。我們的結果表明，當 Caco-2單層中緊密連接完全形成時，TEER 增加達到最大電阻值，並且這些細胞中的果糖處理導致 TEER電阻值依濃度梯度的增高顯著降低、上皮細胞通透性增加、緊密連接結構發生破損且緊密連接蛋白的表現顯著降低。這些結果表明，果糖的長期給予可能會改變緊密連接結構，從而導致上皮屏障功能受損。我們進一步研究高果糖攝取對腸道屏障功能和代謝的影響。C3H/HE小鼠以高果糖飼料(HFrD，60% 果糖)餵食 11 週，並以一般動物飼料(NCD)作為對照組。與NCD組相比，在相似的熱量攝取下，HFrD誘導了小鼠高空腹血糖、高胰島素血症、葡萄糖耐受不佳、高膽固醇血症和胰島素抗性等代謝異常，肝內三酸甘油酯、膽固醇總含量顯著高並在異硫氰酸螢光素葡聚醣(FITC-Dextran)的測試中有顯著高的腸滲透性，其次也發現腸段總長度顯著低並且腸組織切片緊密連接蛋白訊號的缺失。綜觀本篇研究結果，我們證實了果糖的給予對代謝異常的誘導，同時我們的結果也表明了高果糖對腸屏障功能的損害，像是細胞與細胞間連接的不完整性、高通透度、緊密連接結構型態的改變與蛋白表現量的降低。

Fructose is used in most of beverages and processed foods. Excessive fructose intake is reported to cause fructose-induced metabolic syndrome, gut symptoms and intestinal barrier dysfunction, such as mild inflammatory cell infiltration in the mucosal layer and overgrowth of intestinal bacteria. Disruption of the intestinal barrier is also related to many pathological and inflammatory diseases. The aim of this study is to investigate the influence of high- fructose on metabolism and intestinal barrier function, especially intestinal permeability and the expression of tight junction. First, we measured the change of trans-epithelial electrical resistance (TEER) in culture of rat intestinal epithelial cells IEC-6 to ensure the formation of tight junction, and then these cells were treated with different concentration (0.5%, 1%, 1.5%, 2%) of fructose for 24 h to determine the action of fructose on the change of TEER. Our results showed that the increase in TEER to a maximal resistance when tight junction fully formation in IEC-6 monolayers, and fructose treatment in these cells caused significantly decreases in TEER resistance in a dose-dependent manner, increased epithelial cell permeability, disruption of tight junction structure, and a significant decrease in the expression of tight junction proteins. These results suggested that chronic incubation with fructose might alter tight junction structure, which contributing to the impaired epithelial barrier function. We further investigate the effect of high fructose intake on intestinal barrier function and metabolism. C3H/HE mice were fed with either a high-fructose diet (HFrD, 60% fructose) or normal chow diet (NCD) for 11 weeks. Compared with the NCD group, HFrD- induced high fasting blood glucose, hyperinsulinemia, glucose tolerance, hyper- cholesterolemia and insulin resistance in mice under the similar calorie intake.. The total content of intrahepatic triglyceride and cholesterol was significantly increased, and the intestinal permeability was significantly increased. The total length of the intestinal segment was significantly shorter and the tight junction protein signal was absent in intestinal tissue sections, also, the intestinal permeability was significantly higher in the FITC-Dextran test. In summary, the results of this study confirmed that fructose administration induces metabolic abnormalities. Our results also showed that high fructose damages the intestinal barrier function, such as the incompleteness of cell-cell connections, high permeability, changes in the tight junction structure and reduced protein expression.

致謝………………………………………………………………………………………..…..i
中文摘要……………………………………………………………………..……………......ii
英文摘要……………………………………………………………….……….………….....iii
目錄……………………………………………………………………………………………v
圖目錄………………………………………………………………………..........................vii
表目錄……………………………………………………………………………...……..…viii
第一章、前言………………………………………………………………………………....1
第二章、文獻回顧…………………………………………………………………..………..2
2.1代謝症候群 (Metabolic Syndrome) …………………………………..…..….…......2
2.2腸道上皮屏障功能……………………………………………………….………......2
2.3代謝與腸道屏障功能………………………………………………………………...4
2.4果糖代謝及吸收路徑…………………………………………………….………......5
2.5果糖誘導代謝症候群相關代謝異常症狀………………………………….………..5
第三章、研究動機與實驗設計………………………….……………….………………...…7
3.1 研究動機及實驗目標…………………………..…………………………...…….…7
3.2實驗設計………………………………………………………….………………......7
第四章、材料與方法…………………………………………………………….……….….11
4.1實驗小鼠飼養條件及飼料來源……………………………………………….……11
4.2小鼠空腹血糖、胰島素及HOMA-IR代謝生化指標測定………………….…....11
4.3 口服葡萄糖耐受性測試Oral glucose tolerance test(OGTT)……………….…......12
4.4小鼠三酸甘油酯(TG)與總膽固醇(TC)生化值測定…………………….……...….14
4.5腸組織石蠟切片處理及染色……………………………………………….…...….15
4.6腸上皮細胞培養及配方藥品來源………………………………………………….17
4.7細胞跨膜上皮電阻 (Transepithelial Electrical Resistance, TEER)測定…………..17
4.8腸滲透實驗分析(INTESTINAL PERMEABILITY ASSAY)……………..…….....19
4.9細胞免疫螢光染色(Immunocytofluorescence, ICF)…………………………….....19
4.10細胞蛋白質之萃取………………………………………………………...………20
4.11西方墨點法(Western blotting)……………………………………………………..23
4.12統計方法(Statistical analysis)……………………………………………….…….24
第五章、實驗結果……………………………………………………………………….......25
5.1果糖對腸上皮細胞屏障完整性的影響………………………………….…………25
5.2果糖對腸上皮細胞滲透性的影響………………………………………………….25
5.3果糖對腸上皮細胞屏障中緊密連接結構的影響………………………………….25
5.4 果糖對腸上皮細胞緊密連接蛋白表現量的影響…………………………………26
5.5 高果糖飼料對C3H/He小鼠代謝的影響及代謝症候群相關症狀誘導…………26
5.6 高果糖誘導代謝異常之小鼠模型對於腸道的影響…………………………...….28
第六章、討論……………………………………………………………………….………..30
第七章、結論……………………………………………………………………………...…32
參考文獻……………………………………………………….……………………………..33
圖表…………………………………………………………………………………………...38
附錄……………………………………………………………...……………………………51

1.Thorburn AW, Storlien LH, Jenkins AB, Khouri S, Kraegen EW. Fructose-induced in vivo insulin resistance and elevated plasma triglyceride levels in rats. The American Journal of Clinical Nutrition. 49(6): 1155-1163, 1989.
2.Miller A, Adeli K. Dietary fructose and the metabolic syndrome. Current Opinion in Gastroenterology. 24(2):204-9, 2008.
3.Kawabata K, Kanmura S, Morinaga Y, Tanaka A, Makino T, Fujita T, Arima S, Sasaki F, Nasu Y, Tanoue S, Hashimoto S, Ido A. A high‑fructose diet induces epithelial barrier dysfunction and exacerbates the severity of dextran sulfate sodium‑induced colitis. International Journal of Molecular Medicine. 43(3):1487-1496, 2019.
4.Volynets V, Spruss A, Kanuri G, Wagnerberger S, Bischoff S C, Bergheim I. Protective effect of bile acids on the onset of fructose-induced hepatic steatosis in mice. Journal of lipid research. 51(12): 3414-3424, 2010.
5.Miranda PJ, DeFronzo RA, Califf RM, Guyton JR. Metabolic syndrome: definition, pathophysiology, and mechanisms. Am Heart J. 149(1):33-45, 2005.
6.蘇郁文。腸道微菌叢與糖尿病及代謝症候群。臨床醫學月刊。84(1):477-483, 2019。
7.Samson SL, Garber AJ. Metabolic syndrome. Endocrinol Metab Clin North Am. 43:1-23, 2014.
8.Gobato AO, Vasques AC, Zambon MP, Barros Filho Ade A, Hessel G. Metabolic syndrome and insulin resistance in obese adolescents. Rev Paul Pediatr. 32(1):55-62, 2014.
9.Groschwitz KR, Hogan SP. Intestinal Barrier Function: Molecular Regulation and Disease Pathogenesis. J Allergy Clin Immunol. 124:3–20; quiz 21–22, 2009.
10.Schulz O, Pabst O. Antigen Sampling in the Small Intestine. Trends Immunol. 34:155–61, 2013.
11.Nagura H, Sumi Y. Immunological functions of the gut-role of the mucosal immune system. Toxicol Pathol. 16:154-164, 1988.
12.Scaldaferri F, Pizzoferrato M, Gerardi V, Lopetuso L, Gasbarrini A. The Gut Barrier: New Acquisitions and Therapeutic Approaches. J Clin Gastroenterol. 46:S12–7, 2012.
13.Xu, D., & Lu, W. Defensins: A Double-Edged Sword in Host Immunity. Frontiers in immunology. 11: 764, 2020.
14.Riedel Sylvia , Pheiffer Carmen , Johnson Rabia , Louw Johan , Muller Christo J. F. Intestinal Barrier Function and Immune Homeostasis Are Missing Links in Obesity and Type 2 Diabetes Development. Frontiers in Endocrinology. 12:1664-2392, 2022.
15.Jinbao Wu, Jide Wang, Yali Zhang. Research progress on intestinal mucosal barrier. World Chinese Journal of Digestology. 11(5): 619-623, 2003.
16.Gieryńska M, Szulc-Dąbrowska L, Struzik J, Mielcarska MB, Gregorczyk-Zboroch KP. Integrity of the Intestinal Barrier: The Involvement of Epithelial Cells and Microbiota-A Mutual Relationship. Animals (Basel). 12(2):145, 2022.
17.Laissue JA, Chappuis BB, Muller C, Reubi JC, Gebbers JO. The intestinal immune system and its relation to disease. Dig Dis. 11:298-312, 1993.
18.Jafari, Nazila & Rohn, Jennifer. The urothelium: a multi-faceted barrier against a harsh environment. Mucosal Immunology. 15:1-16, 2022.
19.Hu Y-J, Wang Y-D, Tan F-Q, Yang W-X. Regulation of paracellular permeability: factors and mechanisms. Mol Biol Rep. 40:6123–42, 2013.
20.Odenwald MA, Turner JR. Intestinal Permeability Defects: Is it Time to Treat? Clin Gastroenterol Hepatol Off Clin Pract J Am Gastroenterol Assoc. 11:1075–83, 2013.
21.Vogelmann R, Nelson WJ. Fractionation of the epithelial apical junctional complex: reassessment of protein distributions in different substructures. Mol Biol Cell. 16(2):701-716, 2005.
22.Zuo L, Kuo W-T, Turner JR. Tight Junctions as Targets and Effectors of Mucosal Immune Homeostasis. Cell Mol Gastroenterol Hepatol. 10:327–40, 2020.
23.Suzuki T. Regulation of the Intestinal Barrier by Nutrients: The Role of Tight Junctions. Anim Sci J Nihon Chikusan Gakkaiho. 91:e13357, 2020.
24.Ghosh S, Whitley CS, Haribabu B, Jala VR. Regulation of Intestinal Barrier Function by Microbial Metabolites. Cell Mol Gastroenterol Hepatol. 11:1463–82, 2021.
25.Nusrat A, Turner JR, Madara JL. Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Am J Physiol Gastrointest Liver Physiol. 279:G851-G857, 2000.
26.Colgan SP, Dzus AL, Parkos CA. Epithelial exposure to hypoxia modulates neutrophil transepithelial migration. J Exp Med. 184:1003-1015, 1996.
27.Hervé JC, Derangeon M, Sarrouilhe D, Bourmeyster N. Influence of the scaffolding protein Zonula Occludens (ZOs) on membrane channels. Biochim Biophys Acta. 1838(2):595-604, 2014.
28.H. Bauer, J. Zweimueller-Mayer, P. Steinbacher, A. Lametschwandtner, H.C. Bauer. The dual role of zonula occludens (ZO) proteins J. Biomed. Biotechnol. p. 402593, 2010.
29.Foster, D. M., & Kellum, J. A. Endotoxic Septic Shock: Diagnosis and Treatment. International journal of molecular sciences. 24(22):16185, 2023.
30.Skrzypczak-Wiercioch A, Sałat K. Lipopolysaccharide-Induced Model of Neuroinflammation: Mechanisms of Action, Research Application and Future Directions for Its Use. Molecules. 27(17):5481, 2022.
31.Hersoug LG, Moller P, Loft S. Gut microbiota-derived lipopolysaccharide uptake and trafficking to adipose tissue: implications for inflammation and obesity. Obes Rev. 17:297-312, 2016.
32.Bohan R , Tianyu X , Tiantian Z , et al. Gut microbiota: a potential manipulator for host adipose tissue and energy metabolism. J Nutr Biochem. 64:206-17, 2019.
33.Massier Lucas , Blüher Matthias , Kovacs Peter , Chakaroun Rima M. Impaired Intestinal Barrier and Tissue Bacteria: Pathomechanisms for Metabolic Diseases. Frontiers in Endocrinology. 12:1664-2392, 2021.
34.Luissint AC, Parkos CA, Nusrat A. Inflammation and the Intestinal Barrier: Leukocyte-Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair. Gastroenterology. 151(4):616-632, 2016.
35.Massier Lucas , Blüher Matthias , Kovacs Peter , Chakaroun Rima M. Impaired Intestinal Barrier and Tissue Bacteria: Pathomechanisms for Metabolic Diseases. Frontiers in Endocrinology. 12:1664-2392, 2021.
36.Suriano F, Vieira-Silva S, Falony G, et al. Novel insights into the genetically obese (ob/ob) and diabetic (db/db) mice: two sides of the same coin. Microbiome. 9(1):147, 2021.
37.Wang B, Chandrasekera PC, Pippin JJ. Leptin- and leptin receptor-deficient rodent models: relevance for human type 2 diabetes. Curr Diabetes Rev. 10:131–45, 2014.
38.Brun P, Castagliuolo I, Di Leo V, Buda A, Pinzani M, Palu G, et al. Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis. Am J Physiol Gastrointestinal Liver Physiol. 292:G518–25, 2007.
39.Cani PD, Bibiloni R, Knauf C, Waget A, Neyrinck AM, Delzenne NM, et al. Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice. Diabetes. 57:1470–81, 2008.
40.Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, et al. Metabolic Endotoxemia Initiates Obesity and Insulin Resistance. Diabetes. 56:1761–72, 2007.
41.Massier L, Blüher M, Kovacs P, Chakaroun RM. Impaired Intestinal Barrier and Tissue Bacteria: Pathomechanisms for Metabolic Diseases. Front Endocrinol. 12:616506, 2021.
42.Cani PD, Bibiloni R, Knauf C, Waget A, Neyrinck AM, Delzenne NM, et al. Changes in Gut Microbiota Control Metabolic Endotoxemia-Induced Inflammation in High-Fat Diet-Induced Obesity and Diabetes in Mice. Diabetes. 57:1470–81, 2008.
43.Kim K-A, Gu W, Lee I-A, Joh E-H, Kim D-H. High Fat Diet-Induced Gut Microbiota Exacerbates Inflammation and Obesity in Mice via the TLR4 Signaling Pathway. PloS One. 7:e47713, 2012.
44.Million M, Maraninchi M, Henry M, Armougom F, Richet H, Carrieri P, et al. Obesity-Associated Gut Microbiota Is Enriched in Lactobacillus Reuteri and Depleted in Bifidobacterium Animalis and Methanobrevibacter Smithii. Int J Obes. (2012) 36:817–25, 2005.
45.Lecomte V, Kaakoush NO, Maloney CA, Raipuria M, Huinao KD, Mitchell HM, et al. Changes in Gut Microbiota in Rats Fed a High Fat Diet Correlate With Obesity-Associated Metabolic Parameters. PloS One. 10:e0126931, 2015.
46.Mu Q, Kirby J, Reilly CM, Luo XM. Leaky Gut As a Danger Signal for Autoimmune Diseases. Front Immunol. 8:598, 2017.
47.Ying-Cheng Lin, Ta-Jen Wu, Horng-Yih Ou. Fructose and Metabolic Syndrome. Journal of Internal Medicine of Taiwan. 25(6), 2014.
48.Lodge. M., Dykes. R., Kennedy. A. Regulation of Fructose Metabolism in Nonalcoholic Fatty Liver Disease. Biomolecules. 14:845, 2024.
49.Jang C., Hui S., Lu W., Cowan A.J., Morscher R.J., Lee G., Liu W., Tesz G.J., Birnbaum M.J., Rabinowitz J.D. The Small Intestine Converts Dietary Fructose into Glucose and Organic Acids. Cell Metab. 27:351–361, 2018.
50.Merino, B., Fernández-Díaz, C. M., Cózar-Castellano, I., & Perdomo, G.. Intestinal Fructose and Glucose Metabolism in Health and Disease. Nutrients. 12(1):94, 2019.
51.Khitan, Z., & Kim, D. H. Fructose: a key factor in the development of metabolic syndrome and hypertension. Journal of nutrition and metabolism. 2013:682673, 2013.
52.Bhanot S, McNeill J H, Bryer-Ash M. Vanadyl sulfate prevents fructose-induced hyperinsulinemia and hypertension in rats. Hypertension. 23(3): 308 – 312, 1994.
53.Nakagawa T, Hu H, Zharikov S, Tuttle KR, Short RA, Glushakova O, Ouyang X, Feig DI, Block ER, Herrera-Acosta J, Patel JM, Johnson RJ. A causal role for uric acid in fructose-induced metabolic syndrome. American journal of physiology. 290(3): 625-631, 2006.
54.Tobey T.A., Mondon C.E., Zavaroni I., Reaven G.M. Mechanism of insulin resistance in fructose-fed rats, Metabolism. 31(6): 608-612, 1982.
55.Ivana Zavaroni, Susan Sander, Sally Scott, Gerald M. Reaven. Effect of fructose feeding on insulin secretion and insulin action in the rat. Metabolism. 29(10): 970-973, 1980.
56.Carlos Magno M.R. Barros, Rosane Q. Lessa, Mauricio P. Grechi, Tanial L.M. Mouço, Maria das Graças C. Souza, Nicolas Wiernsperger, Eliete Bouskela. Substitution of drinking water by fructose solution induces hyperinsulinemia and hyperglycemia in hamsters. Clinics. 62(3): 327-334, 2007.
57.Wallace, T. M., Levy, J. C., & Matthews, D. R. Use and abuse of HOMA modeling. Diabetes care. 27(6): 1487–1495, 2004.
58.Srinivasan, B., Kolli, A. R., Esch, M. B., Abaci, H. E., Shuler, M. L., Hickman, J. J. TEER measurement techniques for in vitro barrier model systems. Journal of laboratory automation, 20(2): 107–126, 2015.
59.Doridot, L., Hannou, S. A., Krawczyk, S. A., Tong, W., Kim, M. S., McElroy, G. S., Fowler, A. J., Astapova, I. I., & Herman, M. A. A Systems Approach Dissociates Fructose-Induced Liver Triglyceride from Hypertriglyceridemia and Hyperinsulinemia in Male Mice. Nutrients, 13(10): 3642, 2021.
60.Kawabata, K., Kanmura, S., Morinaga, Y., Tanaka, A., Makino, T., Fujita, T., Arima, S., Sasaki, F., Nasu, Y., Tanoue, S., Hashimoto, S., Ido, A. A high‑fructose diet induces epithelial barrier dysfunction and exacerbates the severity of dextran sulfate sodium‑induced colitis. International Journal of Molecular Medicine. 43(3):1487-1496, 2019.
61.Huanlong Qin, Zhiguang Gao. Research progress on the role of intestinal epithelial cell tight junctions in intestinal barrier. World Chinese Journal of Digestology. 13(4): 443-447, 2005.