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研究生:吳靜怡
研究生(外文):Ching-Yi Wu
論文名稱:補充生物素對於改善第2型糖尿病小鼠葡萄糖代謝與調節脂肪及骨骼肌細胞中GLUT4之作用探討
論文名稱(外文):Effect of Biotin Supplementation on Improving Glucose Metabolism and Modulating GLUT4 in Adipose and skeletal Muscle cells of Type 2 Diabetic KK Mice
指導教授:張毓芬張毓芬引用關係
指導教授(外文):Yu-Feng Chang
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:106
中文關鍵詞:生物素第2型糖尿病KK小鼠葡萄糖轉運蛋白
外文關鍵詞:biotintype 2 diabetic KK mice
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中文摘要
胰島素刺激肌肉和脂肪組織對於葡萄糖的利用與代謝能力的下降是第2型糖尿病的主要特徵,也是導致周邊胰島素阻抗性的重要原因。對於胰島素的作用敏感的肌肉與脂肪組織細胞中含有葡萄糖轉運蛋白(glucose transporters, GLUTs)可協助葡萄糖通過細胞膜而進入細胞內,其中葡萄糖轉運蛋白4(glucose transporter 4, GLUT4)是最主要會受胰島素所調節的葡萄糖轉運蛋白。本研究室過去的研究發現,給予生物素補充有助於改善糖尿病動物與人體內的空腹血糖以及葡萄糖耐受性異常,並可增加胰島素標的組織細胞的胰島素敏感性而使胰島素阻抗性下降,然而此作用機制與GLUT4的相關性並不清楚。
因此,本研究目的主要是探討給予生物素補充是否會增加第2型糖尿病小鼠脂肪組織細胞與骨骼肌細胞中GLUT4蛋白質的表現量及位移至細胞膜的作用。本研究以第2型糖尿病KK小鼠作為動物試驗模式,先以高脂飲食誘導糖尿病症狀後,分成3組給予不同劑量的生物素補充,分別為0(對照組)、3及6 mg/Kg of body wt,為期四週。分別在補充前、補充第二週及補充第四週結束時進行空腹血糖值、血清胰島素濃度與葡萄糖耐受性試驗,並在犧牲前30分鐘分成兩種處理方式,即給予或不給予胰島素的刺激。血糖濃度是以standard glucose oxidase的方法分析,血清胰島素以electrochemiluminescence immunoassay(ECLIA)的方法測定。並以西方墨點法(Western blottong)分析脂肪組織及骨骼肌細胞分別在漿膜(plasma membrane, PM)與低密度微粒體(low density microsome, LDM)部份的GLUT4蛋白質表現量。
結果顯示,不論犧牲前是否有胰島素的刺激,兩組生物素補充組脂肪細胞中GLUT4在漿膜(PM)及低密度微粒體(LDM)的表現量均顯著高於對照組(p<0.05),而且GLUT4在漿膜(PM)部份之表現量所佔的比例(PM/(PM+LDM))也顯著較高;至於GLUT4在骨骼肌細胞漿膜(PM)部份之表現量所佔比例(PM/(PM+LDM))也以生物素補充組較高(p<0.05)。因此推論,補充生物素可改善糖尿病KK小鼠的葡萄糖代謝,可能是藉由增加脂肪組織與骨骼肌細胞中GLUT4的位移作用或表現量。
Abstract
Reduced ability of insulin to stimulate glucose uptake and metabolism in muscle and adipose tissue is a major defect in type 2 diabetes mellitus as well as an important causative factor of peripheral insulin resistance in these patients. Insulin-sensitive peripheral tissues, such as muscle and adipose tissues, contain at least two different glucose transporter isoforms, and glucose transporter 4 (GLUT4) is the major insulin-regulated transporter. Our previous study has demonstrated that biotin supplement may improve impaired fasting glucose and glucose tolerance in type 2 diabetes patients and animal models and improve insulin sensitivity in target cells to reduce insulin resistance, but this mechanism related with GLUT4 is not clear.
Thus, we investigated whether biotin supplementation may increase GLUT4 protein expression level and translocation in adipose and skeletal muscle cells of type 2 diabetic KK mice. Forty eight type 2 diabetic KK mice were fed high fat diet to induce diabetic syndrome. Then they were divided into three groups : control group was given 0 mg biotin/kg of body wt per day, 3mg group was given 3 mg biotin/kg of body wt per day, and 6mg group was given 6 mg biotin/kg of body wt per day for 4 weeks. Fasting blood glucose levels, serum insulin levels, insulin resistance, and glucose tolerance test were tested on 0, 2, and 4 weeks after biotin supplementation for all experiment groups. Thirty minutes before sacrificed each groups were divided into insulin-treated or untreated. Blood glucose levels were determined by standard glucose oxidase method. Serum insulin levels were determined by electrochemiluminescence immunoassay (ECLIA). Insulin resistance was determined by “homeostasis model assessment” (HOMA) formula. After 4 weeks, the mice were sacrificed and epididymal adipose tissue and red gastrocnemius muscle were isolated. GLUT4 expression in the plasma membrane (PM) and the low density microsome (LDM) were determined by western blotting.
The results showed that the basal and insulin-induced GLUT4 expression cells in plasma membrane (PM) and the low density microsome (LDM) of the adipose tissue from biotin supplementation groups were significantly increased compared with control group (p<0.05). GLUT4 expression ratio in the plasma membrane (PM/(PM+LDM)) of the adipose and skeletal muscle tissues were also significantly higher in biotin supplementation groups (p<0.05). Therefore, we speculated that biotin supplementation may improve glucose metabolism in type 2 diabetic KK mice by increasing GLUT4 expression and translocation in adipose and skeletal muscle tissues.
目錄
目錄………………………………………………………………………...Ⅰ
圖目錄……………………………………………………………………Ⅴ
表目錄…………………………………………………………………….Ⅸ
縮寫表…………………………………………………………………...ⅩⅠ
中文摘要……………………………………………………………….ⅩⅡ
英文摘要……………………………………………………………….ⅩⅣ
第一章 、緒言……………………………………………………………...1
第二章 、文獻整理………………………………………………………...3
壹、 糖尿病……………………………………………………….3
一、糖尿病的分類………………………………………………3
二、第2型糖尿病與胰島素阻抗性……………………………5
貳、 生物素……………………………………………………….6
一、簡介生物素………………………………………………….6
二、生物素與脂肪及糖類代謝的關係…………………………8
三、生物素對於糖尿病之葡萄糖代謝的影響………………..10
參、 胰島素……………………………………………………...13
一、 胰島素的釋出與訊息傳導…………………………….13
二、 胰島素阻抗…………………………………………….16
肆、 葡萄糖轉運蛋白…………………………………………..22
一、葡萄糖轉運蛋白4的作用機制……………………………23
二、葡萄糖轉運蛋白4與胰島素阻抗性之關係………………25
第三章 、研究目的………………………………………………………28
第四章 、材料方法與試驗設計………………………………………….29
壹、 試驗動物、飼料與飼養環境……………………………..29
一、 試驗動物……………………………………………….29
二、 飼料與墊料…………………………………………….30
三、 飼料成分……………………………………………….30
四、 生物素的補充…………………………………………..32
五、 KK/HIJ品細小鼠之飼養………………………………32
六、 飼養環境條件………………………………………….32
七、 試驗分組………………………………………………..33
貳、 試驗設計…………………………………………………34
參、 測試項目…………………………………………………..31
一、 葡萄糖耐受性試驗……………………………………..37
二、 空腹血糖質之測定………………………………………37
三、 血漿膽固醇、血漿三酸甘油酯之濃度測定……………..38
四、 血清胰島素濃度………………………………………..38
五、 胰島素阻抗性…………………………………………..38
六、 骨骼肌與副睪脂肪組織中GLUT4的測定…………….39
肆、 統計分析…………………………………………………...55
第五章 、結果…………………………………………………………….56
壹、 補充生物素對於體重及血液生化檢測值之變化………56
一、 體重變化………………………………………………..56
二、 血液生化檢測值之變化………………………………..57
貳、 補充生物素對於空腹血糖與胰島素阻抗性之變化……..59
一、 空腹血糖……………………………………………….59
二、 葡萄糖耐受性試驗…………………………………….61
三、 血清胰島素濃度……………………………………….65
四、 胰島素阻抗性…………………………………………..66
參、 補充生物素對於GLUT4蛋白質表現之影響…………..68
一、 副睪脂肪中GLUT4之表現……………………………68
二、 骨骼肌肉中GLUT4之表現…………………………….78
第六章 、討論…………………………………………………………….88
壹、 補充生物素對於空腹血糖及葡萄糖耐受性的影響……..88
貳、 補充生物素對血清胰島素濃度、血漿膽固醇和三酸甘油酯濃度的影響…………………………………….……….….90
參、 補充生物素對胰島素阻抗性與空腹血糖值的影響……...91
肆、 補充生素對脂肪細胞GLUT4的表現與位移作用的影響91
伍、 補充生物素對骨骼肌細胞GLUT4的表現與位移作用的影響…………………………………………………………..94
第七章 、結論…………………………………………………………….97
第八章 、參考文獻…………………………………………………….98
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