| 摘要: | 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. |
