Effect of Kefir on Glucose Uptake in Skeletal Muscle

糖尿病是一種終身性的疾病，可能會導致嚴重的併發症，其中，第二型糖尿病 (Type 2 diabetes mellitus) 佔全球糖尿病患者的 90％，胰島素阻抗為第二型糖尿病的重要特徵，導致血糖升高與脂肪代謝異常。克菲爾具有調節血糖、血脂以及抗發炎等生理活性，但其作用機制與具降血糖之生物活性物質仍不清楚。本研究採用呈現胰島素阻抗之 L6 肌肉細胞作為測試平台，針對傳統克弗爾與乳酸菌、酵母菌共培養之發酵乳，研究其對葡萄糖代謝之調節功效。研究結果顯示，克菲爾發酵時間越長，L6 細胞對葡萄糖的汲取量有顯著增加且具劑量效應，測其成分含量變化可以發現，隨著發酵時間增加，其胜肽鏈長逐漸變短、蛋白質水解率有顯著之提升，再經膜過濾 (M.W≦1 kDa) 處理後，分子量 ≦1 kDa在刺激葡萄汲取能力表現上，發酵後期的顯著差異不及發酵前期來得明顯，推測克弗爾水萃物中具調節血糖的物質可能為小分子蛋白質及胜肽，而這些小分子胜肽隨著發酵時間的增加，產量亦逐漸提升。此外，利用抑制劑試驗之結果顯示，傳統克弗爾於 96 小時發酵水萃物具活化AMPK分子傳導訊息，而傳統克弗爾於 12 及 24 小時發酵之水萃物與胰島素訊息傳導型態是類似的，主要是利用 PI3K 訊息傳導途徑顯著地影響葡萄糖汲取能力，於 37℃共培養克弗爾組別中，於 12 與 96 小時發酵水萃物不僅具有活化 PI3K 訊息分子之能力亦可活化非胰島素訊息傳導途徑 AMPK 訊息分子，使葡萄糖轉運具有加成性。本實驗進一步以陰離子交換層析分離純化克弗爾水萃物，測定區分物對刺激L6細胞對葡萄糖汲取之活性，結果顯示，區分物在 10 µg/ml 有助於刺激細胞汲取葡萄糖的能力，與僅經膜過濾處理之 100 µg/ml 水萃液具有相似之效果。接著，再以抑制劑探討有效區分層之分子傳導訊息，結果顯示，對刺激 L6 細胞汲取葡萄糖具有最佳效果之區分層 (fraction 5) 可藉由活化胰島素依賴型 PI3K 途徑與非胰島素依賴型 AMPK 途徑，提高葡萄糖汲取之能力，使葡萄糖轉運作用具有加成性，表示經陰離子交換層析之區分物具調節血糖之潛力，然而，具有調節血糖之有效成分組成為何，仍需待進一步確定。Diabetes mellitus (DM) is a predominant chronic disease which causes mortality of millions of people yearly. Insulin resistance is a key etiology of Type 2 DM. Kefir is a fermented milk which composed a specific mixture of bacteria and yeast cultures, and have received increasing attention as potential ingredients of health-promoting functional foods. Past studies revealed that kefir can improve diet-related chronic diseases, such as obesity, systemic inflammation, hyperglycemia, hypercholesterolemia and type 2 DM, but the exact mechanism is still unknown. In our study, a free fatty acid induced insulin resistant cell model was first established. We found that when pH is higher than 4.5, the lactic acid bacteria and the yeast in kefir could help each other grow up steadily. With longer fermentation time the glucose uptake activity of L6 cell became higher and along with dose response. According to results of ultrafiltration (1 kDa), it indicated that the active compound might be small molecular protein and peptide. The water extract compound from 12 hours and 96 hours fermentation under 37℃ not only activated AMPK signaling molecules, but also PI3K signaling molecules to induce GLUT4 translocation. The water extract compound from 12 hours and 24 hours fermentation of traditional kefir and 24 hours fermentation under 37℃ primary activated PI3K signaling molecules, the signaling molecules pathway is similar with insulin. The water extract compound from 96 hours fermentation of traditional kefir primary activated AMPK signaling molecules. Furthermore, the water crude extracts was fractionated by anion-exchange chromatography. It was found that the fraction 5 of co-culture kefir at 37℃ has high glucose uptake activity, and it both activated AMPK and PI3K signaling molecules to induce GLUT4 translocation