以C2C12肌肉細胞模式探討神秘果抽出物調控葡萄糖攝入及胰島素訊號路徑之機制

Modulatory effects of the miracle fruit extract on glucose uptake and insulin signaling pathway as elucidated by C2C12 myotubes cells study

韓怡君(Han Yi-Chun)

指導教授 ： 王進崑

中山醫學大學/健康管理學院/營養學系/博士(2020年)

https://doi.org/10.6834/csmu202000277

摘要

糖尿病(Diabetes mellitus) 乃國人常見之慢性代謝症候群，我國盛行率約10%，明顯高於全球盛行率3%，且罹患人數有逐年成長之趨勢，目前根據107及108年衛福部國人十大死因調查，糖尿病高居國人十大死因的第五位，且患病者有年輕化的趨勢。世界衛生組織(WHO) 也預估30年後全球約有近4億人罹患糖尿病。因此，糖尿病之防治仍是目前面臨的重大課題。
傳統西醫治療糖尿病皆以藥物介入為主，但因個體之差異，往往造成適應性不同、副作用等情形，因此近年來，糖尿病研究多著重於替代性或營養品補充療法，如中草藥、天然成分或植物萃取物，以提供糖尿病患者複合治療之選擇，也可做為預防糖尿病之管道。未來，期待能以天然物或植物成分發展出預防或延緩糖尿病病程之介入策略。
神秘果(Synsepalum dulcificum, 又為Miracle fruit)，因含有味覺修飾蛋白神秘果素(Miraculin)，故常作為甜味劑使用，能調整舌頭味覺接受器，使酸性食物在攝取神秘果後，產生甜味味覺。研究指出神秘果具有調控熱量攝取之作用，且含有大量多酚類化合物。而動物實驗則發現，神秘果粉末具有降低胰島素阻抗性、增加胰島素分泌之功能。此外，神秘果果實具有抗氧化、抗糖尿病與調節動物血糖之作用。
本研究欲利用試管實驗及細胞模式探討神秘果對於血糖之調控能力及其中有效成分、可能之作用機制，主要分以兩部分進行：

一、C2C12細胞模式，神秘果乙醇萃取物調控葡萄糖攝入及胰島素之訊號路徑表現
將神秘果分為果皮+果肉及種子兩部位，並利用95%食用級乙醇進行減壓濃縮萃取，測試神秘果汁多酚類成分、抗氧化作用、抗糖化能力，且利用C2C12肌纖維母細胞進行2-NBDG 螢光葡萄糖之攝入試驗。
根據試驗結果顯示，以神秘果部位中果皮及果肉的抗氧化 (antioxidation)、抗糖基化 (antiglycation) 之效果表現最佳。而在肌肉細胞葡萄糖攝入實驗中，也發現不論是果肉或是種子皆有刺激2-NBDG之表現。其中又以果肉之刺激表現更高。在胰島素訊號傳遞之試驗中，也證實神秘果肉具有活化胰島素接受器(IR)、phosphatidylinositide 3-kinases (PI3K)及葡萄糖轉運子4(GLUT4)之表現。
因此，本實驗證實，神秘果果肉具有抗氧化、抗糖化之特性，且推測，神秘果乙醇萃取物可透過胰島素訊號傳遞路徑刺激肌纖維母細胞C2C12攝入葡萄糖之能力，達到調控血糖之效果。

二、探討不同神秘果抽出物調控C2C12肌肉細胞之葡萄糖攝入表現
為探討神秘果中調控葡萄糖攝入之有效活性成分，本試驗利用不同處理方式分離神秘果成分。經蛋白質試驗中顯示，以硫酸銨進行蛋白質鹽析沉澱後，再利用分子濃縮管脫鹽分離出不同分子量之神秘果蛋白質後，神秘果蛋白質純化物多介於10K及30K蛋白質，與神秘果素 (Miraculin,  24 kDa)之分子量相近。而在葡萄糖攝入實驗中，移除多酚類化合物可有利2-NBDG 螢光葡萄糖之攝入。本試驗仍有許多限制及不足，但根據近期研究顯示，食物中多酚類化合物之營養功能可能與蛋白質-多酚類之交互鍵結相關，故本試驗朝探討神秘果中蛋白質-多酚類鍵結之功能與機制之方向。

關鍵字

神秘果 ； 第2型糖尿病 ； 糖化終產物 ； 胰島素訊號傳遞路徑 ； 蛋白質-多酚類交互作用

並列摘要

Diabetes mellitus (DM) is recognized as a chronic metabolic disease, the mainly type of diabetes is type 2 DM, which is also characterized by insulin resistance. The pharmaceutical therapy agents of diabetes have limited dosage because of undesirable side effects and fail to change the process of diabetic complications significantly. Therefore, numerous studies, included this present study have led to screen the novel material or compounds that effectively elevate glucose metabolism without side effects.
Miracle fruit, Synsepalum dulcificum, commonly known as an alternative sweetener, affects the tongue taste receptor to make the sour food subsequently consumed to sweet and its beneficial health effects, such as antioxidant, anti-inflammatory and anti-hyperglycemia. However, the anti-diabetic effects and the mechanism have not cleared yet. Hence, we divided our study into two parts:
PART I. Modulatory Effects of Miracle Fruit Ethanolic Extracts on Glucose Uptake Through the Insulin Signaling Pathway in C2C12 Mouse Myotubes Cells
This study aimed to investigate the anti-diabetic effects of ethanolic extracts of miracle fruit pulp (MF) and seed (MS) on the antiglycation, glucose uptake ability and the underlying mechanisms. Differentiated C2C12 myotubes was treated with MF or MS (1 - 1000 μg/mL) or positive control metformin (1 mM) in the presence or absence of insulin. Compared with metformin group, MF significantly increased the intake of 2- (N- (7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino) -2-deoxyglucose (2-NBDG). Determined by ELISA, MF can also up-regulated the expression of insulin receptor (IR), phosphatidylinositide 3-kinases (PI3K), and glucose transporter 4 (GLUT4). The current results reveal the prevention potential of the ethanolic extraction of miracle pulp on the treatment of type 2 diabetes mellitus in C2C12 muscle cell - type 2 DM model by activation of the IR-PI3K/ GLUT4 insulin pathway. Miracle fruit might be a novel candidate for type 2 diabetes functional food application.
PART II. Effects on Glucose Uptake of Miracle Fruit Protein Extracts with Different Molecular Weights in C2C12 Mouse Myotubes Cells
According to our last study, we would like to confirm the active compound in miracle fruit. Hence, this further study aimed to purified the active compound in miracle fruit which can increase the glucose uptake efficacy and antiglycation activity. Thus, we used polyvinylpolypyrrolidone (PVPP) to remove the polyphenols compounds, ammonium sulfate (AS) to precipitated protein, then disposed salt and low-molecular weight compounds, and separated the miracle fruit protein into different molecular weight parts by centrifugal filters protein concentrator (Molecular Weight Cut Off（MWCO）: 1K, 10K, 30K, 100K). Removal polyphenols of miracle fruit shows better ability to increase the glucose intake in the skeleton cells. After concentration the proteins, the 10K and 30K parts showed higher protein level. Even though this study has several limitations, our study results indicate that the nutritional properties and the functional components of miracle fruit, might be associated with protein-phenol relationships.

並列關鍵字

miracle fruit ； type 2 diabetes mellitus ； advanced glycation end products (AGEs) ； antiglycation ； insulin signal ； glucose uptake ； skeletal muscle ； C2C12 myotubes

參考文獻

李彥、魏秀岩、盧禁、路曉慶、劉艷薇、王金輝 (2011)。神秘果提取物對糖尿病大鼠降血糖作用的研究。廣東藥學院學報，1，p.70-73。

岳慶熙、徐信次 (2004)。神秘果栽培及神秘果素之應用。技術服務，58，p.28-31。

邱毅超、賴宏亮、陳建志 (2010)。神秘果的抗氧化及對STZ誘導的高血糖老鼠血糖調節作用之研究。屏東科技大學生物科技研究所碩士論文，p.1-66。

黃巨波、劉紅、盧聖樓、張浩 (2012)。神秘果種子蛋白質的提取與降糖效用研究。天然產物研究與開發。10，p.1441-1443。

Abate, G., Delbarba, A., Marziano, M., Memo, M., Uberti, D. (2015). Advanced glycation end products (AGEs) in food: focusing on Mediterranean pasta. Journal of Nutrition Food Science, 5(6), p.1