以 FL83B 肝臟細胞株汲取葡萄糖及 HIT-T15 胰臟 β 細胞株胰島素分泌為平台研究山苦瓜之血糖調節活性萃物

Studies on the Extracts of Momordica charantia L. that enhance Glucose Uptake in FL83B Hepatic Cell Line and Insulin Secretion of HIT-T15 Islet Beta Cell Line

馮文嘉(Wen-Chia Feng)

指導教授 ： 黃青真

國立臺灣大學/生命科學院/生化科技學系/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10825

摘要

第二型糖尿病病發病前會經歷一段正常與糖尿病間的過渡時期，稱之為糖尿病前期。目前研究認為，糖尿病前期為『飲食介入』預防糖尿病發生之重要時期。過去本研究室發現，苦瓜經 β-glucosidase 處理後的水萃物和小分子乙酸乙酯萃物比未經酵素處理的樣品更具有降血糖活性，但其詳細降血糖有效成分與機制則尚未被確認。因此，本研究建立以螢光標定葡萄糖，2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4yl) amino]-2-deoxyglucose ( 2-NBDG )，作為偵測肝細胞株 FL83B 汲取葡萄糖模式平台；並以胰臟 beta 細胞之胰島素分泌作為模式平台，來測試苦瓜各式萃物、水解物、區分物及分離物之活性，並探討可能的活性成分與其影響細胞的可能機制。
結果顯示，FL83B 肝臟細胞以處理 15 小時之條件最能顯現苦瓜萃物促葡萄糖汲取之活性。水解苦瓜樣品中以強酸水解產物增加 FL83B 細胞汲取葡萄糖的量較酵素水解產物高。弱酸水解區分物皆顯著抑制細胞對葡萄糖之汲取。胰島beta細胞株部分，唯果汁正丁醇萃物與其經酵素水解的產物可顯著增加細胞分泌胰島素，其餘萃物或區分物沒有顯著影響或有顯著抑制效果。代謝體學分析比較經山苦瓜水萃物處理之細胞與對照組細胞，推測，可能受到樣品影響的代謝路徑，在 FL83B 中為glycerophospholipid、purine、glutathione 及 phenylalanine 的代謝；在 HIT-T15 細胞中則為 purine、glutathione、taurine and hypotaurine、cysteine 及 methionine 的代謝。另外，本研究亦發現adenyl cyclase 抑制劑 SQ22536 可部分抑制HIT-T15 細胞受山苦瓜果汁萃物刺激之胰島素分泌，而 ATP 敏感性鉀通道開放劑 diazoxide、phosphollipase C 抑制劑 U73122 以及 proteinkinase A 抑制劑H89則否。由本研究的結果可知山苦瓜萃物具調節血糖潛力的有效成分似非單一化合物，而其的確可有效的調節肝臟細胞對葡萄糖的汲取並可部分透過影響 adenylyl cyclase 來促進胰臟 beta 細胞分泌胰島素。

關鍵字

糖尿病 ； 肝臟細胞株 ； 胰臟 β 細胞株 ； 山苦瓜 ； 代謝體學

並列摘要

Type 2 diabetes has become a significant public health concern. The “prediabetic” period precede the development of type 2 diabetes are critical for dietary interventions. The water extract(WE) of Bitter gourd (Momordica charantia,MC) (BG) and the ethyl acetate (EA) extract of hydrolyzed small molecules fraction of WE were shown to enhance GLP-1 secretion in STC-1, an enteroendocrine cell line, increase insulin secretion in beta-cell lines, and promote glucose uptake in hepatocyte, adipocytes and myocytes. But specific hypoglycemic active ingredients and mechanisms have not been confirmed. This study thus aimed to examining in more details the activities of hydrolyzed extracts/fractions/isolates of wild BG (WBG) using glucose uptake of hepatocytes ( FL83B hepatic cell line) and insulin secretion of islet beta cells (HIT-T15 beta cell line). Possible mechanisms were preliminarily explored by using metabolomics analysis and specific inhibitor in insulin secretion pathway.
The glucose uptake of FL83B cells was determined by using the uptake of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG), a fluorescent analog of d-glucose. Pre-treatment of FL83B cells with WBG water extracts for 15 hr was found to significantly increased glucose uptake. Among various hydrolyzed extracts/fractions/isolates of WBG, the hexane extract of the 15% HCl hydrolyzeds product, butanol extract of enzyme hydrolyzed product and ethyl acetate extract of 2% HCl hydrolyzed product significantly enhanced glucose uptake of FL83B.In contrast, extracts of remaining hydrolyzed products, and fractions isolated significantly inhibited glucose uptake of FL83B.
Insulin secretion was measured after HIT-T15 cells were treated with 10mM glucose or WBG extract/fractions for 1 hr. Compared to the results of glucose uptake of FL83B cells, insulin secretion was significantly increased only in cells treated with butanol extract of WBG juice, and the EA or butanol extracts of enzymatic hydrolysis products of WBG juice, suggesting different active compounds in WBG acting on different target cells. .
LC-TOF-MS was used to compare metabolome of cells treated with WE, insulin or vehicles. Human metabolome dataset (HMDB) and KEGG database was used for predicting the regulated compounds and mapping possible biochemical pathways involved. The metabolic pathways affected include glycerophospholipid, purine, glutathione and phenylalanine metabolism in FL83B cell and purine, glutathione, taurine and hypotaurine, cysteine and methionine metabolism in HIT-T15 cell.
Finally, the WBG juice stimulated insulin secretion of HIT-T15 cells was partially inhibited by the adenyl cyclase inhibitor SQ22536, but not by ATP-sensitive K channel agonist diazoxide, or phospholipase C inhibitor U73122 or protein kinase A inhibitor H89. The result suggested that WBG juice may stimulate insulin secretion in beta cells through activating adenylyl cyclase, at least in part. In conclusion, this study demonstrated that the hypoglycemic activity of WBG might not be attributed to a single compound and indeed, WBG extracts could enhance glucose uptake in hepatic cell line and increased insulin secretion of islet beta cell line through activating adenylyl cyclase, at least in part.

並列關鍵字

Type 2 diabetes ； hepatic cell line ； islet beta cell line ； wild bitter gourd (Momordica Charantia) ； metabolomics

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