液化澱粉芽孢桿菌的胞外多醣活化苦味接受器亞型之研究

Study on the activation of bitter-taste receptor isoforms by the exopolysaccharides of Bacillus amyloliquefaciens

涂景閎(Jing-Hong Tu)

指導教授 ： 鄭雪玲

國立屏東科技大學/農學院/生物科技系/碩士(2019年)

https://doi.org/10.6346/NPUST202000053

摘要

液化澱粉芽孢桿菌為動物的益生菌，其胞外多醣(exopolysaccharides, EPS) 證實對小鼠有降血糖效果，且能刺激glucagon-like peptide-1 (GLP-1)的分泌，但其作用機制尚不清楚。EPS具有苦味，而有研究證明某些苦味化合物能藉由活化苦味接受器(TAS2R)來誘導GLP-1分泌。而GLP-1有調節血糖的功能。故本論文提出EPS可能因活化苦味接受器而刺激GLP-1分泌的假設，並加以驗證。同時進一步分析EPS活化哪些苦味接受器亞型。人類腸道內分泌細胞株NCI-H716細胞表現人類全部25種TAS2R亞型，並在TAS2R被活化後分泌GLP-1。以EPS刺激NCI-H716細胞，能使該細胞分泌GLP-1，並出現細胞質[Ca2+ ]增加的現象，後者為TAS2R被活化的指標之一。以TAS2R14抗體，或TAS2R訊息傳遞路徑的抑制劑前處理NCI-H716細胞，會抑制EPS誘導的[Ca2+ ]變化及GLP-1分泌。以Gα-gustducin基因轉殖HEK293T細胞，建立Gα-gustducin穩定轉染株，再轉染各TAS2R亞型的基因，建立單一TAS2R的異源表現系統。以此系統進行研究，發現EPS可明顯活化TAS2R14及TAS2R38。這些結果顯示EPS會活化TAS2R，其中TAS2R14及TAS2R38可明顯被EPS活化，且TAS2R14活化與EPS刺激NCI-H716細胞分泌GLP-1有顯著的關聯性。本研究結果提供新的線索解釋有些益生菌具有調節血糖功能的可能分子機制。

關鍵字

液化澱粉芽孢桿菌 ； 胞外多醣 ； 苦味接受器

並列摘要

Bacillus amyloliquefaciens is a probiotic for animals. The exopolysaccharides (EPS) of Bacillus amyloliquefaciens have been shown to have a hypoglycemic effect and increase plasma glucagon-like peptide-1 (GLP-1) level in mice, but the underlying mechanism is unclear. The solution of EPS tastes bitter. Reports demonstrated that some compounds could induce the secretion of GLP-1 via the activation of bitter taste receptor signaling pathway. GLP-1 is known to modulate the level of blood glucose. Therefore, EPS was proposed to stimulate GLP-1 secretion by activating bitter taste receptors (TAS2Rs). This proposal was examined and the isoforms activated by EPS were further elucidated in this study. The human enteroendocrine cell line NCI-H716 cells express all 25 isoforms of human TAS2Rs, and the activation of TAS2Rs can induce GLP-1 secretion from this cell lines. EPS induced GLP-1 secretion and increased cytosolic calcium concentration in NCI-H716 cells, the latter is a biomarker of TAS2R activation. The EPS-induced calcium signal and GLP-1 secretion were inhibited by TAS2R14 antibody and by inhibitors of the TAS2R signaling pathway. Morever, a stable clone of HEK293T cells expressing Gα-gustducin was constructed, and the genes of TAS2R isoforms were transfected into the stable clone individually to establish a heterologous system expressing single TAS2R isoforms. This system was used to study the activation of TAS2R isoforms by EPS. Consequently, EPS was found to obviously activate TAS2R14 and TAS2R38. There results demonstrated that EPS activates TAS2Rs, and TAS2R14 as well as TAS2R38 are obviously activated. Futhermore, the activation of TAS2R14 is closely related to the EPS-induced secretion of GLP-1 from NCI-H716 cells. This study offers a new clue for the mechanisms underlying the effect of glycemic control by some probiotics.

並列關鍵字

Bacillus amyloliquefaciens ； exopolysaccharides ； bitter taste receptors ； glucagon-like peptide-1

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