類黃酮素與大鼠小腸之葡萄糖轉運蛋白作用關係之探討

Abstract

類黃酮素是屬於一種多酚類的化合物，廣泛分佈於各種食物或藥用植物中，大部分的類黃酮素是以β-配醣體(β-glycosides)的型式存在於食物中。在之前的研究中認為這些配醣體不能被小腸所吸收只有非醣體才能穿過腸道細胞膜被吸收。然而，最近的研究指出類黃酮素的吸收似乎和它所接上的醣基有關，但是相關的機轉仍然有待釐清。本實驗的目的是想藉由分離刷狀外緣細胞膜微粒(brush border membrane vesicles；BBMV)和微血管側細胞膜微粒(basolateral membrane vesicles；BLMV)來說明類黃酮素和小腸葡萄糖轉運蛋白之間的作用關係。Wistar大白鼠空腸的BBMV或BLMV是分別利用不同的離心速度或密度梯度去進行分離。實驗則利用快速過濾技術去測量flavones、flavonols、flavanones、isoflavones 和catechins對於氚(3H)標定的葡萄糖在BBMV和BLMV中攝取量的差別。除此之外，更進一步的去探討quercetin-3-O-β-D-glucoside (Q3G)和epicatechin gallate (ECG)的吸收機轉。結果顯示類黃酮素會抑制葡萄糖經由鈉離子依賴型的葡萄糖轉運蛋白(SGLT1)和促進型的葡萄糖轉運蛋白(GLUT2)的攝取。在BBMV中，類黃酮素對於葡萄糖攝取量的抑制程度為ECG > hesperetin ≒ Q3G ≒ naringenin > genistein ≒ genistin >其他類黃酮素。 在BLMV中，類黃酮素對於葡萄糖攝取量的抑制程度為ECG > Q3G ≒ fisetin ≒ gossypin ≒ genistein > naringin ≒ naringenin ≒ quercetin-3-O-β-D-galactoside ≒ daidzin > hesperetin > daidzein ≒ genistin >其他類黃酮素。不同的位置的羥基取代則可能會影響抑制小腸的葡萄糖轉運蛋白運送葡萄糖的效果。更進一步的研究顯示，Q3G和ECG會以競爭型的機轉去抑制BBMV和BLMV的葡萄糖攝取。在BBMV中，Q3G和ECG的Ki值大約分別為499 uM和170 uM；在BLMV中，Q3G和ECG的Ki值大約分別為404 uM和332 uM。所以它們的取代基位置對於葡萄糖轉運蛋白確認這些類黃酮素似乎是相當重要的。而Q3G和ECG也有可能是SGLT1和GLUT2的受質。

Flavonoids are polyphenolic compounds that are widely distributed in foods or herbal medicines. Most flavonoids are present in food as β-glycosides. It was thought that such glycosides could not be absorbed from the intestine, and only aglycons could pass through the gut wall. However, recent evidences indicated that the absorption of flavonoids would be dependent on conjugated sugars, whereas the absorption mechanisms are still far from clear. The objective of this study was to elucidate the interaction mechanisms between flavonoids and intestinal glucose transporters using isolated brush-border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV). BBMV and BLMV were isolated from Wistar rat jejunums by differential centrifugation and density gradient centrifugation, respectively. Uptakes of 3H-glucose by BBMV and BLMV were measured using a rapid filtration technique in the absence or the presence of flavones, flavonols, flavanones, catechins, and isoflavones. In addition, the uptake mechanism of quercetin-3-O-β-D-glucoside (Q3G) and epicatechin gallate (ECG) were further explored. It was showed that flavonoids inhibited glucose uptake by sodium-dependent glucose transporter (SGLT1) and facilitative glucose transporter (GLUT2). In BBMV, the order of inhibitory degree was ECG > hesperetin ≒ Q3G ≒ naringenin > genistein ≒ genistin > other flavonoids. In BLMV, the order of inhibitory degree was ECG > Q3G ≒ fisetin ≒ gossypin ≒ genistein > naringin ≒ naringenin ≒ quercetin-3-O-β-D-galactoside ≒ daidzin > hesperetin > daidzein ≒ genistin > other flavonoids. The substituted positions for the hydroxyl groups in flavonoids could affect the inhibitory activity on glucose uptake by intestinal glucose transporters. Further studies showed that Q3G and ECG competitively inhibited glucose uptake in both BBMV and BLMV. In BBMV, the Ki values for Q3G and ECG were proximately 499uM and 170 uM, respectively. In BLMV, the Ki values for Q3G and ECG were proximately 404 uM and 332 uM, respectively. Their substituted positions would be important for the recognition of flavonoids by intestinal glucose transporter. Also, it was suggested that Q3G and ECG could be substrates of SGLT1 and GLUT2.