Li Wei, Sun Kaiji, Ji Yun, Wu Zhenlong, Wang Weiwei, Dai Zhaolai, Wu Guoyao
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China; and.
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China; and
J Nutr. 2016 May;146(5):964-9. doi: 10.3945/jn.115.228312. Epub 2016 Mar 30.
Glycine traditionally is classified as a nutritionally nonessential amino acid in humans and animals. Because of its abundance in the body and its extensive use via multiple pathways, requirements for glycine are particularly high in neonates. Our recent studies show that dietary glycine supplementation is needed for optimal intestinal development in piglets. Importantly, reduced concentrations of glycine in the lumen of the small intestine are associated with gut dysfunction in low-birth-weight piglets. However, the mechanisms responsible for the beneficial effects of glycine on the intestinal mucosal barrier are largely unknown.
This study tested the hypothesis that glycine may regulate the expression and distribution of tight junction (TJ) proteins, thereby contributing to intestinal mucosal barrier function.
Enterocytes isolated from the jejunum of a healthy newborn pig were propagated to establish a stable cell line. The cells were cultured with 0.05 mmol glycine/L (control; concentration in the small intestinal lumen of low-birth-weight piglets) or 0.25 or 1.0 mmol glycine/L for the indicated periods of time. Epithelial barrier integrity and expression and localization of TJ proteins were analyzed by using monolayer transepithelial electrical resistance (TEER) and paracellular permeability, Western blot, and immunofluorescence imaging.
Compared with controls, cells cultured with 0.25 or 1.0 mmol glycine/L increased TEER (P < 0.05) by 46-53% and 80-111%, respectively, at 60-72 h. Correspondingly, paracellular permeability was reduced (P < 0.05) by 6-21% and 18-27% for 0.25 or 1.0 mmol glycine/L treatment, respectively, at 36-72 h. Compared with controls, protein abundances for claudin-3, claudin-7, and zonula occludens (ZO) 3 were enhanced (25-33%, P < 0.05) by 0.25 and 1.0 mmol glycine/L at 8 h, whereas those for occludin, claudin-1, claudin-4, and ZO-2 were not affected. Compared with controls, 1.0 mmol glycine/L reduced the protein abundance of ZO-1 by 20% at 8 h (P < 0.05), but 0.25 mmol glycine/L had no effect. A glycine concentration of 0.25 mmol/L sustained the localization of claudin-7 and ZO-3 to the interface between enterocytes. Interestingly, 1 mmol glycine/L promoted the distribution of claudin-4 and claudin-7 to the cytosol and nucleus, and the localization of ZO-3 to the plasma membranes, while decreasing the distribution of ZO-1 at cell-cell contact sites, compared with control cells.
Physiologic concentrations of glycine support intestinal mucosal barrier function by regulating the abundance and distribution of claudin-7 and ZO-3 in enterocytes. Supplementation with glycine may provide an effective nutritional strategy to improve intestinal integrity in piglets.
传统上，甘氨酸在人和动物体内被归类为营养非必需氨基酸。由于其在体内含量丰富且通过多种途径广泛利用，新生儿对甘氨酸的需求量特别高。我们最近的研究表明，仔猪最佳肠道发育需要膳食补充甘氨酸。重要的是，低体重仔猪小肠腔内甘氨酸浓度降低与肠道功能障碍有关。然而，甘氨酸对肠道黏膜屏障有益作用的机制在很大程度上尚不清楚。
本研究检验了甘氨酸可能调节紧密连接（TJ）蛋白的表达和分布，从而有助于肠道黏膜屏障功能的假说。
从健康新生猪空肠分离的肠上皮细胞进行传代培养，建立稳定细胞系。将细胞分别用0.05 mmol/L甘氨酸（对照；低体重仔猪小肠腔内浓度）、0.25 mmol/L或1.0 mmol/L甘氨酸培养指定时间。通过单层跨上皮电阻（TEER）、细胞旁通透性、蛋白质印迹法和免疫荧光成像分析上皮屏障完整性以及TJ蛋白的表达和定位。
与对照组相比，在60 - 72小时时，用0.25 mmol/L或1.0 mmol/L甘氨酸培养的细胞TEER分别增加了46% - 53%和80% - 111%（P < 0.05）。相应地，在36 - 72小时时，0.25 mmol/L或1.0 mmol/L甘氨酸处理组的细胞旁通透性分别降低了6% - 21%和18% - 27%（P < 0.05）。与对照组相比，0.25 mmol/L和1.0 mmol/L甘氨酸在8小时时使claudin - 3、claudin - 7和闭合蛋白（ZO）3的蛋白丰度提高了25% - 33%（P < 0.05），而闭合蛋白、claudin - 1、claudin - 4和ZO - 2的蛋白丰度未受影响。与对照组相比，1.0 mmol/L甘氨酸在8小时时使ZO - 1的蛋白丰度降低了20%（P < 0.05），但0.25 mmol/L甘氨酸无此作用。0.25 mmol/L的甘氨酸浓度使claudin - 7和ZO - 3维持在肠上皮细胞之间的界面定位。有趣的是，与对照细胞相比，1 mmol/L甘氨酸促进claudin - 4和claudin - 7向细胞质和细胞核分布，使ZO - 3定位于质膜，同时减少ZO - 1在细胞 - 细胞接触位点的分布。
生理浓度的甘氨酸通过调节肠上皮细胞中claudin - 7和ZO - 3的丰度和分布来支持肠道黏膜屏障功能。补充甘氨酸可能为改善仔猪肠道完整性提供一种有效的营养策略。
