Wang Bin, Wu Zhenlong, Ji Yun, Sun Kaiji, Dai Zhaolai, Wu Guoyao
State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China; and.
State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China; and
J Nutr. 2016 Mar;146(3):501-8. doi: 10.3945/jn.115.224857. Epub 2016 Feb 10.
The tight junctions (TJs) are essential for maintenance of the intestinal mucosal barrier integrity. Results of our recent work show that dietary l-glutamine (Gln) supplementation enhances the protein abundance of TJ proteins in the small intestine of piglets. However, the underlying mechanisms remain largely unknown.
This study was conducted to test the hypothesis that Gln regulates TJ integrity through calcium/calmodulin-dependent kinase 2 (CaMKK2)-AMP-activated protein kinase (AMPK) signaling which, in turn, contributes to improved intestinal mucosal barrier function.
Jejunal enterocytes isolated from a newborn pig were cultured in the presence of 0-2.0 mmol Gln/L for indicated time points. Cell proliferation, monolayer transepithelial electrical resistance (TEER), paracellular permeability, expression and distribution of TJ proteins, and phosphorylated AMPK were determined.
Compared with 0 mmol Gln/L, 2.0 mmol Gln/L enhanced (P < 0.05) cell growth (by 31.9% at 48 h and 11.1% at 60 h). Cells treated with 2 mmol Gln/L increased TEER by 32.2% at 60 h, and decreased (P < 0.05) TJ permeability by 20.3-40.0% at 36-60 h. In addition, 2.0 mmol Gln/L increased (P < 0.05) the abundance of transmembrane proteins, such as occludin, claudin-4, junction adhesion molecule (JAM)-A, and the plaque proteins zonula occludens (ZO)-1, ZO-2, and ZO-3 by 1.8-6 times. In contrast, 0.5 mmol Gln/L had a moderate effect on TJ protein abundance (20.2-70.5%; P < 0.05) of occludin, claudin-3, claudin-4, JAM-A, and ZO-1. 2.0 mmol Gln/L treatment led to a greater distribution of claudin-1, claudin-4, and ZO-1 at plasma membranes compared with 0 mmol Gln/L. This effect of Gln was mediated by the activation of CaMKK2-AMPK signaling, because either depletion of calcium from the medium or the presence of an inhibitor of CaMKK2 abrogated the effect of Gln on epithelial integrity.
Our findings indicate that activation of CaMKK2-AMPK signaling by Gln is associated with improved intestinal mucosal barrier function through enhancing the abundance of TJ proteins and altering their intracellular localization in intestinal porcine epithelial cells.
紧密连接（TJ）对于维持肠道黏膜屏障完整性至关重要。我们近期的研究结果表明，日粮补充L-谷氨酰胺（Gln）可提高仔猪小肠中TJ蛋白的丰度。然而，其潜在机制仍 largely 未知。
本研究旨在验证以下假设，即Gln通过钙/钙调蛋白依赖性激酶2（CaMKK2）-AMP激活的蛋白激酶（AMPK）信号通路调节TJ完整性，进而有助于改善肠道黏膜屏障功能。
从新生仔猪分离的空肠肠上皮细胞在含有0 - 2.0 mmol Gln/L的条件下培养指定时间点。测定细胞增殖、单层跨上皮电阻（TEER）、细胞旁通透性、TJ蛋白的表达和分布以及磷酸化AMPK。
与0 mmol Gln/L相比，2.0 mmol Gln/L增强了（P < 0.05）细胞生长（48 h时增长31.9%，60 h时增长11.1%）。用2 mmol Gln/L处理的细胞在60 h时使TEER增加32.2%，在36 - 60 h时使TJ通透性降低（P < 0.05）20.3 - 40.0%。此外，2.0 mmol Gln/L使跨膜蛋白如闭合蛋白、Claudin-４、连接黏附分子（JAM）-A以及斑块蛋白闭锁小带（ZO）-1、ZO-2和ZO-3的丰度增加（P < 0.05）1.8 - 6倍。相比之下，0.5 mmol Gln/L对闭合蛋白、Claudin-3、Claudin-4、JAM-A和ZO-1的TJ蛋白丰度有中等程度影响（20.2 - 70.5%；P < 0.05）。与0 mmol Gln/L相比，2.0 mmol Gln/L处理导致Claudin-1、Claudin-4和ZO-1在质膜上的分布更多。Gln的这种作用是由CaMKK2-AMPK信号通路的激活介导的，因为培养基中钙的耗尽或CaMKK2抑制剂的存在消除了Gln对上皮完整性的影响。
我们的研究结果表明，Gln激活CaMKK2-AMPK信号通路与通过增加TJ蛋白丰度并改变其在猪肠道上皮细胞中的细胞内定位来改善肠道黏膜屏障功能相关。
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