Department of Nutrition, Food Science and Physiology, University of Navarra, Pamplona 31008, Spain.
Cytokine. 2013 Oct;64(1):181-7. doi: 10.1016/j.cyto.2013.07.004. Epub 2013 Jul 31.
During intestinal inflammation TNFα levels are increased and as a consequence malabsorption of nutrients may occur. We have previously demonstrated that TNFα inhibits galactose, fructose and leucine intestinal absorption in animal models. In continuation with our work, the purpose of the present study was to investigate in the human intestinal epithelial cell line Caco-2, the effect of TNFα on sugar transport and to identify the intracellular mechanisms involved.
Caco-2 cells were grown on culture plates and pre-incubated during different periods with various TNFα concentrations before measuring the apical uptake of galactose, α-methyl-glucoside (MG) or fructose for 15 min. To elucidate the signaling pathway implicated, cells were pre-incubated for 30min with the PKA inhibitor H-89 or the PKC inhibitor chelerythrine, before measuring the sugar uptake. The expression in the apical membrane of the transporters implicated in the sugars uptake process (SGLT1 and GLUT5) was determined by Western blot.
TNFα inhibited 0.1mM MG uptake after pre-incubation of the cells for 6-48h with the cytokine and in the absence of cytokine pre-incubation. In contrast, 5mM fructose uptake was stimulated by TNFα only after long pre-incubation times (24 and 48 h). These effects were mediated by the binding of the cytokine to its specific receptor TNFR1, present in the apical membrane of the Caco-2 cells. Analysis of the expression of the MG and fructose transporters at the brush border membrane of the cells, after 24h pre-incubation with the cytokine, revealed decrease on the amount of SGLT1 and increase on the amount of GLUT5 proteins. Short-term inhibition of MG transport by TNFα was not modified by H-89 but was blocked by chelerythrine.
SGLT1 and GLUT5 expression in the plasma membrane is regulated by TNFα in the human epithelial cell line Caco-2 cells, leading to alteration on sugars transport, suggesting that TNFα could be considered as a physiological local regulator of nutrients absorption in response to an intestinal inflammatory status.
在肠道炎症期间，TNFα 水平升高，因此可能会发生营养物质吸收不良。我们之前已经证明，TNFα 在动物模型中抑制了半乳糖、果糖和亮氨酸的肠道吸收。基于我们的工作，本研究的目的是在人肠道上皮细胞系 Caco-2 中研究 TNFα 对糖转运的影响，并确定涉及的细胞内机制。
将 Caco-2 细胞在培养板上生长，并在不同时期用不同浓度的 TNFα 预孵育，然后在 15 分钟内测量半乳糖、α-甲基-葡萄糖苷（MG）或果糖的顶端摄取。为了阐明所涉及的信号通路，在测量糖摄取之前，用 PKA 抑制剂 H-89 或 PKC 抑制剂白屈菜红碱预孵育细胞 30 分钟。通过 Western blot 确定参与糖摄取过程的转运蛋白（SGLT1 和 GLUT5）在顶端膜上的表达。
TNFα 抑制了细胞用细胞因子预孵育 6-48 小时后的 0.1mM MG 摄取，并且在没有细胞因子预孵育的情况下也是如此。相比之下，只有在长时间预孵育（24 和 48 小时）后，5mM 果糖摄取才被 TNFα 刺激。这些作用是由细胞因子与其在 Caco-2 细胞顶膜上的特定受体 TNFR1 结合介导的。在用细胞因子预孵育 24 小时后，分析细胞刷状缘膜上的 MG 和果糖转运体的表达，发现 SGLT1 的量减少，GLUT5 蛋白的量增加。TNFα 对 MG 转运的短期抑制不受 H-89 影响，但被白屈菜红碱阻断。
在人肠道上皮细胞系 Caco-2 细胞中，TNFα 调节 SGLT1 和 GLUT5 在质膜上的表达，导致糖转运的改变，提示 TNFα 可被视为对肠道炎症状态下营养物质吸收的生理局部调节剂。
