Institute of Physiology and Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland.
NCCR Kidney.CH, Zurich, Switzerland.
PLoS One. 2020 May 29;15(5):e0233863. doi: 10.1371/journal.pone.0233863. eCollection 2020.
Adaptive regulation of epithelial transporters to nutrient intake is essential to decrease energy costs of their synthesis and maintenance, however such regulation is understudied. Previously we demonstrated that the transport function of the basolateral amino acid uniporter LAT4 (Slc43a2) is increased by dephosphorylation of serine 274 (S274) and nearly abolished by dephosphorylation of serine 297 (S297) when expressed in Xenopus oocytes. Phosphorylation changes in the jejunum of food-entrained mice suggested an increase in LAT4 transport function during food expectation. Thus, we investigated further how phosphorylation, expression and localization of mouse intestinal LAT4 respond to food-entrained diurnal rhythm and dietary protein content. In mice entrained with 18% protein diet, LAT4 mRNA was not submitted to diurnal regulation, unlike mRNAs of luminal symporters and antiporters. Only in duodenum, LAT4 protein expression increased during food intake. Concurrently, S274 phosphorylation was decreased in all three small intestinal segments, whereas S297 phosphorylation was increased only in jejunum. Interestingly, during food intake, S274 phosphorylation was nearly absent in ileum and accompanied by strong phosphorylation of mTORC1 target S6. Entraining mice with 8% protein diet provoked a shift in jejunal LAT4 localization from the cell surface to intracellular stores and increased S274 phosphorylation in both jejunum and ileum during food anticipation, suggesting decreased transport function. In contrast, 40% dietary protein content led to increased LAT4 expression in jejunum and its internalization in ileum. Ex vivo treatments of isolated intestinal villi fraction demonstrated that S274 phosphorylation was stimulated by protein kinase A. Rapamycin-sensitive insulin treatment and amino acids increased S297 phosphorylation, suggesting that the response to food intake might be regulated via the insulin-mTORC1 pathway. Ghrelin, an oscillating orexigenic hormone, did not affect phosphorylation of intestinal LAT4. Overall, we show that phosphorylation, expression and localization of intestinal mouse LAT4 responds to diurnal and dietary stimuli in location-specific manner.
上皮转运蛋白对营养摄入的适应性调节对于降低其合成和维持的能量成本至关重要，但这种调节仍未得到充分研究。我们之前证明，在 Xenopus 卵母细胞中表达时，碱性氨基酸载体 LAT4（Slc43a2）的转运功能可通过丝氨酸 274（S274）去磷酸化增加，而通过丝氨酸 297（S297）去磷酸化几乎完全消除。在食物诱导的小鼠空肠中磷酸化变化表明，在食物预期期间 LAT4 转运功能增加。因此，我们进一步研究了食物诱导的昼夜节律和膳食蛋白质含量如何影响小鼠肠道 LAT4 的磷酸化、表达和定位。在摄入 18%蛋白质饮食的小鼠中，LAT4mRNA 不受昼夜调节，而腔侧转运体和反向转运体的 mRNA 则不受昼夜调节。只有在十二指肠中，LAT4 蛋白表达在进食期间增加。同时，在所有三个小肠段中 S274 磷酸化减少，而 S297 磷酸化仅在空肠中增加。有趣的是，在进食期间，回肠中的 S274 磷酸化几乎不存在，并且伴随着 mTORC1 靶标 S6 的强烈磷酸化。用 8%蛋白质饮食饲养的小鼠会引起空肠 LAT4 定位从细胞表面转移到细胞内储存，并在食物预期期间增加空肠和回肠中的 S274 磷酸化，表明转运功能降低。相比之下，40%的膳食蛋白质含量导致空肠中 LAT4 表达增加，回肠中 LAT4 内化。分离的肠绒毛部分的离体处理表明，蛋白激酶 A 刺激 S274 磷酸化。雷帕霉素敏感的胰岛素处理和氨基酸增加 S297 磷酸化，表明对食物摄入的反应可能通过胰岛素-mTORC1 途径调节。胃饥饿素，一种波动的食欲激素，不影响肠道 LAT4 的磷酸化。总的来说，我们表明，肠道小鼠 LAT4 的磷酸化、表达和定位以位置特异性的方式响应昼夜和膳食刺激。
