Multiple retinal cells harbor a circadian oscillator, including retinal pigment epithelial cells (RPE). However, little is known about the functions that are regulated by the RPE clock. The aim of this study was to investigate whether the circadian clock in the RPE regulates the transport of glucose and its glycolytic metabolic by-product - lactate. To that end, we first characterized the mRNA expression profile of glucose and monocarboxylate transporters in ARPE-19 cells. We found that SLC2A1 and SLC16A1 were, respectively, the most abundantly expressed glucose and lactate (monocarboxylate) transporters. We further observed that the protein products of SLC2A1 (encoding GLUT1) and SLC16A1 (encoding MCT1) localize on the apical membrane of ARPE-19 monolayers. In a subsequent time-course experiment, we found that SLC2A1 and SLC16A1 mRNA oscillated in ARPE-19 monolayers, but not in dispersed cells, suggesting that monolayer cellular organization is necessary for rhythmic regulation of these transporters. In these monolayers, we found that MCT1 proteins varied over time, in contrast to GLUT1 proteins which did not vary over time. Spectrophotometric measurements of supernatants sampled from ARPE-19 monolayer cultures revealed that glucose concentrations did not significantly differ between apical (Api) supernatants and basolateral (BL) ones. In addition, we did not find rhythms in Api or BL glucose concentrations. Conversely, we found higher lactate concentrations in Api supernatants than BL ones. Further, we found that Api lactate concentrations were rhythmic. Pearson's r revealed that the concentration gradients (Api - BL) of glucose and lactate correlated with the gene expression of respective SLC2A1 and SLC16A1 transporters. Incubation with photoreceptor outer segments (POS) affected the mRNA expression of SLC16A1 and SLC2A1 in ARPE-19 monolayers in a time-dependent manner, thus suggesting that the retina might modulate the RPE clock-controlled expression of transporters via interactions with POS. In conclusion, this work provides evidence that the transport of lactate is regulated by the circadian clock in the RPE.

中文翻译：

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昼夜节律时钟通过SLC16A1（MCT1）调节RPE介导的乳酸运输。

多个视网膜细胞带有昼夜节律振荡器，包括视网膜色素上皮细胞（RPE）。但是，对于RPE时钟所调节的功能知之甚少。这项研究的目的是研究RPE中的生物钟是否调节葡萄糖的运输及其糖酵解代谢副产物乳酸。为此，我们首先表征了ARPE-19细胞中葡萄糖和单羧酸盐转运蛋白的mRNA表达谱。我们发现SLC2A1和SLC16A1分别是表达最丰富的葡萄糖和乳酸（单羧酸盐）转运蛋白。我们进一步观察到SLC2A1（编码GLUT1）和SLC16A1（编码MCT1）的蛋白质产物位于ARPE-19单层膜的顶膜上。在随后的时程实验中，我们发现SLC2A1和SLC16A1 mRNA在ARPE-19单层中振荡，但在分散细胞中没有振荡，这表明单层细胞组织对于这些转运蛋白的节律性调节是必需的。在这些单层中，我们发现MCT1蛋白随时间变化，而GLUT1蛋白则不随时间变化。从ARPE-19单层培养物中取样的上清液的分光光度测量显示，顶端（Api）上清液和基底外侧（BL）上清液中的葡萄糖浓度没有显着差异。另外，我们没有发现Api或BL葡萄糖浓度的节律。相反，我们发现Api上清液中的乳酸浓度高于BL上清液。此外，我们发现乳酸Api的浓度具有节律性。皮尔逊 sr表明，葡萄糖和乳酸的浓度梯度（Api-BL）与各自SLC2A1和SLC16A1转运蛋白的基因表达相关。感光外段（POS）的孵育以时间依赖的方式影响ARPE-19单层中SLC16A1和SLC2A1的mRNA表达，因此表明视网膜可能通过与POS相互作用来调节RPE时钟控制的转运蛋白表达。总之，这项工作提供了证据，证明乳酸的运输受RPE中生物钟的调节。因此表明视网膜可能通过与POS相互作用来调节RPE时钟控制的转运蛋白表达。总之，这项工作提供了证据，证明乳酸的运输受RPE中生物钟的调节。因此表明视网膜可能通过与POS相互作用来调节RPE时钟控制的转运蛋白表达。总之，这项工作提供了证据，证明乳酸的运输受RPE中生物钟的调节。