Anticipation of food intake induces phosphorylation switch to regulate basolateral amino acid transporter LAT4 (SLC43A2) function

Abstract

Key points Amino acid absorption requires luminal uptake into and subsequent basolateral efflux out of epithelial cells, with the latter step being critical to regulate the intracellular concentration of the amino acids. The basolateral essential neutral amino acid uniporter LAT4 (SLC43A2) has been suggested to drive the net efflux of non‐essential and cationic amino acids via parallel amino acid antiporters by recycling some of their substrates; its deletion has been shown to cause defective postnatal growth and death in mice. Here we test the regulatory function of LAT4 phosphorylation sites by mimicking their phosphorylated and dephosphorylated states in Xenopus laevis oocytes and show that dephosphorylation of S274 and phosphorylation of S297 increase LAT4 membrane localization and function. Using new phosphorylation site‐specific antibodies, we observe changes in LAT4 phosphorylation in mouse small intestine that correspond to its upregulation at the expected feeding time. These results strongly suggest that LAT4 phosphorylation participates in the regulation of transepithelial amino acid absorption. Abstract The essential amino acid uniporters LAT4 and TAT1 are located at the basolateral side of intestinal and kidney epithelial cells and their transport function has been suggested to control the transepithelial (re)absorption of neutral and possibly also cationic amino acids. Uniporter LAT4 selectively transports the branched chain amino acids leucine, isoleucine and valine, and additionally methionine and phenylalanine. Its deletion leads to a postnatal growth failure and early death in mice. Since LAT4 has been reported to be phosphorylated in vivo, we hypothesized that phosphorylation regulates its function. Using Xenopus laevis oocytes, we tested the impact of LAT4 phosphorylation at Ser274 and Ser297 by expressing mutant constructs mimicking phosphorylated and dephosphorylated states. We then investigated the in vivo regulation of LAT4 in mouse small intestine using new phosphorylation site‐specific antibodies and a time‐restricted diet. In Xenopus oocytes, mimicking non‐phosphorylation of Ser274 led to an increase in affinity and apparent surface membrane localization of LAT4, stimulating its transport activity, while the same mutation of Ser297 decreased LAT4's apparent surface expression and transport rate. In wild‐type mice, LAT4 phosphorylation on Ser274 was uniform at the beginning of the inactive phase (ZT0). In contrast, at the beginning of the active phase (ZT12), corresponding to the anticipated feeding time, Ser274 phosphorylation was decreased and restricted to relatively large patches of cells, while Ser297 phosphorylation was increased. We conclude that phosphorylation of small intestinal LAT4 is under food‐entrained circadian control, leading presumably to an upregulation of LAT4 function at the anticipated feeding time.

A guide to plasma membrane solute carrier proteins

“…SLC43A2 has a broader expression profile than SLC43A1, being expressed in the placenta, kidney, leukocytes, and at lower levels in many other tissue types [192]. The localization of this protein to the plasma membrane follows feeding patterns in mice, with increased plasma membrane expression of SLC43A2 occurring cyclically in anticipation of food intake, attesting to a role for SLC43A2 in amino acid absorption [196]. This protein is also required for proper mouse development, with knockout models exhibiting significant growth retardation and low amniotic fluid amino acid levels [197].…”

Time-of-Day-Dependent Physiological Responses to Meal and Exercise

“…These reports suggest that absorption of peptides in small intestine exhibits diurnal variation via the DBPand PPARα-mediated circadian control of PEPT1 expression. In recent years, time-dependent intestinal absorption of amino acids has been reported (56,57). Jando et al reported that isoleucine absorption is higher in the active phase than in the rest phase, although the protein levels of intestinal amino acid transporter B0AT1 in the rat intestine are not changed between the two time points (57).…”

“…The branched-chain amino acids, such as leucine, valine, and isoleucine, are absorbed via LAT4 (SLC43A2), a basolateral neutral amino acid transporter (58). LAT4 phosphorylation at Ser274 is higher at the beginning of the rest phase than at the beginning of the active phase in mice (56). LAT4 shows high activity under dephosphorylated condition, suggesting that post-translational modulation such as phosphorylation could be involved in the time-dependent amino acid absorption.…”

Tissue‐specific deletion of mouse basolateral uniporter LAT4 (Slc43a2) reveals its crucial role in small intestine and kidney amino acid transport

“…We can nevertheless not exclude a compensatory change at the level of other transporters or even at the level of the tested ones, if occurring by post‐transcriptional modifications, such as LAT4 that itself was shown to undergo post‐translational regulation in the context of food anticipation, specifically by serine (de)phosphorylation (Oparija et al . 2019).…”

“…The primary antibodies used were: rabbit anti-LAT4, guinea pig anti-B 0 AT1 (Guetg et al 2015), rabbit anti-LAT2 (Vilches et al 2018) and mouse anti-ß actin (Oparija et al 2019) as described previously. The secondary antibodies used include: Alexa Fluor 594 anti-rabbit (catalogue no.…”