Essential amino acid transporter Lat4 (Slc43a2) is required for mouse development

Abstract

Amino acid (AA) uniporter Lat4 (Slc43a2) mediates facilitated diffusion of branched-chain AAs, methionine and phenylalanine, although its physiological role and subcellular localization are not known. We report that Slc43a2 knockout mice were born at expected Mendelian frequency but displayed an ∼10% intrauterine growth retardation and low amniotic fluid AAs, suggesting defective transplacental transport. Postnatal growth was strongly reduced, with premature death occurring within 9 days such that further investigations were made within 3 days of birth. Lat4 immunofluorescence showed a strong basolateral signal in the small intestine, kidney proximal tubule and thick ascending limb epithelial cells of wild-type but not Slc43a2 null littermates and no signal in liver and skeletal muscle. Experiments using Xenopus laevis oocytes demonstrated that Lat4 functioned as a symmetrical low affinity uniporter with a K₀.₅ of ∼5 mm for both in- and efflux. Plasma AA concentration was decreased in Slc43a2 null pups, in particular that of non-essential AAs alanine, serine, histidine and proline. Together with an increased level of plasma long chain acylcarnitines and a strong alteration of liver gene expression, this indicates malnutrition. Attempts to rescue pups by decreasing the litter size or by nutrients injected i.p. did not succeed. Radioactively labelled leucine but not lysine given per os accumulated in the small intestine of Slc43a2null pups, suggesting the defective transcellular transport of Lat4 substrates. In summary, Lat4 is a symmetrical uniporter for neutral essential AAs localizing at the basolateral side of (re)absorbing epithelia and is necessary for early nutrition and development.

Figure 1

Confirmation of Lat4 deletion and validation of Lat4 antibody by immunofluorescence on tissue of wild-type and Lat4 KO pups A basolateral signal is seen with Lat4 antibody (green) in the kidney (A) and small intestine (C) of 3-day-old wild-type pups, whereas no such signal is visible on sections from KO littermates (B and D). No signal (green) is observed in skeletal muscle (E) and liver (G) of wild-type pups in addition to the background also visible in the KO mice (F and H). As a luminal marker, Tmem27 (red) was used for kidney proximal tubule cells (A and B) and MDR1 was used for the canalicular membrane of liver hepatocytes (red) (G and H). Scale bars = 20 μm.

Figure 2

Localization of Lat4 on small intestine and kidney sections by immunofluorescence A, on this cross-section of duodenal villi, Lat4 signal (green) localizes to the basolateral membrane of the enterocytes that are counterstained for the luminal transporter B⁰AT1 (red). B, on a sagittal section of duodenal villi, Lat4 signal (green) is visible all along the villi (V) but not in crypts (C). In both (A) and (B), 4′,6-diamidino-2-phenylindole (blue) was used to label the nuclei. On kidney sections, Lat4 (green) signal is detectable in the proximal tubule (PT) which is identified in (C) by the luminal protein Tmem27 and by the basolateral uniporter Tat1 (pink) on consecutive sections (D and E). F, labelling of the luminal transporter NCC (red) identifies the distal convoluted tubule (DCT), which displays a weaker Lat4 signal compared to the proximal tube. G and H, a strong Lat4 signal (green) is visible in the thick ascending limb (TAL), which is identified in consecutive sections by the luminal transporter NKCC2 (red). Scale bar = 20 μm.

Figure 3

Concentration-dependence of l-phenylalanine uptake and efflux in X. laevis oocytes expressing human LAT4 Oocytes were injected with 25 ng of hSLC43A2 cRNA and incubated for 3 days. A, concentration-dependent uptakes were performed for 10 min with nine different concentrations of phenylalanine; n = 8–24 oocytes pooled from three independent experiments. B, for the efflux experiments, oocytes were injected with 50 nl of phenylalanine at five different concentrations; n = 12–14 oocytes pooled from three independent experiments. Data are the are mean ± SEM. Curves corresponding to the Michaelis–Menten equation were fitted to the experimental data using a non-linear regression routine (GraphPrism, version 5.0).

Figure 4

Effect of Slc43a2 deletion on intrauterine growth and AA concentrations in amniotic fluid at E18 A, representative image of E18 Slc43a2^+/+ and Slc43a2^−/− fetuses with corresponding weights. Scale bar = 5 mm. B, AA concentrations in amniotic fluids (C). Results were normalized to Slc43a2^+/+ (1.0) and represent the mean ± SEM of data pooled from two independent experiments: n = 7 for Slc43a2^+/+ mice, n = 15 for Slc43a2^+/− mice and n = 4 for Slc43a2^−/− mice. Two-way ANOVA and Dunnet's multiple comparison post hoc test for (B) and Bonferroni post hoc test for (C): **P < 0.01. NS, non-significant.

Figure 5

Postnatal growth retardation and premature death of Slc43a2^−/− mice A, representative image of a Slc43a2^−/− mouse (top) versus a Slc43a2^+/+mouse (bottom) at day 7 after birth. B, growth curves for Slc43a2^+/+, Slc43a2^+/- and Slc43a2^−/− mice; represented as the mean ± SEM: n = 7 for Slc43a2^+/+, n = 22 for Slc43a2^+/− and n = 7 for Slc43a2^−/−; comparison of Slc43a2^+/+ and Slc43a2^−/−. Two-way ANOVA and Bonferroni post hoc test: **P < 0.01, ***P < 0.001; the cross represents the death of all Slc43a2^−/− mice. C, survival curves for the same mice as (B).

Figure 6

Impact of Slc43a2 deletion on plasma AAs, acylcarnitines and glucose Plasma AAs (A), long-chain unsaturated acylcarnitines (C) and dicarboxylacylcarnitines (DAs) (D). Represented are the mean ± SEM: n = 3 for Slc43a2^+/+ (black bars) and n = 6 for Slc43a2^−/− (white bars). Two-way ANOVA and Bonferroni post hoc test: *P < 0.05, **P < 0.001. B and E, blood glucose measurement from mice at day 2 without (B) and with 30 min of starvation (E) prior to sample collection. Represented are the mean ± SEM. Student's t test: **P < 0.01.

Figure 7

Effect of Slc43a2 deletion on liver and kidney A, heat map summary of differential gene expression pattern in Slc43a2^−/− liver. RNA was isolated from mice at day 2 and each column represents a different mouse. Upregulated genes are indicated in red; downregulated genes are indicated in blue. B, heamatoxylin and eosin staining of liver. Left: liver section of Slc43a2^−/− KO mouse. Right: liver section of Slc43a2^+/+ mouse. *Leucocyte infiltration in the periportal region of Slc43a2^−/− mouse liver. Scale bar = 50 μm. C, gene expression of selected AA transporters in the kidney of Slc43a2^+/+ (black bars) and Slc43a2^−/− (white bars). Represented are the mean ± SEM: n = 3. Two-way ANOVA and Bonferroni post hoc test: **P < 0.01.

Figure 8

Distribution of radiolabelled l-leucine and l-lysine in different organs after oral administration Mice were starved 30 min prior to oral administration of radiolabelled AA solution. Leucine (A) and (C) lysine accumulation in different organs normalized by weight. Leucine (B) and (D) lysine concentration in plasma 30 min after oral administration (Slc43a2^+/+: black bars, Slc43a2^−/−: white bars). The small intestine of the pups was divided into three equals parts from proximal to distal: SI1, SI2 and SI3. Represented are the mean ± SEM pooled from three independent experiments: n = 6 for Slc43a2^+/+ mice and n = 8 for Slc43a2^−/− mice. Two-way ANOVA and Bonferroni post hoc test: ***P < 0.001.

Figure 9

Schematic representation of apical and basolateral AA transporter cooperation in proximal tubule kidney cell Neutral AAs are imported apically mostly via the symporter B⁰AT1 (SLC6A19) and are effluxed across the basolateral membrane mostly by the anti-porter LAT2–4F2hc and the uniporters TAT1 and LAT4. We postulate that essential neutral AAs are equilibrated between the intracellular compartment and the extracellular space via the two low affinity uniporters TAT1 and LAT4 and may recycle into the cell via anti-porters and thereby drive the efflux of other AAs, in particular of (non)-essential neutral AAs via LAT2–4F2hc and cationic AAs via y⁺LAT1–4F2hc. Therefore, a defect in the uniporter(s) LAT4 and/or TAT1 is suggested to impact not only on the basolateral efflux of their own AA substrates, but also on that of the other substrates of the anti-porters LAT2–4F2hc and y⁺LAT1–4F2hc.