Role of neutral amino acid transporter LAT4 in mouse epithelia

Abstract

Amino acids (AAs) are not only building blocks of proteins, but also play key roles in energy metabolism and as (precursors of) neurotransmitters. To maintain amino acid homeostasis, small intestine (SI) and kidney play a major role by absorbing amino acids from food and preventing their loss in the urine, respectively. Given their zwitterionic nature, neutral AAs require transport proteins to cross cell membranes. LAT4 (Slc43a2), a facilitated diffusion uniporter specific for essential neutral amino acids (Leu, Ile, Val, Met and Phe) was first identified by Bodoy et al., 2005 and then shown by us to be highly expressed in the basolateral membranes of SI and kidney tubule epithelia. LAT4 global knockout caused a postnatal malnutrition‐like condition leading to death within 10 days, which was hypothesized to be due to a severe defect in intestinal absorption of AAs (Guetg et al. 2015). To test this hypothesis, we generated an intestine‐specific LAT4 knockout mouse model using Cre‐LoxP system. Intestine specific LAT4 knockout pups were viable and did not show any significant changes in body weight gain as compared to their control littermates even up to 2 months after birth. Quantitative PCR, western blot and immunofluorescence data showed a nearly 95% efficient knockout of LAT4 throughout the SI (n=4). However, these mice displayed no visible phenotype. Amino acid transport along the SI was then assessed by gavaging an AA mix containing radiolabelled LAT4 substrate 3H‐Leu. Blood samples were collected from the tail vein 10′ before and 2′,5′, 10′, 20′ and 60′ after gavage. Mice were then euthanized, SI divided into 4 equal parts and the intestinal tissue and its contents collected separately. Measuring the distribution of 3H‐Leu using a β‐counter revealed an accumulation of Leu in the initial segment of the SI of knockout mice (n=5, P<0.001 by ANOVA), accompanied by a delayed bolus movement. Thus, we show that LAT4 knockout in SI is not the sole or major reason for the lethal phenotype of LAT4 global knockout pups, although it could have contributed to the lethality. In parallel, we have generated a doxycycline‐inducible kidney tubule‐specific LAT4 knockout mouse model to assess the effects of LAT4 deletion in kidney, both in young and adult animals. Results obtained with this new model will allow us to determine whether the urinary loss of amino acids may have strongly contributed to the lethal phenotype of LAT4 global knockout mice or whether it was caused by the defect of LAT4 in other tissues.Support or Funding InformationThis project is funded by a grant of the Swiss National Foundation (SNF) to FV.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.