Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA110

Poster Communications

Role of neutral amino acid transporter LAT4 in mouse epithelia

A. RAJENDRAN1, N. Poncet1, L. Oparija1, F. Verrey1

1. Institute of Physiology, University of Zurich, Zurich, Switzerland.


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 (Guetg et al. 2015). To test whether this phenotype was due to a defect in intestinal absorption, we generated an inducible intestine-specific LAT4 knockout mouse model using a Cre-LoxP system. After induction of recombination by I.P. injection of tamoxifen (3mg/ml per 25g body weight) for 5 days just after weaning or at adult stage, body weight and food intake were monitored for 10 days before mice were euthanized by cervical dislocation and intestines collected. Quantitative PCR, western blot and immunofluorescence data showed a nearly 99% efficient knockout of LAT4 throughout the SI (n=20). However, these mice displayed no visible phenotype. Amino acid transport along the SI was assessed by gavaging an AA mix containing radioactively labelled LAT4 substrate 3H-Leu. Blood samples were collected from the tail vein 10′ before and 3′, 10′ 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 ß-counter revealed only a slightly impaired transport with accumulation of 3H-Leu in the initial segment of the SI of knockout mice (n=3, p<0.05 by ANOVA). Furthermore, expression of other AA transporters studied at the RNA level did not seem to be altered to compensate for the lack of intestinal LAT4 (n=10). Thus, we showed that LAT4 knockout in SI past the postnatal phase does not affect the general well-being of mice that displayed only a mild absorption defect. To test whether this defect could be more deleterious in earlier development, we are breeding constitutional intestine-specific LAT4 knockout mice. 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 these new models will allow us to determine whether the lethal phenotype of LAT4 global knockout mice was due to a defect in intestinal absorption and/or urinary loss of amino acids or whether it was caused by the defect of LAT4 in other tissues.

Where applicable, experiments conform with Society ethical requirements