Abstract
Amino acids play central roles for living organisms being the monomer building blocks of proteins and having a variety of other vital physiological functions. Thus, amino acids are important nutrients that are absorbed by the small intestine and reabsorbed by the kidney proximal tubule through their epithelia. An important characteristic of epithelial cells is their polarity, and hence, different sets of proteins are expressed apically and basolaterally to allow the vectorial transport of nutrients. Various amino acid transporters perform passive (uniport) or active (cotransport and exchange) transport. A well characterized amino acid transporter expressed in the small intestine and the kidney basolateral membrane is LAT2-4F2hc (SLC7A8-SLC3A2) which has a broad selectivity for neutral amino acids, including aromatic amino acids. LAT2 acts as an obligatory exchanger and thus cannot contribute alone to the net vectorial flux of amino acids. In order to fulfil the directional transport of specific amino acids, LAT2 needs to exchange them against other amino acids that are recycled via a directional transporter inserted in the same membrane, for instance by a uniporter(s) such as TAT1 (SLC16A10) which transports aromatic amino acids by facilitated diffusion. A functional cooperation between the uniporter TAT1 and the exchanger LAT2 was demonstrated in X. laevis oocytes (Ramadan, Camargo et al. 2007). Moreover, mice lacking TAT1 were shown to excrete in urine not only aromatic amino acids but also other neutral amino acids that are substrates of LAT2 (Mariotta, Ramadan et al. 2012) and LAT2 knockout mice to display a modest increase of Slc16a10 (TAT1) mRNA in their kidneys (Braun, Wirth et al. 2011). These studies indicate the functional importance of cooperation between LAT2 and TAT1. To further investigate their role in vivo a double knockout (dKO) mouse lacking both TAT1 and LAT2 transporters was generated.
For my dissertation, the dKO mouse model was used to answer the question whether the lack of both TAT1 and LAT2 transporters changes the amino acid balance between cells, blood and urine more dramatically than the single transporter defects. Our main focus was to investigate the absorptive function of small intestine and kidney, and to test for compensatory mechanism(s) in dKO mice.
In contrast to the single KOs, dKO mice had lower body weight and reduced breeding performance. However, the intestinal function of dKO animals was mildly disrupted and
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overall amino acid absorption was not retarded. The lack of both TAT1 and LAT2 induced compensatory mechanisms for intestinal absorption, and our data suggest that the paracellular pathway is involved in this process. Further investigation is needed to elucidate this mechanism of regulation. Unlike the intestinal function, the kidney absorption was highly affected in dKO animals. Urinary hyperexcretion of all neutral amino acids revealed synergistic cooperation between TAT1 and LAT2 for renal reabsorption. Furthermore, overexpression of the amino acid transporter y+LAT1 (Slc7a7) on mRNA and protein levels demonstrated a possible compensatory mechanism to the loss-of-function of TAT1 and LAT2.
In the course of dKO mouse characterisation I noticed higher incidence of cataract formation in these animals. As a result, a second project was initiated that is part of my dissertation. The main goal of this project was to investigate cataract development in our mouse population by screening dKO, TAT1 KO, LAT2 KO, and wild type (WT) eye lenses. Mice at different ages (between 2 and 19 months of age) were analyzed. Overall we found a statistically significant increase of cataract formation in LAT2 KO (p=0.009) and much more in dKO mice (p<0.0001), while TAT1 KO alone did not seem to influence cataract formation. When looking at age and gender in a combinatorial analysis, we found out that old females had the highest occurrence of cataract when both LAT2 and TAT1 were abolished (p=0.017) and also when LAT2 alone was knocked out (p=0.0187). Furthermore, genetic screening of patients with cataract was performed. Mutations in SLC7A8 were found in 30 out of 570 cases. Taken together, we conclude that a defect in LAT2 favors cataract formation and that this deleterious effect is further stimulated by a defect of TAT1. This latter observation further supports the notion that the cooperation between the exchanger LAT2 and the uniporter TAT1 is of functional importance in physiology.
Boiadjieva, Emilia Borislavova