Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA122

Poster Communications

The amino acid transporter Lat1 (SLC7A5) resides in intestinal crypts and affects villus development

N. Poncet1, J. Jando1, C. Feuerstacke1, P. M. Taylor2, F. Verrey1

1. Institute of Physiology, University of Zurich, Zurich, Switzerland. 2. School of Life Sciences, University of Dundee, Dundee, United Kingdom.

Lat1 is a sodium-independent obligatory amino acid (AA) exchanger composed of two subunits: the catalytic permease SLC7A5 and its accessory glycoprotein SLC3A2. Lat1 transports large neutral AA including the essential AA leucine, which exerts a permissive effect on the mTORC1 pathway. By transporting leucine, Lat1 couples nutrient sensing and growth signals. To study the impact of Slc7a5 deletion in adult mice, we used a tamoxifen-inducible (Rosa26-CreERT2) mouse model. Tamoxifen (4mg/25g body weight) was administered orally for 3 consecutive days. This procedure produced >90% reduction in Slc7a5 mRNA expression in peripheral tissues (spleen, lung, small intestine, skeletal muscle, pancreas, adipose tissue), and resulted in a progressive body weight loss over a 12-day period. Reduced skeletal muscle (~5%) and white adipose tissue mass (~50% epididymal fat pads) substantially contributed to this loss of weight. Consistently, the Slc7a5 knockout mice exhibited a decreased respiratory exchange rate (RER) and higher remaining energy in faeces. These results prompted us to study intestinal absorption. An intestinal ring uptake approach showed reduction of leucine and glucose absorption in the ileum of Slc7a5 knockout mice. This functional phenotype was associated with decreased mRNA levels for B0AT1 and SGLT1, respectively AA and glucose luminal transporters. We used qPCR and in situ hybridization methods to show that Slc7a5 mRNA was expressed within the crypts and co-localised with the stem cell markers LGR5 and Olfm4. Labelling ileum sections with Ki67 (cellular marker for proliferation) revealed an extended domain of transit/amplifying cells within the Slc7a5 knockout crypts. Similarly, the BrdU incorporation method showed a higher proliferation rate in this cell population. To further dissect the molecular mechanisms underlying this phenotype, we established an ex-vivo culture model. However, organoids derived from Slc7a5fl/fl Rosa26-CreERT2 intestines and treated transiently in vitro with 4-Hydroxytamoxifen (4-OHT, 1µM), failed to develop stable crypt-like structures and displayed a decreased 5-ethynyl-2'-deoxyuridine (EdU) incorporation despite an increase in Ki67 positive cells. Taken together these data suggest that Lat1 (SLC7A5) expression within the intestinal crypts is required for adequate villus development. Further experiments including an RNAseq approach to compare wildtype and Slc7a5 knockout crypts and villi will help clarify the role Lat1 plays for the control of the proliferation/differentiation balance in intestinal epithelium.

Where applicable, experiments conform with Society ethical requirements