System L amino acid transporters and their relation to cellular energy status and glucose

Intracellular availability of the amino acid L-leucine in pancreatic β cells is thought to regulate cell growth and modulate D-glucose-induced insulin secretion (Data published in Cheng et al. 2016). System L amino acid transporters (LATs) control transport of L-leucine across the plasma membrane and may therefore influence intracellular L-leucine concentration. The aim of this project was to test the hypothesis that D-glucose supply to the cultured rat β cell line INS1E regulates leucine transport by acting on LATs. Incubation of cells in medium containing 0 versus 5 mM (control) D-glucose for 6 hours increased L-[3H]-leucine transport influx by 70% and increased intracellular L-leucine concentration by 150% in INS1E. In contrast, very variable responses were seen on exposure of INS1E to 25 mM D-glucose. Treating INS1E with the AMP-activated kinase (AMPK) agonist AICAR also increased L-[3H]-leucine influx by 70% suggesting that D-glucose deprivation regulates transport by lowering energy status which then activates the energy status sensor AMPK. D-glucose deprivation increased mRNA expression for the LAT1 (SLC7A5) isoform of LAT transporter about 3.5-fold when assessed by RT-Q-PCR. The effect showed very variable magnitude and was not observed for LAT2 (SLC7A8) or LAT4 (SLC43A2) mRNA, which were also strongly expressed in INS1E. In spite of strong LAT2/4 expression, siRNA silencing of LAT1 mRNA by 85% was sufficient to decrease basal L-[3H]-leucine influx by 35%. However, blockade of the LAT1 mRNA increase on D-glucose deprivation by treating with transcription inhibitor actinomycin-D failed to block the accompanying increase in L-[3H]-leucine influx, suggesting that activation of LAT transporter proteins by low energy status was largely occurring through an additional non-transcriptional mechanism. To test this, a human LAT1-eGFP-tagged cDNA construct completely lacking the normal LAT1 promoter sequence was transfected into HEK293A cells and successfully expressed eGFP fluorescence. Even though wild-type HEK293A showed high basal L-[3H]-leucine influx and no stimulation of this flux by AICAR, and LAT1-eGFP transfection alone failed to give statistically significant stimulation of L-[3H]-leucine influx, a significant 24% increase in transport was obtained in cells subjected to combined LAT1-eGFP transfection + AICAR. These results suggest that D-glucose starvation and AMPK activation in INS1E cells lead to a previously undescribed stimulation of L-leucine transport, which is similar in magnitude to that previously described for glucose transport in response to AMPK activation in muscle. It is concluded that declining energy status sensed through AMPK activates LAT1 (and maybe other isoforms of LAT L-leucine transporters), possibly through an AMPK-dependent translocation of LAT proteins between sub-cellular pools analogous to that previously described for GLUT monosaccharide transporters in muscle.