Slc4a11 Facilitates Glutamine Metabolism, Mitochondrial Function and ROS Prevention

Abstract

PurposeSLC4A11 codes for an electrogenic NH3‐sensitive H+ transporter. Slc4a11−/− mice show significant oxidative damage within corneal endothelial cells and an alteration of glutamine metabolism. Here, we asked if NH3 derived from glutamine (Gln) catabolism induces oxidative stress and whether Slc4a11 is protective.MethodsCorneal endothelial tissue from Slc4a11−/− mice was evaluated for oxidative stress (MitoSox, protein nitrosylation and OHdGuanine), mitochondrial morphology (MitoTracker Green) and autophagy (LC3B). Conditionally immortalized cell lines of Slc4a11−/− and WT mouse corneal endothelial cells (MCEC) expressing temperature sensitive Large T antigen were generated. In MCEC, cell death was quantified by AnV‐FITC + PI, mitochondrial anion superoxide (O2−) by MitoSox, mitochondrial membrane potential (MMP) measured by TMRE, and ATP was determined by luciferin‐luciferase assay. NH3 production was measured by colorimetric assay. Relative glucose and glutamine flux was quantified by GC‐MS.ResultsKO corneal endothelium from 12 and 40 week old mice show greater production of mitochondrial O2−, oxidized DNA and protein, induction of autophagy, and alteration of mitochondrial morphology compared to WT. In WT MCEC, Gln (0.5 mM) increases [ATP] concomitant with increased mitochondrial O2−, and increased apoptosis (compared to Gluc only). In contrast, in KO MCEC Gln decreases [ATP], with significantly greater mitochondrial O2− and apoptosis (compared to WT) that can be rescued by MitoQ. Application of NH3 alone increased mitochondrial O2−, depolarized MMP and increased apoptosis significantly more in KO. Gln derived NH3 toxicity can be partially reduced by GLS1 inhibitors BPTES or CB839 and totally inhibited by supplementation with Dimethyl‐α‐Ketoglutarate (α‐KG) or by Cyclosporin A (inhibitor of the opening of the mitochondrial transition pore). In MCEC stained with TMRE, 2 population of were observed: a population of smaller cells with depolarized MMP (TMRE− cells) and a population of normal size cells with hyperpolarized MMP (TMRE+ cells). Even though Gln increases the percentage of TMRE− cells the remainder TMRE+ cells are hyperpolarized as expected as Gln fuels the TCA cycle. This hyperpolarization is much higher in KO and suggest that it is the cause of O2− production followed by PTP opening and then secondary MMP depolarization. Gln induced hyperpolarization and NH3 derived Gln are the cause of high O2− production in KO cells.ConclusionWe conclude that Slc4a11 protects cells from Gln and NH3 induced toxicity.Support or Funding InformationNIH RO1 EY008834This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.