Taurine transporter (TauT) deficiency impairs ammonia detoxification in mouse liver Natalia Qvartskhavaa, Cheng Jun Jina, Tobias Buschmanna, Ute Albrechta, Johannes Georg Bodea, Niloufar Monhaserya, Jessica Oenartoa, Hans Jürgen Bidmonb, Boris Görga, and Dieter Häussingera,1 aClinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich Heine University, 40225 Düsseldorf, Germany; and bCécile and Oskar Vogt Institute for Brain Research, Heinrich Heine University, 40225 Düsseldorf, Germany Edited by Arthur J. L. Cooper, New York Medical College, Valhalla, NY, and accepted by Editorial Board Member David J. Mangelsdorf February 11, 2019 (received for review July 30, 2018) Hepatic ammonia handling was analyzed in taurine transporter The amino acid taurine is highly abundant in brain, liver, and (TauT) KO mice. Surprisingly, hyperammonemia was present at an other organs (9). Intracellular taurine concentrations are main- age of 3 and 12 months despite normal tissue integrity. This was tained at high levels through uptake by the taurine transporter accompanied by cerebral RNA oxidation. As shown in liver perfusion TauT (10). Functions of taurine in the body are remarkably di- experiments, glutamine production from ammonia was diminished verse: taurine acts as an osmolyte, an antioxidant, and a molecular in TauT KO mice, whereas urea production was not affected. In chaperone, and promotes antiinflammatory effects and modulates – livers from 3-month-old TauT KO mice protein expression and neurotransmission (11 13). Studies from TauT KO mice showed that low taurine levels cause a variety of malfunctions in different activity of glutamine synthetase (GS) were unaffected, whereas – the ammonia-transporting RhBG protein was down-regulated by organs in an age-dependent way (14 20). Within 1 y after birth about 50%. Double reciprocal plot analysis of glutamine synthesis TauT KO mice lose vision, suffer from auditory, olfactory, and versus perivenous ammonia concentration revealed that TauT KO muscle dysfunction, and show altered synaptic transmission in brain (for review see ref. 21). At higher age (beyond 15 mo), liver had no effect on the capacity of glutamine formation in 3-month- manifestations develop, such as fibrosis, unspecific hepatitis, and old mice, but doubled the ammonia concentration required for half- tumor formation (17, 21). However, the pathogenetic events that maximal glutamine synthesis. Since hepatic RhBG expression is precede such structural liver dysfunction in aged TauT KO mice restricted to GS-expressing hepatocytes, the findings suggest that remained unclear until now. MEDICAL SCIENCES an impaired ammonia transport into these cells impairs glutamine In the present study, we analyzed effects of TauT deficiency on synthesis. In livers from 12-, but not 3-month-old TauT KO mice, hepatic ammonia handling in 3- and 12-mo-old TauT KO mice. RhBG expression was not affected, surrogate markers for oxidative Our results suggest that TauT deficiency impairs ammonia de- stress were strongly up-regulated, and GS activity was decreased by toxification by perivenous scavenger cells in the liver, thereby in- 40% due to an inactivating tyrosine nitration. This was also reflected ducing systemic hyperammonemia. This is explained by down- by kinetic analyses in perfused liver, which showed a decreased regulation of the ammonium transporter RhBG at 3 mo of age and glutamine synthesizing capacity by 43% and a largely unaffected a tyrosine nitration-induced inactivation of GS at 12 mo of age due ammonia concentration dependence. It is concluded that TauT de- to oxidative/nitrosative stress. The data also suggest that RhBG ficiency triggers hyperammonemia through impaired hepatic gluta- expression can be a site of control of hepatic glutamine synthesis. mine synthesis due to an impaired ammonia transport via RhBG at 3 months and a tyrosine nitration-dependent inactivation of GS in 12- Results month-old TauT KO mice. Taurine Transporter Knockout Triggers Hyperammonemia Without Affecting Liver Integrity. TauT deficiency was shown to trigger glutamine | hyperammonemia | taurine | scavenger cells | oxidative stress liver fibrosis in mice beyond an age of 15 mo (17). However, as shown by Sirius red staining (SI Appendix,Fig.S1A) and immu- nofluorescence analysis of collagens I and IV (SI Appendix,Fig. he liver plays a central role for ammonia homeostasis in the B body. A sophisticated structural and functional organization S1 ), no signs of fibrosis were found in livers from 3- and 12-mo-old T TauT KO and WT mice and expression of these surrogate markers of urea and glutamine synthesis as well as glutaminase has evolved, which not only allows for efficient ammonia detoxication by the liver, but also to adjust urea synthesis to the needs of sys- Significance temic acid base homeostasis (1, 2). In this organization, gluta- minase augments urea synthesis by amplifying the ammonia Taurine transporter deficiency in mice results in hyperammonemia concentration in the periportal compartment, whereas glutamine and reduced glutamine formation in hepatic scavenger cells due synthetase (GS), which is restricted to a small perivenous cell to an impaired ammonia transport at 3 months of age and a ty- population (3) acts as a high-affinity detoxification system for the rosine nitration-dependent inactivation of glutamine synthetase ammonia, which escapes periportal urea synthesis (1, 2, 4). Due to in 12-month-old mice. The data suggest that down-regulation of this important function, the small perivenous subpopulation of theammoniatransporterRhBGcanberatelimitingforthesyn- hepatocytes has been termed “scavenger cells” (2). These cells thesis of glutamine in mouse liver. exclusively express GS, the glutamate transporter 1 (Glt1) and the + Author contributions: D.H. designed research; N.Q., C.J.J., T.B., U.A., N.M., J.O., H.J.B., and NH3/NH4 transporter Rhesus type glycoprotein B (RhBG) as B.G. performed research; J.G.B. contributed new reagents/analytic tools; N.Q., C.J.J., T.B., well as uptake systems for dicarboxylates (3, 5, 6). Pioneer work U.A., N.M., J.O., H.J.B., B.G., and D.H. analyzed data; and B.G. and D.H. wrote the paper. with the isolated perfused rat liver suggested already decades ago The authors declare no conflict of interest. that intact scavenger cells are essential for maintenance of phys- This article is a PNAS Direct Submission. A.J.L.C. is a guest editor invited by the iologically low ammonia concentrations in the blood (1, 2, 7), and Editorial Board. liver-specific GS deletion results in systemic hyperammonemia in Published under the PNAS license. mice (8). This study also confirmed that chronic hyperammonemia 1To whom correspondence should be addressed. Email: [email protected]. in the absence of liver damage is sufficient to induce cerebral This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. oxidative stress and to impair behavior and locomotion, which are 1073/pnas.1813100116/-/DCSupplemental. hallmarks of hepatic encephalopathy (HE) (8). Published online March 12, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1813100116 PNAS | March 26, 2019 | vol. 116 | no. 13 | 6313–6318 Downloaded by guest on October 3, 2021 by about 40% in protein lysates of livers from 12- but not from 3- mo-old TauT KO mice compared with the respective wild-type mice (Fig. 4B). Glt1 protein levels were significantly increased by about 80% in livers from 3-mo-old and about fourfold in livers from 12-mo- old TauT KO mice as shown by Western blot and immunoflu- orescence analysis (SI Appendix, Fig. S3 B and C). Mechanism of Impaired Glutamine Synthesis in Livers from 3-Mo-Old Fig. 1. Blood ammonia levels in TauT KO and WT mice. Ammonia levels in TauT KO Mice. As shown above, in perfused livers from 3-mo-old blood from 3- and 12-mo-old WT and TauT KO mice were measured as de- TauT KO mice, glutamine synthesis from ammonia at concen- scribed in Materials and Methods. *Statistically significantly different com- trations up to 0.5 mmol/L is diminished (Fig. 2A), although GS pared with WT. n = 3–8 animals for each condition. expression (Fig. 4A) and activity (Fig. 4B) were not affected compared with 3-mo-old wild-type mice. We therefore analyzed the ammonia concentration dependence of glutamine pro- of fibrosis was not different from respective wild-type controls. duction by the liver (VGln) using a double-reciprocal plot analysis However, a strong Sirius red staining was noted in liver slices from in analogy to Lineweaver–Burk blotting. In this approach, the bile duct ligated mice (21 d postsurgery), which served as a positive perivenous intrasinusoidal ammonia concentration available for control (SI Appendix,Fig.S1A). These data suggest that taurine + GS-positive scavenger hepatocytes {[NH4 ]scav} can be esti- transporter knockout does not induce liver fibrosis or structural mated as the sum of the ammonia concentration in effluent + liverdamageinTauTKOmiceuptoanageof12mo.However, {[NH ] } and the ammonia consumed by glutamine synthesis 4 effl + unexpectedly, blood ammonia levels were significantly elevated to and being recovered in effluent perfusate {[Gln] )}: [NH μ + effl 4 about 115 mol/L in both, 3- and 12-mo-old TauT KO mice com- ] = [NH ] + 2 [Gln] . As shown in Fig. 5A double re- pared with WT mice, which presented blood ammonia levels of scav 4 effl + effl μ ciprocal blotting of [NH4 ]scav versus VGln showed that the max- about 70 mol/L (Fig. 1). imal rates of hepatic glutamine production were 0.50 μmol/g/min and 0.48 μmol/g/min in 3-mo-old wild-type and TauT KO mice, Effects of TauT KO on Hepatic Glutamine and Urea Production from respectively, indicating that the capacity for glutamine synthesis Ammonia in Perfused Mouse Liver. The effect of TauT deficiency was not affected by TauT deficiency. On the other hand, the + on hepatic ammonia handling was analyzed in perfused livers ammonia concentrations {[NH ] } required for half-maximal from 3- and 12-mo-old WT and TauT KO mice.