Glutaminolysis-ammonia-urea Cycle Axis, Non-alcoholic Fatty Liver Disease Progression and Development of Novel Therapies

Copy igh : © 2022 The Au ho (s). This a icle has been published unde he e ms o C ea i e Commons A ibu ion-Noncomme cial 4.0 In e na ional License (CC BY-NC 4.0), which pe mi s noncomme cial un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided ha he ollowing s a emen is p o ided. “This a icle has been published in Jou nal o Clinical and T ansla ional Hepa ology a h ps://doi.o g/10.14218/JCTH.2021.00247 and can also be iewed on he Jou nal’s websi e a h p://www.jc hne .com ”. Re iew A icle Jou nal o Clinical and T ansla ional Hepa ology 2022 ol. 10(2) | 356–362 DOI: 10.14218/JCTH.2021.00247 Glu aminolysis-ammonia-u ea Cycle Axis, Non-alcoholic Fa y Li e Disease P og ession and De elopmen o No el The apies Ángela Rojas1,2,3# , Ma ía Rosa io Ga cía-Lozano1,2,3,4# , An onio Gil-Gómez1,2,3# , Manuel Rome o-Gómez1,2,3 and Ja ie Ampue o1,2,3* 1Depa men o Uni o Diges i e Diseases, Vi gen del Rocío Uni e si y Hospi al, Se ille, Spain; 2SeLi e g oup a he Ins i- u e o Biomedicine o Se ille (IBIS), Vi gen del Rocío Uni e si y Hospi al/CSIC/ Uni e si y o Se ille, Se ille, Spain; 3Cen e o he S udy o Li e and Gas oin es inal Diseases (CIBERehd), Ca los III Na ional Ins i u e o Heal h, Mad id, Spain; 4De- pa men o O ganic and Medicinal Chemis y, Facul y o Pha macy, Uni e si y o Se ille, E-41071, Se ille, Spain Recei ed: 27 June 2021 | Re ised: 29 July 2021 | Accep ed: 14 Oc obe 2021 | Published: 4 Janua y 2022 Abs ac The p e alence o non-alcoholic a y li e disease (NAFLD) is inc easing wo ldwide, e lec ing he cu en epidemics o obesi y, insulin esis ance, ype 2 diabe es melli us, and me abolic synd ome. NAFLD is cha ac e ized by he accu- mula ion o a in he li e , and is known o be a cause o ci - hosis. Al hough many pa hways ha e been p oposed, he cause o NAFLD-linked ib osis p og ession is s ill unclea , which posed challenges o he de elopmen o new he a- pies o p e en NASH- ela ed ci hosis and hepa ocellula ca cinoma. Ci hosis is associa ed wi h ac i a ion o hepa ic s ella e cells (HSC) and accumula ion o excess ex acellula ma ix p o eins, and inhibi ing he ac i a ion o HSCs would be expec ed o slow he p og ession o NAFLD-ci hosis. Mul iple molecula signals and pa hways such as oxida i e s ess and glu aminolysis ha e been epo ed o p omo e HSC ac i a ion. Bo h mechanisms a e plausible an i ib o ic a ge s in NASH, as he ac i a ion o HSCs he p oli e a ion o myo ib oblas s depend on hose p ocesses. This e iew summa izes he ole o he glu aminolysis-ammonia-u ea cycle axis in he con ex o NAFLD p og ession, and shows how he axis could be a no el he apeu ic a ge . Ci a ion o his a icle: Rojas Á, Ga cía-Lozano MR, Gil- Gómez A, Rome o-Gómez M, Ampue o J. Glu aminolysis- ammonia-u ea Cycle Axis, Non-alcoholic Fa y Li e Disease P og ession and De elopmen o No el The apies. J Clin T ansl Hepa ol 2022;10(2):356–362. doi: 10.14218/JCTH. 2021.00247. In oduc ion Non-alcoholic a y li e disease (NAFLD) is a se ious pub- lic heal h p oblem a ec ing mo e han hal a billion people wo ldwide,1 being a leading cause o li e ansplan a ion and hepa ocellula ca cinoma (HCC).2,3 I is de ined as a li e disease wi h e idence o lipid accumula ion by hepa o- cy es (hepa ic s ea osis) con i med by his ology, imaging, o nonin asi e biochemical es s in he absence o signi i- can alcohol in ake, de ined as 30 g/day o women and 40 g/day o men, long- e m use o s ea ogenic medica ion, o monogenic he edi a y diso de s.4 In mos pa ien s, NAFLD is associa ed wi h me abolic co- mo bidi ies like o e weigh o obesi y, dyslipidemia, a e ial hype ension, and ype 2 diabe es melli us (T2DM). App oxi- ma ely 90% o pa ien s wi h NAFLD ha e a his o y o leas one me abolic synd ome, which means ha NAFLD com- p ises a g oup o condi ions and is he eason ha unde - lies es ima es ha he wo ldwide p e alence will con inue o inc ease. In ha se ing, a g oup o expe s has ecen ly p oposed a change in name o MAFLD (me abolic-associa ed a y li e disease) and a diagnosis based on he p esence o me abolic dys unc ion and no on he absence o o he condi- ions.5 The pa hogenesis o NAFLD is hus mul i ac o ial, and changes in se e al physiological sys emics ha e been impli- ca ed. Insulin esis ance is one o mos ele an mechanisms, and is ul ima ely associa ed wi h lipo oxici y, endoplasmic e- iculum s ess, and au ophagy. Also, a complex in e ac ion o en i onmen al ac o s, changes in he mic obio a, and p e- disposing gene ic a ian s con ibu e o NAFLD.6 The his o- logical spec um o NAFLD anges om simple s ea osis wi h accumula ion o iglyce ides by hepa ocy es o non-alcoholic s ea ohepa i is (NASH), cha ac e ized by apop osis, nec osis, in lamma o y cell in il a ion, ballooning, and ib osis agains Keywo ds: Non-alcoholic a y li e disease; Ci hosis; Fib osis; Glu aminoly- sis; Ammonia; U ea. Abb e ia ions: α-SMA, alpha-smoo h muscle ac in; ARG1, a ginase-1; ASAL, a ginigosuccina e lyase; ASS, a ginigosuccina e syn he ase; CD-HFD, choline- de icien high- a die ; CPS1, ca bamoyl Phospha e Syn he ase I; FXR, Fa nesoid X ecep o ; GAB, glu aminase B; GAC, glu aminase C; GLS, glu aminase; GLUD1, glu ama e dehyd ogenase 1; GPR91, cogna e G p o ein-coupled ecep o 91; GS, glu amine syn he ase; HCC, hepa ocellula ca cinoma; HSC, hepa ic s el- la e cells; KGA, kidney- ype glu aminase; LGA, li e - ype glu aminase; LOLA, L-o ni hine L-aspa a e; MCD, choline-de icien die ; MCDD, me hionine die and choline-de icien die ; MF, myo ib oblas s; NAFLD, non-alcoholic a y li e dis- ease; NASH, non-alcoholic s ea ohepa i is; OP, o ni hine phenylace a e; OCT, o - ni hine ansca bamoylase; ROS, eac i e oxygen species; T2DM, ype 2 diabe es melli us; TCA, ica boxylic acid cycle; VLDL, e y low-densi y lipop o ein. #These au ho s ha e equally con ibu ed o his wo k and sha e co- i s au ho - ship. *Co espondence o: Ja ie Ampue o, Diges i e Disease Depa men and CIBERehd, Vi gen del Rocio Uni e si y Hospi al, A enida Manuel Siu o s/n, Se- illa 41013, Spain. ORCID: h ps://o cid.o g/0000-0002-8332-2122. Tel: +34- 955-015761, Fax: +34-955-015899, E-mail: [email p o ec ed] Jou nal o Clinical and T ansla ional Hepa ology 2022 ol. 10(2) | 356–362 357 Rojas Á. e al: Glu aminolysis-ammonia-u ea axis in NAFLD a s ea o ic backg ound.7 NASH is conside ed he mos ha m- ul pa o he spec um o NAFLD, being an impo an isk ac o o ci hosis and li e cance de elopmen as well as ex ahepa ic e en s.8 Abou 20–30% o NAFLD pa ien s p o- g ess o NASH, which is mo e likely o p og ess o ad anced ib osis, ci hosis, and HCC.9 No ably, HCC can also a ise in he NASH s age be o e ci hosis occu s (Fig. 1).10 Managemen o NASH should ocus on he es i u ion o me abolic de angemen s and hal ing in lamma o y, ib o- genic, and ca cinogenic pa hways.11 Li es yle in e en ion, based on he Medi e anean die and inc easing physical ac- i i y, is he i s -line he apy.12 Se e al d ugs a e unde in- es iga ion in phase II and III clinical ials in pa ien s wi h unsuccess ul li es yle in e en ions. Howe e , he e a e cu - en ly no d ugs app o ed o he managemen o his en i y. The causes and me abolic ep og amming unde lying he pa hogenesis o NAFLD-d i en ci hosis and HCC emain o be disco e ed. In his e iew, we summa ize he ole o he glu aminolysis-ammonia-u ea cycle in he pa hogenesis o NAFLD and i s ole as a po en ial no el he apeu ic a ge . Glu aminolysis and he u ea cycle P e ious s udies ha e epo ed a s ong associa ions be ween NAFLD and he glu amine pa hway and u ea cycle13,14 ha a e in ol ed in in acellula a y acid accumula ion and ib o- genesis. L-glu amine is he mos abundan ee amino acid in he human body. I s hepa ic me abolism has p e iously been implica ed in egula ing cellula edox eac ions and en- e gy balance as glu amine can be used as a subs a e in he ica boxylic acid cycle (TCA) o suppo mi ochond ial me- abolism.15 Glu amine me abolism pa hways and he se e al o he equi ed enzymes a e shown in Figu e 2. Glu aminase ca alyzes he i s s ep o glu amine ca abolism in he li e by con e ing glu amine o glu ama e and ammonia, and esul - ing in wo phospha e-ac i a ed glu aminase iso o ms, GLS2 and GLS. The GLS2 gene, loca ed in ch omosome 12, en- codes wo splice a ian s wi h low ac i i y and allos e ic egu- la ion, li e - ype glu aminase (LGA, sho ansc ip iso o m), and glu aminase B (GAB, long ansc ip iso o m), which a e highly exp essed in he no mal adul li e .16 Likewise, he GLS gene, loca ed in ch omosome 2, encodes wo splice a ian s wi h high ac i i y and low subs a e a ini y, kidney- ype glu- aminase (KGA, long ansc ip iso o m) and glu aminase C (GAC, sho ansc ip iso o m), which a e mainly exp essed in he kidney unde heal hy condi ions.17 P e ious s udies ha e shown ha GLS is up egula ed in cells wi h inc eased p oli e a ion a es and accoun s o mos glu aminase ac i i y in some human umo cells, whe eas GLS2 exp ession is as- socia ed wi h es ing o quiescen cell s a es.18,19 Ammonia is a gu -de i ed ni ogen compound p oduced by hyd olysis o u ea by bac e ial o ganisms, and by glu amine ca abolism.20 Changes in he mic obio a and he gu –li e axis a ec he me abolism o lipids and ca bohyd a es, impac he balance o in lamma o y media o s, and cause me abolic de- egula ion ha p omo e NAFLD p og ession. The mic obio a and i s me aboli es, such as ammonia, play di ec and indi ec oles in gu ba ie unc ion and ib osis de elopmen , bu u - he s udies a e needed.21 In no mal condi ions, ammonia is elimina ed by he li e h ough he hepa ic u ea cycle, a p o- cess known as u eagenesis. The main ole o his pa hway is o ni ogen balance in he whole body and equi es i e u ea cycle enzymes, ca bamoyl phospha e syn hase I (CPS1), o ni hine ansca bamylase (OCT), a gininosuccina e syn- hase (ASS), a gininosuccina e lyase (ASAL), and a ginase-1 (ARG1). I he u ea cycle is dis up ed, he concen a ion o ammonia in he li e inc eases, and glu amine syn he ase (GS) ac s o p omo e glu amine syn hesis. Newly syn hesized glu amine is eleased in o he blood, en e s he li e and is me abolized by GLS o GLS2 o glu ama e and ammonia. Glu amine, glu aminase ac i i y, and ammonia me abo- lism ha e been desc ibed in li e diseases, mainly in acu e li e ailu e and ci hosis,22 hepa ic encephalopa hy23,24 and HCC.25,26 Howe e , i s ole in he p og ession o li e dis- eases like NAFLD has no been desc ibed. Glu aminolysis, mainly GLS ac i i y, has been associa ed wi h (a) he ac- cumula ion o in acellula lipids by hepa ocy es,13 (b) HSC ac i a ion and ib osis p og ession,14,27 and (c) me abolic ep og amming ha is needed o uel cance cell g ow h.28 Glu amine me abolism and NAFLD The de ailed mechanism o NAFLD p og ession o NASH e- mains as a challenge. I is well known ha insulin esis - ance and s ea osis p omo e NASH p og ession.29 In addi- ion, he accumula ion o excess lipids in he li e p omo es oxida i e s ess, inc eases a y acid oxida ion, and eleases p oin lamma o y cy okines ul ima ely esponsible o mi o- chond ial and hepa ocy e damage, in lamma ion, and ac i- a ion o ib ogenesis pa hways.30 The e idence sugges s ha NASH is a mul i ac o ial p ocess in ol ing nume ous condi ions, including abe a ions o lipid me abolism, oxida- i e and endoplasmic e iculum s ess, insulin esis ance, mic obio a imbalance, immune esponses, gene ic suscep- ibili y, and genomic ins abili y.31 The p ocesses appea o modi y some signaling pa hways in which he glu aminoly- sis-ammonia-u ea cycle is included. The e a e se e al hy- po heses abou he ni ogen imbalance in NAFLD. Al hough u he s udies a e needed o con i ma ion, hey include (a) in es inal dysbiosis o mic obio a changes;21 (b) inc eased glu aminolysis,13 (c) u ea cycle dys unc ion,32 and (d) de- c ease o glu amine syn hesize ac i i y.33 Fig. 1. Spec um o non-alcoholic a y li e disease (NAFLD). Jou nal o Clinical and T ansla ional Hepa ology 2022 ol. 10(2) | 356–362358 Rojas Á. e al: Glu aminolysis-ammonia-u ea axis in NAFLD S ea osis and he glu amine-ammonia-u ea cycle axis Recen ly, Simon e al.13 showed ha GLS was o e exp essed in he li e s o NASH pa ien s and in mice in die -induced NASH models, p o iding e idence ha glu amine me abolism is a po en ial he apeu ic a ge in NASH p og ession. I is well known ha dys unc ion o e y-low-densi y lipop o ein (VLDL) syn hesis and sec e ion occu s in NASH.34 Mice eed a choline-de icien die (MCD), he main componen o VLDL memb anes, esul s in a li e accumula ion.35 Li e al.36 showed in an animal model ha a me hionine and choline-de- icien die esul ed in signi ican changes in se um glu amine le els, and o he s udies epo ed a swi ch om he GLS2 o he GLS iso o m in he end s ages o ch onic li e disease.18,27 Bo h mRNA and p o ein exp ession o GLS we e signi ican ly up egula ed, whe eas GLS2 exp ession dec eased in gene ic Ma 1a−/− mice ed a 0.1% me hionine- and choline-de icien die (MCDD) o 6 weeks. The e idence he hepa ic GLS was inc eased in p eclinical NASH models, led o s udies in li e issue om NASH pa ien s. Hepa ic GLS le els we e inc eased in NASH pa ien s compa ed wi h heal hy con ols. In addi ion, i Gls was silenced in mice ed a 0.1% MCDD and choline- de icien high- a die (CD-HFD) hen he a con en o he li e dec eased because o he es o a ion o VLDL syn hesis pa hways and educ ion o oxida i e me abolism.13 Li e ib osis and he glu amine-ammonia-u ea cycle axis Recen epo s ha e desc ibed he in ol emen o glu ami- nolysis in li e disease, mainly in he ib ogenesis pa hway and HCC de elopmen .27,37 Ac i a ion o HSCs is a key s ep in li e ib ogenesis and ca cinogenesis. Mul iple molecu- la signals and pa hways ha e been epo ed o modula e HSC ac i a ion, including oxida i e s ess38 and cell me- abolism.39 Induc ion o glu aminolysis is a key componen o ib ogenesis p og ession and is necessa y o cance cell g ow h.27,40 Indeed, some cance cells a e glu amine de- penden , and he inhibi ion o glu aminolysis has been p o- posed as a po en ial he apy o some cance s. Hepa ic mi ochond ial dys unc ion has a majo ole in NASH p og ession. Se e al icious cycles in ol ing TNF-α, eac i e oxygen species (ROS), and chemically eac i e li- pid pe oxida ion p oduc s al e mi ochond ial espi a o y chain polypep ides and mi ochond ial DNA in hepa ocy es con aining a acuoles. In his se ing, mi ochond ial ROS o ma ion esul s in biologically ac i e lipid pe oxida ion p oduc s and cy okine elease, which combine o igge hepa ic damage.41 ROS o igina ing om an impai ed TCA cycle, a y acid oxida ion, oxida i e phospho yla ion, lipo- oxici y.38,39 and in il a ing in lamma o y cells du ing li e inju y induces ansdi e en ia ion o HSCs o MF-HSCs and he consequen syn hesis o ex acellula ma ix.42 Recen ly, Chia a e al.33 epo ed inc eased ammonia and p og essi ely dec eased GS concen a ions in NASH pa- ien s compa ed wi h hose wi h simple s ea osis because o a educ ion in he exp ession o se e al u ea cycle genes. In addi ion, ch onic exposu e o high ammonia le els was ound o ha e p o ound e ec s on HSC ac i a ion and ib o- genesis.14 Du e al.27 epo ed inc eased bioene ge ic and biosyn he ic demands by ac i a ed HSCs ha we e suppo - ed by an inc ease in glu aminolysis.43 In addi ion, ac i a ed HSCs in cul u e and in i o we e shown o ha e inc eased u iliza ion o glu amine and exp ession o genes in ol ed in Fig. 2. Glu amine me abolism pa hway and i s associa ion wi h NASH- ib ogenesis. Up egula ed pa hways a e shown in ed. CPS, ca bamoyl phospha e dehyd ogenase; FXR, a nesoid X ecep o ; GLS, glu aminase; GLUD, glu ama e dehyd ogenase; GS, glu amine syn he ase; NASH, non-alcoholic s ea ohepa i is; OCT, o ni hine ansca bamoylase; SDH, succina e dehyd ogenase. Jou nal o Clinical and T ansla ional Hepa ology 2022 ol. 10(2) | 356–362 359 Rojas Á. e al: Glu aminolysis-ammonia-u ea axis in NAFLD glu amine me abolism, such as GLS, glu amic-oxaloace ic ansaminase, and glu ama e dehyd ogenase 1 (GLUD1).44 In he same way, succina e, which is con e ed o uma a e in he TCA cycle by succina e dehyd ogenase, is in ol ed in HSCs ac i a ion binding o and ac i a ing i s cogna e G p o- ein-coupled ecep o 91 (GPR91). The induc ion o GPR91 p omo ed he up egula ion o ib ogenic ma ke s such as alpha-smoo h muscle ac in (α-SMA), ans o ming g ow h ac o -be a (TGF-β), and collagen ype I, sugges ing ha he succina e-GPR91 pa hway may also be a po en ial he apeu- ic a ge in li e ib osis.45,46 The a ailable esul s con i m ha glu amine me abolism is inc eased in NASH, and ela ed o s ea osis, oxida i e s ess, and ib ogenic pa hways, which makes i a po en ial he apeu ic a ge o NASH ea men . Role o d ugs on he glu aminolysis axis in NAFLD No d ugs ha e been licensed and app o ed o ea ing NAFLD bu he e ec s o many d ugs on a numbe o pa h- ways a e unde in es iga ion.47 In his scena io, adminis- e ing an ammonia-lowe ing d ug in NAFLD could educe disease p og ession by sca enging non-u ea ammonia and he eby educing he ac i a ion o HSCs.14 Despi e inc eas- ing o he ole o he glu aminolysis-ammonia-u ea cycle axis in he pa hogenesis o NAFLD, no d ug is being de eloped o speci ically a ge his mechanism. The oles o some glu- aminase inhibi o s in NAFLD, such as BPTES and CB-839, emain unclea . Ins ead, ammonia-lowe ing d ugs, such as L-o ni hine L-aspa a e (LOLA) o o ni hine phenylace a e (OP), ini ially indica ed o o he diseases (e.g., hepa ic en- cephalopa hy)48 a e being es ed o NAFLD. In addi ion, some d ugs ini ially de eloped o NAFLD ha e o - a ge e - ec s on he glu aminolysis-ammonia-u ea cycle axis. Speci ic NAFLD- a ge ed d ugs Obe icholic acid is a Fa nesoid X ecep o (FXR) agonis ha has shown e icacy in p ima y bilia y cholangi is49 and p omis- ing esul s o NAFLD in Phase II and III clinical ials.50 FXR, in addi ion o ha ing an essen ial ole in ca bohyd a e and lipid me abolism, exe s e ec s on some genes ha egula e en- zymes in he u ea cycle and ac s as a ansc ip ional egula o o ammonia de oxi ica ion ia u eagenesis and glu amine syn- hesis in he li e .51 Inac i a ion o FXR leads o he hepa ic ac- cumula ion o some u ea cycle p ecu so s, and ac i a ion e- sul s in an inc ease in he exp ession o glu amine syn he ase GS) and u ea cycle- ela ed genes ha p omo es glu amine syn hesis and u ea p oduc ion. Obe icholic acid induces he exp ession o Glul ia ac i a ion o FXR, which is an al e na i e way o dispose o excess ni ogen because o he con e sion o glu ama e in o glu amine in pe icen al hepa ocy es.52 Repu posing nonspeci ic NAFLD d ugs Me o min is a biguanide used o ea T2DM. No da a sup- po s i s use o NAFLD despi e ini ial en husiasm ela ed o i s mechanism o ac ion, because o a lack o e icacy in educing ib osis.53 Howe e , me o min appea s o be e ec i e as a chemop e en i e agen o HCC and o he umo s.54 A p e i- ous s udy has epo ed pa ial inhibi ion o glu aminase ac i - i y (abou 20%), bo h in chemical and cell assays compa ed wi h con ols.55 On he o he hand, piogli azone, a pe oxisome p oli e a o -ac i a ed ecep o (PPAR)-γ agonis , is ano he an idiabe ic d ug ha could in e e es wi h he glu aminoly- sis-ammonia-u ea cycle axis by es ic ing he con e sion o glu amine o glu ama e by educing he exp ession o GLS1, he a e-limi ing enzyme.56 Thus, in a pe sonalized medicine scena io, some an idiabe ic d ugs a e e ec i e o NAFLD pa- ien s in whom he glu aminolysis-ammonia-u ea cycle axis is he p edominan pa hogenic insul o li e disease. LOLA is a well-known d ug o ea hype ammonemia in ci hosis,57 acu e li e ailu e, and hepa ic encephalopa hy58 by syn hesizing u ea and glu amine ia he enzyme GS. In he se ing o NAFLD, a signi ican imp o emen in ALT le - els has been obse ed, oge he wi h a signi ican dec ease o iglyce ides, in a andomized con olled ial including 72 pa ien s ecei ing LOLA o 12 weeks.59 In ano he ial, LOLA also imp o ed hepa ic mic oci cula ion in 78 pa ien s wi h NASH.60 The e idence suppo s he bene i s o LOLA o pa ien s wi h NAFLD. OP is an ammonia-lowe ing agen ha p omo es glu- amine syn hesis om ammonia in skele al muscle and ex- c e es he o ni hine- ela ed glu amine om he kidneys.61 OP is a sa e and e ec i e ammonia sca enge o ea ing hype ammonemia in condi ions including ci hosis,62 acu e- on-ch onic and acu e li e ailu e.63 OP was shown o e- duce plasma ammonia, issue ma ke s o HSC ac i a ion, and po al p essu e in bile duc -liga ed a s wi h ad anced ib osis and hype ammonemia.64 Rega ding he NAFLD scena io, OP appea s o p e en hepa ocy e dea h and o educe he de elopmen o ib osis, hepa ic ammonia, and in lamma ion in an animal model, by es o ing o u ea cycle enzyme ac i i y and unc ion.14 P eclinical disco e y o glu aminoly ic ammonia- a ge ing he apies D ug disco e y is an in ensi e p ocess ha equi es ex en- si e p eclinical and clinical s udies (Fig. 3).65 Success ul d ug disco e y equi es in-dep h knowledge o a disease, he sig- ni ican elemen s in pa hogenesis. In NAFLD, may be a ele- an enzyme because o i s ole in ammonia p oduc ion (Fig. 4). Essen ial ini ial s eps a e he de elopmen o p o o ypes o “hi s“ agains he p ede e mined a ge (e.g., GLS has been demons a ed as a alida ed a ge )13 and op imiza ion o imp o e po ency and educe side e ec s, o “leads”. Once a lead compound is ound, he candida e has o be assessed in he p eclinical s age o es ablish he sa e y and e icacy, in- cluding in o ma ion abou p ope ies such as abso p ion, dis- ibu ion, me aboliza ion, and exc e ion, as well as he bes dosage and adminis a ion ou e, and side e ec s/ad e se e en s. P eclinical ials equi e in i o, in i o, and ex i o assays in li ing o ganisms o cells in animals and humans, o using nonli ing o ganisms o issue ex ac s. In silico assays based on compu e simula ions can accele a e he p ocess. Conclusions NAFLD is a complex, mul i ace ed condi ion he molecula and cellula le els, which is e lec ed by he di icul y o de- eloping new he apeu ics, bu he need o de elop p e en- i e ea men s ha slow he p og ession o o e en e e se ib osis is clea . In ha se ing, he glu aminolysis-ammo- nia-u ea cycle axis eme ges as a undamen al pa hogenic p ocess in NAFLD. Fu u e s udies should ocus on i o b ing pe sonalized medicine close . Funding This p ojec was unded in pa by he Spanish Minis y o Economy, Inno a ion and Compe i ion, Ins i u o de Salud Ca - los III (PI19/00589, PI19/01404, PI16/01842, PI17/00535 and GLD19/00100). Ángela Rojas has ecei ed he Sa a Bo - Jou nal o Clinical and T ansla ional Hepa ology 2022 ol. 10(2) | 356–362360 Rojas Á. e al: Glu aminolysis-ammonia-u ea axis in NAFLD Fig. 3. D ug disco e y p ocess. Fig. 4. Ta ge ing glu aminolysis: examples o selec ed glu aminase inhibi o s. 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