Glutamate dehydrogenase, hyperammonemia, and HI/HA syndrome: Study on the contribution by the liver

Thesis

Reference

Glutamate dehydrogenase, hyperammonemia, and HI/HA syndrome:

Study on the contribution by the liver

LUCZKOWSKA, Karolina

Abstract

Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of glutamate to α-ketoglutarate. This mitochondrial enzyme is regulated by negative cooperativity and a wide array of allosteric effectors. Among them, most potent inhibitor GTP and most potent activator ADP. The importance of GDH regulation has been highlighted by the discovery of the hyperinsulinism-hyperammonaemia (HI/HA) syndrome. It is caused by dominant activating mutations that abrogate GTP inhibition, resulting in dangerously high serum levels of insulin and ammonium.

LUCZKOWSKA, Karolina. Glutamate dehydrogenase, hyperammonemia, and HI/HA syndrome: Study on the contribution by the liver. Thèse de doctorat : Univ. Genève, 2020, no. Sc. Vie 69

DOI : 10.13097/archive-ouverte/unige:145909 URN : urn:nbn:ch:unige-1459093

Available at:

http://archive-ouverte.unige.ch/unige:145909

Disclaimer: layout of this document may differ from the published version.

UNIVERSIT ´E DE GEN `EVE

D´epartement de Physiologie Cellulaire et M´etabolisme

FACULT´E DE M´EDECINE Prof. Pierre Maechler

Glutamate dehydrogenase, hyperammonemia, and HI/HA syndrome:

Study on the contribution by the liver

TH` ESE

pr´esent´ee `a la facult´e des sciences de l’Universit´e de Gen`eve pour obtenir le grade de Docteur `es sciences, mention biochemie

par

Karolina Luczkowska de

Gdynia (Pologne)

Th`ese N<sup>o</sup> 69 Gen`eve, 2020

Abstract

Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of gluta- mate toα-ketoglutarate. This mitochondrial enzyme is regulated by negative cooperativity and a wide array of allosteric effectors. Among them, most potent inhibitor GTP and most potent activator ADP. The importance of GDH regulation has been highlighted by the discovery of the hyperinsulinism-hyperammonaemia (HI/HA) syndrome. It is caused by dominant activating mutations that abrogate GTP inhibition, resulting in dangerously high serum levels of insulin and ammonium.

The present study provides the first molecular characterisation of GDH-G446V variant with a point mutation located on the descending helix in the antenna region of GDH, lim- iting its rotational capacity. Enzymatic assessment, performed in lymphoblastoid cell line (LCLs) derived from an adult HI/HA patient, pointed to altered responses to both major allosteric modulators of GDH: ADP and GTP. In the assessment of metabolic rate, these alterations translated into enhanced glutaminolysis and higher mitochondrial oxidative capacity in comparison to control cell line.

Ammoniagenesis and gluconeogenesis are essential metabolic functions of the liver.

Hepatic GDH constitutes 1% of the total cellular protein content and is positioned at the interface between amino acid pool and carbohydrate metabolism. The bidirectional reaction catalyzed by GDH allows either ammonium assimilation through the synthesis of glutamate or conversely its oxidation with the concomitant generation of ammonium.

In this study we used inducible liver-specific GDH knockout mice (HepGlud1-/-) to in- vestigate metabolic compensations resulting from hyperammonaemia, occurring at lungs and kidney levels. In order to maintain blood pH, hyperammonaemia is accompanied by increased bicarbonate and lactate levels with increased CO₂ partial pressure. To chal- lenge our model, mice were exposed to diets with varying amount of protein contents.

Accompanying the increase in protein load, we observed in both control and HepGlud1-/- mice linear changes in plasma and urine urea levels. With high protein intake, plasma glutamine levels remained high in HepGlud1-/- mice, while they decreased substantially in control animals. Surprisingly, chronically elevated ammonium levels in HepGlud1-/-

mice decreased with the consumption of high protein diet. Urine and blood pH remained remarkably stable regardless of the amount of protein intake.

The source of hyperammonaemia of HI/HA syndrome is not yet defined. Liver, as a crucial place for nitrogen metabolism and ammonium detoxification, is foreseen as the source of hyperammonaemia in HI/HA patients. We tackled this issue by establishing a model where we can dissect the contribution of the liver expressing GDH_S445L variant, the most frequently seen in HI/HA patients. We used HepGlud1-/- mice transduced with adenoviral vector carrying the GDHS445L variant and we characterised its nitrogen metabolism.

R´esum´e

La glutamate d´eshydrog´enase (GDH) catalyse la d´esamination oxydative r´eversible du glu- tamate enα-c´etoglutarate. Cette enzyme mitochondriale est r´egul´ee par une coop´erativit´e n´egative et un large ´eventail d’effecteurs allost´eriques : l’inhibiteur le plus puissant ´etant le GTP et l’activateur le plus efficace l’ADP. L’importance de la r´egulation de la GDH a ´et´e mise en ´evidence par la d´ecouverte du syndrome d’hyperinsulin´emie-hyperammon´emie (HI/HA). En effet, des mutations activatrices de la GDH, exprim´ees de fa¸con domi- nante et qui suppriment l’inhibition du GTP, entraˆınent des niveaux s´eriques d’insuline et d’ammonium dangereusement ´elev´es.

La pr´esente ´etude fournit la premi`ere caract´erisation mol´eculaire du variant G446V de la GDH avec une mutation situ´ee sur l’h´elice descendante dans la r´egion d’antenne, limi- tant sa capacit´e de rotation. L’´evaluation enzymatique, r´ealis´ee dans une lign´ee cellulaire lymphoblasto¨ıde (LCL) d´eriv´ee d’une patiente adulte HI/HA, indique une modification de la r´eponse aux deux principaux modulateurs allost´eriques de la GDH: l’ADP et le GTP.

Dans l’´evaluation du taux m´etabolique cellulaire, cela se traduit par une glutaminolyse accrue et une oxydation mitochondriale plus ´elev´ee dans la lign´ee LCL portant la mutation G446V par rapport `a la lign´ee t´emoin.

La d´etoxification de l’ammonium en ur´ee et la glucon´eogen`ese sont des fonctions m´etaboliques essentielles du foie. La GDH h´epatique constitue 1% des prot´eines cellu- laires totales et est positionn´ee `a l’interface de contrˆole entre le pool d’acides amin´es et le m´etabolisme des glucides. La r´eaction bidirectionnelle catalys´ee par la GDH permet soit l’assimilation d’ammonium par la synth`ese de glutamate, soit inversement la produc- tion d’α-c´etoglutarate `a partir de glutamate avec la g´en´eration concomitante d’ammonium.

Dans cette ´etude, nous avons utilis´e des souris invalid´ees pour le g`ene de la GDH sp´ecifiquement dans le foie et de mani`ere inductible (HepGlud1-/-) afin d’´etudier les compensations m´etaboliques r´esultant de l’hyperammon´emie associ´ee. En particulier au niveau des poumons et des reins. Afin de maintenir le pH sanguin, l’hyperammon´emie s’accompagne d’une augmentation des taux de bicarbon‘ate et de lactate avec une augmentation de la pression partielle de CO2. Pour tester notre mod`ele, les souris ont ´et´e expos´ees `a des

r´egimes alimentaires avec une quantit´e variable de prot´eines. Associ´es `a des teneurs en prot´eines accrues, nous avons observ´e chez les souris t´emoins et HepGlud1-/- des change- ments lin´eaires d’ur´ee dans le plasma et l’urine. Les taux plasmatiques de glutamine sont rest´es ´elev´es chez les souris HepGlud1-/- avec un apport ´elev´e en prot´eines, tandis que chez les souris contrˆoles, ces taux diminuaient consid´erablement. ´Etonnamment, les niveaux chroniquement ´elev´es d’ammonium des souris HepGlud1-/- ont diminu´e avec la consommation d’un r´egime riche en prot´eines. Le pH de l’urine et du sang sont rest´es remarquablement stables, quelle que soit la quantit´e d’apport en prot´eines.

La source de l’hyperammon´emie li´ee au syndrome HI/HA n’est toujours pas d´efinie.

Le foie, en tant qu’organe crucial pour le m´etabolisme de l’azote et de d´etoxification de l’ammonium, est suspect´e d’ˆetre `a l’origine de l’hyperammon´emie chez les patients HI/HA.

Dans cette perspective, nous avons ´etabli un mod`ele murin o`u nous pouvons diss´equer la contribution du foie avec le variant S445L de la GDH, le variant le plus fr´equemment observ´e chez les patients HI/HA. Nous avons g´en´er´e des souris HepGlud1-/- transduites avec un vecteur ad´enoviral portant le variant GDH_S445L et nous avons initi´e l’´etude de son m´etabolisme azot´e.

Acknowledgements

This thesis would not be possible without the help and support of my supervisor Pierre Maechler, his extensive knowledge has been inspirational in all these years. His insightful comments widen my research from various perspectives and improve my writing.

I would also like to thank Sophie De Seigneux and Enzo Nisoli for having accepted to evaluate my thesis and be part of my jury. Their fair judgment is precious to me.

A lot of people helped me during this journey, I’ll name just a few, but I sincerely remem- ber and thank you all.

Thierry Brun for the stimulating discussions and his contagious love for kidneys.

Eric Feraille for always finding time and his unique sense of humour.

To Dominique, for interesting discussions on history and shared love for cats.

To Lucie to be the gentle creature she is, always, regardless the circumstances.

A Gaelle et Florian pour leur travail consid´` erable. Je ne serais pas ici sans vous. Votre travail et votre humour m’ont aid´e beaucoup.

To Yen, for our shared love for Kasugai peas. To Cecilia for bringing Spanish in the lab, I miss to hear it.

Dla Moniki za to ze jestes zawsze obok mnie. Za to ze moge powiedziec ci duzo, za duzo, a nawet wszystko.

Dla Patryka, za to ze walczysz o siebie, o nas i ze pokochales gory. Jestem z Ciebie dumna.

Dla mojego taty, tesknie za Toba wielkoludzie, Pochodzilabym z Toba po Tatrach.

A Stella e Domenico, per farmi sentire sempre a casa, siete belli!

To Mochi in Kyoto, to Bise in Lausanne. For their vocal contribution in my daily routine.

Marco,

luce della mia vita.

Questa tesi `e per te!

Contents

Abstract iv

R´esum´e vi

Acknowledgements vii

Table of Abbreviations x

1 Introduction 1

1.1 Nitrogen metabolism . . . 1

1.2 Glutamine . . . 3

1.3 Glutamate . . . 4

1.4 Alanine . . . 5

1.5 Metabolic profile of hepatocytes . . . 7

1.6 Ammonium detoxifying systems . . . 8

1.7 Urea cycle . . . 9

1.8 Glutamine synthesis . . . 12

1.9 Kidney ammonium homeostasis . . . 13

1.10 Glutamate dehydrogenase . . . 15

1.11 Thesis Aims . . . 19

2 Results 21 2.1 Influence of protein intake on nitrogen metabolism in mice lacking liver glutamate dehydrogenase . . . 22

2.1.1 Abstract . . . 23

2.1.2 Introduction . . . 23

2.1.3 Materials and methods . . . 25

2.1.4 Results . . . 27

2.2 Hyperinsulinism associated with GLUD1 mutation: allosteric regulation and functional characterization of p.G446V glutamate dehydrogenase . . . . 37

2.3 Establishing a mouse model to investigate the hyperamoneamia of HI/HA syndrome patients . . . 46

2.3.1 Introduction . . . 47

2.3.2 Materials and methods . . . 47

2.3.3 Results . . . 49

3 Discussion 53 3.1 Liver GDH knock outin vivo . . . 53

3.2 GDH-G446V mutationin vitro . . . 57

3.3 Establishing a liver-targeted mouse model to investigate the hyperammon- aemia of HI/HA patients . . . 59

3.4 Perspectives regarding the liver-targeted mouse model of HI/HA patients . 60 3.5 Conclusions . . . 60

Bibliography 61

Table of Abbreviations

Abbreviation Full description

AA Amino Acid

ADP Adenosine diphosphate

ALAT Alanine transaminase ANOVA Analysis of variance

ASAT Aspartate aminotransferase ATP Adenosine triphosphate AUC Area under the ROC curve

BCAT Branched-chain amino acid aminotransferase BCH 2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid

BSA Bovine serum albumin

CNS Central nervous system

CPS Carbamoyl phosphate synthase DMEM Dulbecco’s Modified Eagle Medium DNA Deoxyribonucleic acid

ERT Enzyme replacement therapy

FCS Fetal calf serum

GDH Glutamate dehydrogenase

GLS Glutaminase

GLUT Glucose transporters

GS Glutamine synthetase

GTP Guanosine-5’-triphosphate

Abbreviation Full description

HA Hyperammonemia

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HI hyperinsulinism

KRBH Krebs-Ringer bicarbonate HEPES buffer LCL Lymphoblastoid Cell lines

NAD Nicotinamide adenine dinucleotide NADH Nicotinamide adenine dinucleotide

NAG N-Acetylglucosamine

PEPCK Phosphoenolpyruvate carboxykinase RIPA Radioimmunoprecipitation assay buffer

RNA Ribonucleic acid

RT Room temperature

SEM Standard error of the mean SNAT Serotonin-N-acetyl transferase TCA Tricarboxylic acid cycle

WB Western blot

CHAPTER 1

Introduction

1.1 Nitrogen metabolism

Nitrogen is a common element in the universe and dinitrogen forms about 78% of Earth atmosphere. While being common on Earth, it is not abundant on other neighbouring planets. In order to be usable by plants, nitrogen gas (N2) present in all its abundance in Earth atmosphere must be reduced to ammonia (NH₃) by nitrogen-fixing bacteria. Of which, two kinds are associated with plants: non-symbiotic Cyanobacteria and symbiotic Rhizobium. Nitrogen fixation is carried out by bacterial nitrogenases forming reduced nitrogen, ammonium (NH⁺₄), which in turn can be assimilated by all organisms to form amino acids. In eucaryotes, its assimilation occurs through the action of glutamate de- hydrogenase (GDH) and glutamine synthetase (GS). Glutamate, as a donor or acceptor of nitrogen in these reactions, is playing a central role in mammalian nitrogen flow. Ad- ditionally, ammonium might be transferred to other carbon skeletons by transamination reactions, carried out by transaminases.

In aqueous solutions, NH3 is a base, forming a conjugated pair with NH⁺₄. The pKa of the reaction is 9.3, i.e. at this pH the equilibrium is reached and the concentrations of NH3 and NH⁺₄ are equal. With the drop of pH below pKa, the ionized form predominates.

Since the physiological plasma and intracellular pH values oscillate around 7.4, 99% of NH₃ is in the ionized form. Consequently, changes in pH cause exponential changes in NH3 and almost no change in NH⁺₄.

NH3 is a small, uncharged molecule. This feature misled into believing that NH3 can diffuse across lipid membrane. However, ammonia is a molecule with trigonal pyramid geometry resulting from asymmetric electrostatic potential of partially negative nitrogen and partially positive hydrogens. This arrangement makes ammonia a relatively polar molecule and of a dipole moment of 1.47 D. Consequently, NH₃ has a very limited plasma membrane permeability, similar to its conjugated pair NH⁺₄, at least in the absence of specific transport proteins. In human, the plasma concentration of NH⁺₄ ranges between 11-50µM, varying slightly depending upon the site of vasculature. Values are the highest in the portal vein since ammonium produced in gastrointestinal tract - due to protein digestion and microbial metabolism - diffuses through the intestinal mucosa and is carried out by the portal circulation to the liver. In ureotelic organisms, ammonium is converted into urea through the stream of reactions called urea cycle, found exclusively in the liver.

Glutamine synthesis is an alternative pathway of ammonium detoxification in the liver and may act as a sink for excess NH⁺₄ through glutamine synthetase action. In hepatic vein, the blood distributed further to different organs is rich in nitrogen carriers, like urea and glutamine, and poor in free ammonium. Glutamine constitutes 50% of the body’s free amino acid pool and it has an important role in inter-tissue physiology. In addition to its role as a nitrogen donor, glutamine is an important vehicle for the transport of carbon between different organs. During mild hyperammonaemia, due to liver failure, muscles act as a buffer, taking up NH⁺₄ and temporarily detoxifying it into glutamine [Olde Damink et al., 2002]. In kidney, glutamine metabolism plays a crucial role in an acid-base homeostasis. Its catabolism results in ammonium production which disposal into urine restores physiological pH of blood. Ammonia in its gaseous form can be detected in human skin [Noseet al., 2005] and in exhaled air of the lungs, indicating that these organs may marginally participate to nitrogen elimination. This interorgan ammonium traffick- ing have many constituents with intestines and kidney as main NH⁺₄ producers, liver and muscle as main NH⁺₄ consumers. It is an ongoing dynamic process of free ammonium ions continually produced (breakdown of purine and pyrimidine bases and amino acids) and consumed (glutamine and carbamoylphosphate synthesis). The enzymes involved in the metabolism of ammonium ions are the cytosolic enzyme GS and mitochondrial enzymes:

glutaminase (GLS), GDH and carbamoylphosphate synthetase-1 (CPS-1). Aminotrans- ferases are both cytosolic and mitochondrial enzymes involved in transferring of amino group between amino acids and their paired keto acids without generating nor consuming NH⁺₄. The ones of major physiological significance are aspartate transferase (ASAT), ala- nine transferase (ALAT) and branched-chain amino acids transferase (BCAT). Systemic

1.2. Glutamine

ammonium homeostasis is dependent upon the activity and distribution of these enzymes in different organs. Ammonium at high levels is toxic, leading to functional disturbance of the central nervous system, predominantly affecting the astrocytes, which in turn can lead to coma and death [Norenberget al., 2004].

Abbreviation Name Gene Reaction catalysed

GDH Glutamate dehydrogenase GLUD1 Catalyses the oxidative deamination of glutamate to α-ketoglutarate and ammonium

GLS Glutaminase GLS Catalyses the hydrolysis of glutamine

to glutamate and ammonium

GS Glutamine synthetase GLUL Catalyses the synthesis of glutamine from glutamate and ammonium in an ATP-dependent reaction

ALAT Alanine aminotransferase GPT Catalyses the reversible transamination between alanine and 2-oxoglutarate to generate pyruvate and glutamate ASAT Aspartate aminotransferase GOT1 Catalyses the reversible transfer of an

α-amino group between aspartate and glutamate

Table 1.1: Table of ammonium metabolizing enzymes of interest.

1.2 Glutamine

Apart from glucose, glutamine is a primary nutrient for the cells [Bodeet al., 2002]. Its importance derives from the characteristics it shares with glucose. Both nutrients sat- isfy two essential needs of the cell: ATP production and the provision of intermediates for biosynthetic pathways [DeBerardinis and Cheng, 2010]. Accordingly, plasmatic glu- tamine concentration is relatively high ∼0,5mM [Norenberg et al., 2004]. While diet is an important source of this amino acid, glutamine is synthetized by most tissuesde novo from glutamate, mainly in the liver and muscles. The enzymes involved in glutamine biosynthesis are GDH and GS. The action of the latter is an ATP-dependent addition of ammonium to glutamate. Glutamine may also be freed from proteins by proteolysis.

Its degradation involves double deamination toα-ketoglutarate in reactions catalysed by phosphate-dependent GLS and then GDH localized in mitochondrial matrix. Glutamine-

derivedα-ketoglutarate replenishes the TCA cycle and gives rise to: two ammonium and two reducing equivalents (NADH). Glutamine’s metabolic fate in proliferating cells serves primarily as source of energy, i.e. mostly oxidized in mitochondria. Furthermore, carbon skeleton of glutamine might be used as gluconeogenic substrate in the liver and kidneys.

During starvation, glucose is mostly produced in the liver, but the kidneys can account for about 20% of this circulating substrate [Stumvollet al., 1999]. In the liver, glutamine provides nitrogen for the urea cycle through the action of GLS and GDH. Additionally, glutamine is a building block for proteins, precursor of purines, pyrimidines, glutathione, glutamate andγ-aminobutyrate. Skeletal muscles constitute the primary reservoir of glu- tamine, acting as an ammonium scavenger. Glutamine is essential for acid-base home- ostasis in the kidney: used by tubular epithelial cells as a source of NH⁺₄ for disposal into urine during metabolic acidosis [Busque and Wagner, 2009]. Besides, glutamine rep- resents a major substrate used by intestinal cells. It promotes enterocytes proliferation, regulates tight junctions, supresses pro-inflammatory signalling pathways in the intestines [Kim and Kim, 2017]. The involvement of glutamine metabolism in immune and cancer cells has been documented thoroughly [Calder, 1994; Kim and Kim, 2013]. Moreover, one of the hallmarks of cancer and immune cell is their dependence upon glutamine and increased glutaminolysis. In tumor cells, glutamine throughα-ketoglutarate, malate, and pyruvate is transformed into lactate that acidifies the cellular environment. Even though glutamine can be produced de novo by the cells, most of them rely upon extracellular glutamine supply at least to some extent. This is particularly the case for highly prolif- erative cells such as immune cells, intestine cells and cancer cells [Bhutia and Ganapathy, 2016]. Glutamine, classified early on as a non-essential amino acid, might instead become rate-limiting and conditionally essential in various pathologic states. Particularly under catabolic conditions, the consumption of glutamine by the kidney, gastrointestinal track and immune cells rises dramatically [Felig, 1973; Cahill, 2006; Owenet al., 1967].

1.3 Glutamate

Glutamine is quantitatively the most important precursor of glutamate. The intracellular glutamate pool relies heavily upon glutamine provision and GLS action [DeBerardinis and Cheng, 2010]. Glutamate is a non-essential amino acid; it can be synthetized in the body through different metabolic pathways and is in the centre of all transamination reactions in the body. Metabolism of arginine, ornithine, proline and histidine also gives rise to glutamate. Once incorporated into glutamate, nitrogen can be dispatched into different pools of amino acids through the action of aminotransferases: ASAT and ALAT. These

1.4. Alanine

enzymes are catalysing the reversible transfer of amino group between glutamate and alanine or aspartate. The biological versatility of glutamate might be partially explained by its close association with the TCA cycle and straight conversion toα-ketoglutarate by GDH. This amino acid fulfils unique functions in different tissues. In liver, glutamate serves as a precursor molecule for the synthesis of N-acetyl-L-glutamate, which is an allosteric activator of the CPS-1 – 1^st enzyme of urea cycle. Its presence triggers the production of carbamoylphosphate, the rate-limiting step of urea cycle. In the brain, glutamate is a crucial excitatory neurotransmitter acting through G-coupled glutamate receptors that modify neuronal and glial excitability. In pancreaticβ-cells, glutamate is participating in the amplifying pathway of insulin secretion as well as an extracellular feedback inhibitor of insulin secretion [Maechler, 2017].

1.4 Alanine

The average concentration of alanine in the plasma is 0.35mM [DeBerardinis and Cheng, 2010]. During fasting, the release of alanine from the muscles increases but paradoxi- cally blood alanine concentration decreases, suggesting enhanced extraction by the liver [MacDonaldet al., 1976]. ASAT, the enzyme initiating alanine metabolism, is located pre- dominantly in liver, muscles and intestines [Yanget al., 2009]. It catalyses the reversible transfer of amino group from glutamate to pyruvate, producingα-ketoglutarate and ala- nine. Since the reaction is reversible and pyruvate is both a substrate and a product, it establishes a close link with metabolic pathways related to glycolysis, gluconeogenesis and the TCA cycle. Furthermore, as the reaction is at near equilibrium, the direction is determined by the relative concentrations of the available substrates and products. As mentioned, alanine can be synthetized from pyruvate and from branched chain amino acids as leucine, valine and isoleucine. Alanine is quantitatively the most abundant amino acid released by muscles in prolonged fasting. Its production from extrahepatic tissue not only derives from the catabolism of cellular proteins but is also contributed by itsin situ synthesis from glucose-derived pyruvate through transamination. This requires an appro- priate source of amino group to transfer it to pyruvate, provided by branched chain amino acids [5]. Alanine released from the muscles is transported through systemic circulation to the liver where it is deaminated to pyruvate. The resulting carbon skeleton is then used in gluconeogenesis with subsequent ammonium entrance in urea cycle. During overnight fasted conditions, circulating glucose levels fluctuate only a little due to hepatic glucose production keeping up with peripheral glucose utilization [DeBerardinis and Cheng, 2010].

This cycling of nutrients between skeletal muscle and the liver refers to the glucose-alanine

cycle. The gluconeogenic pathway progressively takes over as starvation lasts and becomes vital for the glucose homeostasis. It seems that glutamine is the most important glucose precursor in the kidney, whereas alanine-induced gluconeogenesis is essentially confined to the liver [DeBerardinis and Cheng, 2010].

Dietary protein

Glutamine Alanine GLS

Glutamate NH4+ ALAT

NH4+

α-KT

Alanine NH4+

UREA

Glutamate ALAT

Pyruvate

glucose GDH

GDH

ASAT Glutamine

GLS NH4+ GS

NH4+

Aspartate ASAT Glutamine α-KT

GLS NH4+

Glutamate GDH NH4+

α-KT

URINARY EXCRETION

Val,Leu,Ile α-KT Glutamate α-keto acid

Alanine Pyruvate

glucose ALAT

GS NH4+

Glutamine

AT

Figure 1.1: The inter-organ flow of metabolites in nitrogen metabolism.Release and up- take of nitrogen carriers by various organs in humans and their subsequent metabolism in different tissues.

1.5. Metabolic profile of hepatocytes

1.5 Metabolic profile of hepatocytes

Hepatic balance between glucose utilization, storage and production is finely tuned by wide array of hormonal signals and nutritional status of the body. The net hepatic pro- duction is the sum of the main fluxes through gluconeogenesis, glycogenesis, glycogenolysis and glycolysis. In the fasted state, liver maintains euglycemia to provide fuel for obligate glucose consuming cells such as neurons and red blood cells. Starvation time initiates a series of adaptations to enable the continuous production of nutrients for critical organs [Chunget al., 2015]. This crucial survival response is coordinated by the liver which first liberates glucose from its glycogen stores, backed up byde novo glucose production later on. Liver glucose production accounts for 90% of endogenous production. In addition, ke- tone bodies are produced to provide alternative substrate to glucose as a source of energy for highly oxidative tissues such as the CNS. Postprandially, GLUT2 transporter permits the internalization of glucose surplus from the circulation before blood is redistributed and reaches other organs. Upon enteral glucose entrance, hepatocytes increase glycogen synthesis and suppress hepatic glucose output. During short-term fasting, glycaemia is the result of both glycogenolysis and gluconeogenesis, while upon longer fasting period glucose production relies solely on gluconeogenesis [Exton, 1979]. Gluconeogenesis is the de novosynthesis of glucose from non-carbohydrate precursors such as lactate and amino acids (released from muscles), glycerol (derived from triglycerides stored in adipocytes).

Since the non-glucose precursors need to be mobilized and consecutively transported to the liver, it does not participate to the immediate response to hypoglycaemia (unlike glycogen mobilisation). Gluconeogenesis takes place predominantly in the liver (system- ically important) and to a lesser extent in the cortex of the kidney (locally important).

The pathway uses glycolytic enzymes, except for the irreversible reactions catalysed by pyruvate kinase, 6-phospho-fructokinase and hexokinase. Those are bypassed by alternate reactions in gluconeogenesis pathway: pyruvate carboxylase, fluctose-1,6-phosphatase and phosphoenolpyruvate carboxykinase (PEPCK), which is the rate-limiting reaction of the pathway. Glycolysis is reciprocally regulated to avoid futile cycle. Gluconeogenesis is stimulated by diabetogenic hormones such as glucagon, growth hormone, epinephrine and cortisol. The pathway is energy-dependant and requires 4 ATP and 2 GTP in order to generate glucose-6-phosphatase from pyruvate. Among the amino acids transported from the muscles to the liver in energy-challenging conditions, alanine is the most abundant while glutamine is a preferential substrate for gluconeogenesis in the kidney [Stumvoll et al., 1998]. The in vivo study by Karaca et al. showed that in order to metabolize alanine in the liver, hepatic GDH is crucial for this substrate to be glucogenic [Karaca et al., 2018a].

1.6 Ammonium detoxifying systems

The increased availability of precursors for gluconeogenesis is paralleled by increase in sub- strates for the urea cycle resulting from protein catabolism and concomitant build-up of ammonium due to associated deamination. The rate of ureagenesis determines the rate of NH⁺₄ elimination and consequently might impact the amino acid entry into gluconeogenic pathway [Ohtake and Clemens, 1991]. There are two essential pathways for ammonium detoxification in the liver: glutamine synthesis and ureagenesis. Hepatocytes located along the porto-venous axis are characterized by zone-specific differences in enzymatic activities, embedded within sophisticated structural and functional liver organization [23]. The spe- cial organisation of the various metabolic pathways - metabolic zonation - forms the basis for efficient adaptation of hepatic metabolism to different nutritional requirements of the body in different metabolic states [Gebhardt, 1992]. Metabolic zonation allows efficient interactions between hepatocyte subpopulations for ammonium homeostasis maintenance:

periportal hepatocytes located near sinusoid inflow receive blood highly oxygenated and rich in nutrients, while perivenous hepatocytes near sinusoid outflow have the poorest sup- ply of oxygen and nutrients. The borderline between periportal perivenous hepatocytes is not anatomically defined but is based on comparative-functional aspects, based on the metabolic pathways in consideration: the glutamine and urea synthesis pathways. These are mutually exclusive in terms of function in the cell. Urea synthesis and glutaminase are present in periportal zone, whereas glutamine synthetase is found exclusively in the subpopulation surrounding the terminal hepatic venule, which is virtually free of CPS-1 [Haussinger, 1990a]. Preferential amino acid uptake is linked to this enzymatic compart- mentalization: glutamate is taken up by perivenous zone, limited to 7% of the acinus hepatocytes; while proline, alanine and glutamine are taken up by the highly ureagenic and gluconeogenic periportal zone [Haussinger, 1990a]. Additionally, periportal hepato- cytes display higher ASAT enzymatic activity [Yang et al., 2009]. Exclusive distribution of urea cycle enzymes or GS is complemented by U shaped distribution of GDH (higher in periportal and perivenous hepatocytes), playing role in each of the ammonium system dis- posal [Haussinger, 1990a]. It is possible that in periportal hepatocytes GDH is catalysing the deamination reaction in order to provide ammonium for the urea cycle meanwhile in the perivenous zone it could produce glutamate for GS [Brosnan and Brosnan, 2009].

The liver’s blood flow is of certain peculiarity having dual supply from portal vein and hepatic artery [Eipelet al., 2010]. Previously published data suggest that ammonium reaching perivenous hepatocytes are of intestinal origin, delivered through the portal vein [Haussinger, 1990a]. This in turn suggests that GS system works as a scavenger for the ammonium that escaped urea detoxification in periportal zone. Study by Cooper et al.

1.7. Urea cycle

addressed the question of short-term metabolic fate of ammonium in livers of adult male rats using labelled NH⁺₄ bolus injections [Cooper et al., 1987]. Labelled nitrogen was exchanged between mitochondrial and cytoplasmic ASAT, GDH and ALAT with only a small fraction being metabolized to glutamine and a large fraction being incorporated into the hepatic glutamate pool. This indicates conditions in the liver which largely favour one-way incorporation of ammonium into glutamate.

1.7 Urea cycle

Urea cycle evolved as a response to changing environment: from organism thriving in Devonian seas to a terrestrial existence of amphibian progenitors. In the fish, generated ammonium can easily defuse into the surrounding water but air-breathing first amphib- ians imposed a conversion of ammonium into somewhat non-toxic product – urea. In 1932, Krebs proposed urea to be a cyclic process where ornithine, citrulline and arginine are the carriers of nitrogen. The function of urea cycle is to capture NH⁺₄ while permit- ting the formation of carbon skeleton for oxidative metabolism without facing the building up of ammonium. Safe disposal of ammonium is carried out by the hepatic metabolism which plays a crucial role in the adaptation of whole-body nitrogen homeostasis to dietary protein variations. It is an anabolic, energy requiring system where the synthesis of one mole of urea requires expenditure of 3 moles of ATP [Shambaugh, 1977]. The compo- nents of the urea cycle are closely related to the intermediates of the TCA cycle, namely fumarate and obtained through transamination aspartate. The metabolism of nearly all amino acids involves aminotransferases where glutamate andα-ketoglutarate are reaction partners. Of crucial importance is the fact that GDH and aminotransferases are catalysing reversible reactions, which enables hepatic nitrogen metabolism to tailor the ammonium and aspartate production to the needs and capacities of urea cycle. Glutamate family of amino acids comprises glutamine, histidine, arginine, ornithine and proline; all of them being potentially transformed into glutamate. Furthermore, converted toα-ketoglutarate their catabolism increases the sum of TCA cycle intermediates. Taking advantage of this anaplerotic input, PEPCK is catalysing cataplerotic reaction of oxaloacetate to phospho- enolpyruvate, allowing gluconeogenic pathway [Brosnan and Brosnan, 2009]. Addition- ally, glutamate plays a special role in the regulation of urea cycle since N-acetylglutamate (obligatory activator of carbamoylphosphate synthetase) is produced from acetyl-CoA and glutamate by NAG synthetase. The urea cycle starts in the mitochondria and ends in the cytoplasm of hepatocytes. Its first rate-limiting step involves the metabolism of carbon monoxide and ammonium to carbamoyl phosphatevia CPS-1. Catabolism of glutamine

by glutaminase provides the 1^st ammonium for urea cycle. High activity of glutaminase is essential to reach NH⁺₄ concentration to match Kmof CPS-1 for ammonium - around 13µM as established in vitro in intact mitochondria [Cohen et al., 1985]. Moreover, glutami- nase is regulated on feed-forward mechanism of ammonium signal. [Buttroseet al., 1987].

CPS-1 and GLS share interesting evolutionary history: crystal structure of CPS-1 from Escherichia coli shows that CPS-1 has a domain with the glutaminase activity linked by a molecular tunnel to other active sites for further reaction to carbamoyl phosphate [Hewagama et al., 1999]. This shows the crucial role played by glutaminase in CPS-1 activity. After formation of carbamoylphosphate, the latter reacts with ornithine to form citrulline that is transported from the mitochondria to the cytoplasm. ASAT transfers glutamate amino group to oxaloacetate to produce aspartate which is used as 2^ndnitrogen introduced to the urea cycle by the action of arginosuccinate synthetase, which requires ATP and produces arginosuccinate. The final step is to convert the latter into arginine with the concomitant release of fumarate by arginosuccinate lyase.

Urea cycle is challenged with two main sources of amino acids and their subsequent catabolism: postprandially from dietary intake and f aitmaison amino acids released by the muscles during starvation. The ingested proteins of animal and plant origins are of different amino acids composition. During digestion, amino acids are released in the GI track and subjected to different absorption kinetics and transamination of first pass metabolism, hence it is difficult to evaluate its systemic circulation concentration after a meal [Boset al., 2003]. Study by Stoll et al. tried to address this question using piglets hourly fed with milk [Stoll et al., 1998]. They confirmed previous observations on the impact of first pass metabolism on glutamate and aspartate blood concentrations: the near complete removal of glutamine and substantial net synthesis of alanine by the intestinal tissue. Dietary excess of amino acids, resulting from the ingestion of high protein diet, stimulates their oxidative catabolism for energy production and concomitantly gives rise to substantial release of NH⁺₄. Urea cycle is a tightly controlled system to prevent losses of valuable amino acids when theirs source is scarce and also a way to excretion in case of their excess. Due to the responsiveness of this mechanism, daily dietary variations can be counteracted, and toxic circulating levels of ammonium avoided. When dietary protein level is low, free amino acids are used for protein synthesis, paralleled by inhibition of catabolism, while diets with high protein content induce extensive catabolism of amino acids. In the liver of animals consuming a high protein diet ALAT, ASAT and GDH activities are increased [Colomboet al., 1992]. Diets from 7.5% to 60% protein contents do not impact on plasma concentrations of alanine but alter substantially glutamine levels.

In the liver of fed rats, gluconeogenic amino acids, alanine and glutamine, are low with

1.7. Urea cycle

high protein diet whereas low protein diet lead to their accumulation [Moundras et al., 1993]. Upon fasting, glutamine and alanine released from peripheral tissues constitute the most prominent fraction of amino acid in blood [Mcet al., 1957].

Alanine

Pyruvate Glutamate

Acetyl CoA Oxalocetate

Citrate

α-ketoglutarate Fumarate

Aspartate

Arginino- Succinate

Arginine Ornithine

Carbamoyl Phosphate CO2 + NH4+

Citrulline

Fumarate Aspartate

Glutamate GDH

ALAT

ASAT

GlutamineGLS Urea

Glucose

ENTRY ENTRY

ENTRY

Figure 1.2: Urea cycle and TCA cycle. The urea cycle is shown on the left and the TCA cycle on the right. Alanine is transaminated to glutamate, which is further transaminated to aspartate, enters the urea cycle to form arginosuccinate, which is then cleaved to arginine and fumarate.

The latter re-enters the TCA cycle. GDH: glutamate dehydrogenase; GLS: glutaminase; ALAT:

alanine aminotransferase; ASAT: aspartate aminotransferase.

1.8 Glutamine synthesis

Glutamine synthetase (GS) is catalysing the condensation of glutamate and ammonium (amidation) to form glutamine in an ATP-dependant manner. Liver is not a major net producer of glutamine, even though the GS activity in perivenous hepatocytes is very high [Watford, 2000]. The explanation for this paradox lies in high glutamine turnover within metabolic zonation of the liver: glutaminase catalysing deamination by periportal hepato- cytes and GS catalysing re-synthesis in perivenous hepatocytes [Watford, 2000; Haussinger and Schliess, 2007]. Together, they carry out reciprocal reactions with glutamine as either a product or a substrate depending on substrate availability and acid-base homeostasis.

Measurements of portal-arterial-venous differences in rats showed slight net output and intake of glutamine by the liver depending upon metabolic conditions: net glutamine pro- duction by rat liver is increased in metabolic acidosis and during some phases of fasting [Matsutakaet al., 1973]. Hepatic GS knockout causes only mild hyperammonaemia but a substantial decrease in whole body muscle/fat ratio [Hakvoortet al., 2017]. Muscle GS is required for extrahepatic detoxification during energy challenging conditions as for fast- ing. Indeed, normal muscle can detoxify 2.5µmol/g muscle/h in a GS-dependent manner indicating that during fasting muscle GS is part of an adaptive response to ammonium handling [He et al., 2010]. Interestingly, infusion in mice of ammonium bicarbonate in the portal vein stimulates glutamine uptake and glutamate and urea release from the liver [Buttrose et al., 1987]. Studies on ammonium metabolism showed that ∼35% of ammo- nium from portal vein is converted into urea,∼35% into glutamine and 30% is going back to systemic circulation in NH⁺₄ form [Weineret al., 2015]. Moreover, glutamine emerged as a good precursor of urea nitrogen and concomitantly an oxidizable substrate to provide ATP for ureagenesis, which is heavily energy dependent.

1.9. Kidney ammonium homeostasis

Periportal hepatocytes Perivenous hepatocytes NH4⁺

RhBG AA (Glutamine)

SNAT3/5

Glutamine NH4+

Glutamate

α-ketoglutarate NH4+

NH4+

Urea cycle Urea

GLS

GDH

NH4+ RhBG NH4+

Glutamine

SNAT3/5 Glutamine Glutamate

UT-A Urea

NH4+

α-ketoglutarate NH4+ GDH Blood flow direction

GS

Urea

Figure 1.3: Nitrogen metabolism in periportal and perivenous hepatocytes. Periportal hepatocytes, receive blood from the portal vein, which is high in nutrients, and the hepatic artery, which is highly oxygenated. Here, the blood is rich in amino acids and ammonium, transported from the intestines, which are metabolized into urea. Perivenous hepatocytes are exposed to lower oxygen tension as well as nutrient and hormone levels. Ammonium that escapes conversion to urea is transported along the liver acini to be converted into glutamine by perivenous hepatocytes.

1.9 Kidney ammonium homeostasis

Physiological pH of blood is 7.35-7.45. Below this range, condition is defined as acidosis and above as alkalosis. The primary pH buffer system of blood is the bicarbonate (HCO⁻₃) /carbon dioxide (CO₂) equilibrium. The increase in HCO⁻₃ concentration or decrease in CO2 partial pressure renders blood alkalotic while a decrease in HCO⁻₃ and an increase in CO₂ makes blood more acidic. CO₂ levels are regulated by the pulmonary system through respiration, while HCO⁻₃ level is regulated by kidneys. Acid-base balance is critical for health and is achieved by ammonium excretion, reabsorption of filtered bicarbonate and generation of new bicarbonate in the kidney. In homeostasis, renal nitrogen excretion equals nitrogen intake and it consists almost exclusively of urea and ammonium. The other nitrogen compounds are making up to 1% of total renal nitrogen excretion [Weiner and Verlander, 2013]. A wide range of conditions, as acid-base homeostasis and urine concentrating process, controls ammonium and urea handling in the kidneys. Urea is the largest circulating pool of nitrogen (except the one in circulating proteins) and its blood concentration ranges between 2.5-7.1 mM. Its production changes in parallel to the protein intake and degradation of endogenous proteins.

Glutamine is synthesizedde novoin the liver and reaches kidney proximal tubular cells through systemic circulation where its uptake is mediated by two transporters: the apical Na⁺-dependent neutral amino acid transporter-1 and the basolateral sodium-coupled neu- tral amino acid transporter-3 (SNAT3) [Weiner and Verlander, 2013]. Once internalized, glutamine can serve as a precursor for gluconeogenesis, being metabolized by phosphate dependent GLS and GDH. The net outcome of this pathway is the production of 2 NH⁺₄ and 2 HCO⁻₃- per glutamine. Produced bicarbonate is transported across basolateral membrane and returns to the systemic circulation where it buffers endogenous and exoge- nous acids. Around 50% of ammonium produced in the kidney is excreted in urine, the remaining ammonium, along with HCO⁻₃-, enters the systemic circulation. In the peri- portal hepatocytes, ammonium along with HCO⁻₃- is metabolized predominantlyvia the urea cycle to generate urea. Therefore, ammonium metabolized in the liver to urea has no net acid-base benefit [Weiner and Verlander, 2013]. Since urinary ammonium is pro- duced in the kidney, renal venous ammonium exceeds arterial ammonium and consequently increases systemic ammonium concentration [Weiner, 2017]. The fraction of renal ammo- nium excreted in urine versus the one transported into the circulation can shift quickly in response to acid-base disbalance, such as metabolic acidosis or alkalosis. Metabolic acidosis is characterized by a drop in blood pH due to a reduction in serum bicarbonate concentration. Acidosis stimulates glutamine uptake into the proximal tubular cells and upregulates the expression of key ammonium producing enzymes: glutaminase and GDH [Lim, 2007]. In parallel to higher glutamine uptake, renal ammoniagenesis and gluconeo- genesis are increased. Glutamate deaminated toα-ketoglutarate can feed the TCA cycle for further oxidation or used in the gluconeogenic pathway to be converted to glucose and bicarbonates. The thus formed bicarbonates alleviate acidosis, while ammonium excreted in urine restores the systemic pH [Busque and Wagner, 2009]. GDH catalyses deamina- tion of glutamate to control acidosis [Spanaki and Plaitakis, 2012]. Both production and transport of ammonium are directly linked to changes in systemic pH [DuBose, 2017]. The enhanced glutamine metabolism in the kidneys is compensated by increased release from the muscles during starvation and metabolic acidosis [Chang and Goldberg, 1978]. The major acid-base disturbance that mammals face daily results from metabolism of proteins [Weiner and Verlander, 2013]. The typical diet yields 20-40 mM of sulfuric acid and phos- phoric acid daily. These acids dissociate into hydrogen ions, which in turn are buffered by bicarbonates. As a result, the organism is challenged daily by 50-80 mEq of hydrogen ions. Since protein intake is a major determinant of endogenous acid production, the net acid excretion varies during protein restriction [Leeet al., 2015]. The protein restric- tion is followed by changes in expression of various enzymes involved in renal ammonium

1.10. Glutamate dehydrogenase

metabolism, including the ammonium-generating enzymes phosphate-dependent GLS and PEPCK and the ammonium-metabolizing enzyme GS [Leeet al., 2015].

1.10 Glutamate dehydrogenase

Glutamate dehydrogenase is an enzyme that catalyses the reversible conversion of gluta- mate toα-ketoglutarate and ammonium while reducing NAD⁺to NADH. The latter action is linked to redox homeostasis. Specifically, reduction ofα-ketoglutarate generates gluta- mate and enables oxidation of NADH to NAD⁺, needed for mitochondrial metabolism. In the opposite direction, oxidative deamination of glutamate fuels the TCA cycle, providing carbon skeleton. Therefore, GDH is bridging protein and carbohydrate metabolism. GDH reaction is reversible, at least in the test tube, and its predominant flux is dictated by substrates provision as well as by ADP and GTP allosteric regulations [Riceet al., 1987].

α-ketoglutarate + NH4++ NADH

GDH

GTP ADP Leucine

SIRT4 SHAD

glutamate + NAD⁺

Figure 1.4: Reaction catalyzed by GDH.Allosteric regulators of GDH, inhibitors shown as red lines, activators shown by green arrows.

The atomic structure of nonmammalian and mammalian forms have been determined [Peterson and Smith, 1999; Li et al., 2014]. GDH is a dimer of trimer stacked on top of each other. Each subunit is composed of three domains. The bottom domain has extensive contacts with the subunit from the adjacent trimer. The top domain is the NAD binding domain, raising above it, is a long antenna-like protrusion. These antennas protruding from each of three subunits are intertwined with each other. During catalytic cycle when enzymatic cleft opens, they rotate in a clockwise motion and the core of the hexamer

expands [Rice et al., 1987]. This homohexameric enzyme is composed of ∼500 residues in mammals and 450 residues in bacteria. The bovine GDH shows 27% sequence identity with GDH fromClostridium symbiosumand 90% sequence homology with human GDH [Smith and Stanley, 2008]. The largest difference between mammalian and bacterial GDH is a 48 amino acid insertion - antenna. As opposed to the extensive allosteric regulation in mammalian GDH, bacterial forms of GDH are relatively unregulated. It is thought that antenna enables allosteric regulation by a wide range of modulators [Smith and Stanley, 2008]. The two major allosteric regulators are ADP and GTP, which appear to exert their opposing effect via abortive complexes [Rice et al., 1987]. The latter ones are formed as ternary complex of GDH-glutamate-NADH in the oxidative deamination reaction and GDH-α-ketoglutarate-NAD in the reductive amination reaction. Once they are formed, coenzymes bind to the ternary complex and exhibit their allosteric effects: ADP is an activator working by destabilization of the abortive complex, while GTP is an inhibitor stabilizing the abortive complex. Additionally, GTP and ADP bind in an antagonistic manner due to steric competition on GDH. GTP binds to the side of each subunits, re- inforcing the closed state of the enzyme, which explains why GTP increases the affinity of the enzyme for its product. In contrast, ADP binds behind the NAD-binding domain, activating the enzyme by keeping it in an open state. GDH allosteric regulation by ADP and GTP bridges enzyme regulation with energy status of the cell. GTP is generated in the TCA cycle at the step of succinyl-CoA. It is a direct inhibitory mechanism for GDH under the conditions of high energy state, rich in triphosphate. Conversely, ADP is a signal of low energy state in the cell, activating GDH in oxidative direction enables TCA cycle replenishment and generation of energy from amino acids. Comparative analyses of GDH amino acid sequences from three mammalian species showed that the enzyme is highly conserved [Liet al., 2014]. Extensive kinetic analysis has elucidated many aspects of GDH catalysis. It is thought that the thermodynamic equilibrium of mammalian GDH favours glutamate synthesis, but it is still unclear which directionin vivo enzyme oper- ates. GDH can be either a source of ammonium or an ammonium scavenger. Some studies suggest that, due to high concentrations of glutamate and low concentrations of free am- monium ions in mitochondria, under baseline conditions GDH operates predominantly in the deaminating direction [Adevaet al., 2012]. Since the GDH reaction is reversible, the predominant flux is dictated by the concentration of the substrates and its affinity for those substrates. Because of relatively high Km for ammonium, GDH was expected to operate towards the oxidative deamination direction. In particular in tissues with low ammonium concentration. In the liver, where sufficiently high mitochondrial ammonium concentration can be reached from the glutaminase activity, glutamate formation might

1.10. Glutamate dehydrogenase

be favoured. GDH flux is also linked to production or removal of ammonium depending on which direction predominantly operates [Treberget al., 2014].

GLUD1 is the gene encoding for glutamate dehydrogenase. Its chromosomal location is on the long (q) arm of chromosome 10 at position 23.2. Most of the non-lethal activating mutations of GLUD1 are de novo single nucleotide missense mutations occurring within exon 6, 7, 11, and 12 causing the hyperinsulinism/hyperammonaemia (HI/HA) syndrome [Kapooret al., 2009; Suet al., 2018]. Patients are heterozygous, possessing both a mutated and a normal allele. Accordingly, the respective contributions of wild type versus mutant subunits in the operating GDH hexamer is unknown, while the functionally defective allele appears to be dominant. Indeed, one can hypothesise that HI/HA patients carry heterohexamers composed of both wild type and mutated mers. The familial forms that have been reported have autosomal dominant pattern of inheritance. Phenotypically, reported GDH mutations result in an attenuation of GTP-mediated inhibitory action on GDH. Consequently, increased GDH activity in pancreatic β-cells increases the α- ketoglutarate entry into TCA cycle which maintains a high ATP/ADP ratio triggering the insulin-secretion cascade [Rice et al., 1987; Palladino and Stanley, 2010]. Moreover, HI/HA patients show much more acute insulin response to leucine vs. the control subjects [Weinzimer et al., 1997]. This would account for the hyperinsulinism part of HI/HA syndrome, but the source of the accompanying hyperammonaemia is yet to be defined.

Hyperammonaemia is a feature of urea cycle defects (UCD), fatty acid oxidation de- fects, prematurity, etc. UCD patients display symptoms associated with high plasmatic level of ammonium: vomiting, seizures or coma. Interestingly, HI/HA patient’s hyperam- monaemia seems asymptomatic. It has been previously postulated by Charles A. Stanley that gain-of-function activity of GDH might result in increased flux towards oxidative deamination and free ammonium generation. Simultaneously, excessive glutamate oxida- tion might lead to a depletion of intrahepatic pool of glutamate, which could negatively affect the production of N-acetylglutamate (NAG) in the liver [Palladino and Stanley, 2010]. This might in turn influence urea synthesis since NAG is an essential allosteric activator of carbamoyl-phosphate synthase. The symptomatic landscape of HI/HA pa- tients in terms of hyperammonaemia is heterogenous with values elevated 3-5 times above the upper limit of the control range. Additionally, ammonium levels do not change with fasting [Palladino and Stanley, 2010; Weinzimeret al., 1997]. When challenged with stan- dard oral protein tolerance test, HI/HA patient’s plasma glucose decrease rapidly (nearly 5 times more than controls) meanwhile plasma ammonium raise twice as much as the controls [Kelly et al., 2001]. The levels of plasma amino acids are within control range for HI/HA patients except slightly higher glutamine, which is common for all types of hy-

perammonaemia [Smith and Garg, 2017]. An interesting angle about the source organ of hyperammonaemia was provided by a study by Treberget al.that investigated the effects of GDH allosteric activator BCH, an analogue of leucine, on ammonium metabolism. This intervention resulted in mild hyperammonaemia in BCH-treated animals due to increased renal ammonium output, while hepatic output remained unchanged. This points to renal GDH and the kidney as a potential source of hyperammonaemia [Treberget al., 2010].

Figure 1.5: Glutamate dehydrogenase structure. Two trimers of subunits stacked directly on top of each other, hexameric GDH with six subunits displayed in different colours. Adapted from Nassaret al., 2018

1.11. Thesis Aims

1.11 Thesis Aims

Liver GDH knock out in vivo

The metabolic effects of consuming varying amounts of proteins have been extensively investigated [Peretet al., 1981; Peters and Harper, 1985]. In the present study, we focused on changes in the liver because of its high degree of plasticity to adapt and tailor en- zymes activities according to nutritional and physiological conditions. We used inducible liver-specific GDH knock out mice (HepGlud1-/-), knowing that the lack of hepatic GDH induces disturbance in nitrogen metabolism resulting in mild hyperammonaemia [Karaca et al., 2018a]. We investigated changes in acid-base homeostasis secondary to the increased levels of plasma ammonium in HepGlud1-/- mice, while such hyperammonaemia did not induce visible symptoms. Additionally, we challenged our mouse model with varying pro- tein intakes to characterize the dynamic of changes in ammonium; focusing on the main nitrogen pools, i.e. peripheral plasma urea and glutamine levels. Because the elevation of substrate availability for ureagenesis goes hand-in-hand with increased substrates for gluconeogenesis during starvation, we performed both in vitro and in vivo complemen- tary experiments to investigate acute responses to amino acid loads in starved mice or on isolated hepatocytes.

GDH-G446V mutation in vitro

Most of the GDH mutations are characterized by decreased sensitivity of the enzyme to the allosteric inhibitory action of GTP. GDH-G446V is a rare mutation and carriers of this mutant form suffer from HI/HA syndrome and mental retardation. The goal of this project was to characterize the GDH mutation G446V in in vitro conditions based on a HI/HA patient-derived lymphoblastoid cell line and to assess the responses to the main allosteric modulators of GDH,i.e. the activator ADP and the inhibitor GTP. In parallel, we performed computational modelling to evaluate the steric changes introduced by the substitution of glycine to valine in the overall complex structure of GDH.

Establishing a mouse model to investigate the hyperammonaemia of HI/HA patients

The HI/HA syndrome is a complex disease with a phenotype contributed by multiple organs. HI/HA patients suffer from a combination of hyperinsulinism (HI) inducing hy- poglycemia, hyperammonemia (HA), and often epilepsy or mental retardation. The organ responsible for the hyperammonemia remains unclear. To dissect the contribution of selec- tive tissue in this complex syndrome, we established a mouse model targeting specifically

the liver. We used inducible liver GDH knock out (HepGlud1-/-) mice, then transduced with an adenovirus carrying mutant GDH_S445L. This way, only the liver expressed the GDH mutant form, allowing assessment of hepatic contribution to HI/HA.

CHAPTER 2

Results

2.1 Influence of protein intake on nitrogen metabolism in mice lacking liver glutamate dehydrogenase

Authors:

Karolina Luczkowska and Pierre Maechler.

Contribution:

Karolina Luczkowska designed and performed all experiments except for metabolic cages and measurements of ammonium and urea which were performed in the clinical unit of HUG. K L analyzed all the data except those from metabolic cages. K L participated to the writing of the manuscript. K L will be the first author of the paper. Pierre Maechler designed and supervised the research, analyzed the data and edited the manuscript.

This chapter is a preprint of a manuscript.

In the present study we investigated the changes of nitrogen metabolism upon different dietary protein load in a mouse model. Using liver-specific GDH knockout mice we char- acterized changes in blood acid-base homeostasis secondary to hyperammonaemia. We evaluated changes in hepatic detoxification of ammonium into two disposal systems: the urea cycle and glutamine synthesis along with the capacity for nitrogen excretion through urine. We investigated GDH- dependent gluconeogenesis from amino acids during starva- tion both inin vitro and in vivo.

2.1. Influence of protein intake on nitrogen metabolism in mice lacking liver glutamate dehydrogenase

2.1.1 Abstract

In our body, the liver is at the anatomic and metabolic cross road, buffering and control- ling the amount and nature of nutrients available for peripheral tissues. Ureagenesis and gluconeogenesis are essential metabolic functions of the liver to maintain ammonium and glucose plasma concentrations within their respective narrow ranges. Hepatic GDH plays a crucial role in nitrogen metabolism being positioned at the interface of amino acid and carbohydrate metabolisms. It is implicated in ammonium assimilation and amino acid deamination, releasing free ammonium and carbon skeleton, closely influencing gluconeo- genesis. In this study we used inducible liver-specific GDH knockout mice (HepGlud1-/-) to assess the associated hyperammonaemia and its metabolic compensations, occurring through respiration by the lungs and excretion by the kidneys. We evaluated changes in hepatic detoxification of ammonium into two disposal systems, i.e. the urea cycle and glutamine synthesis tested at varying amounts of ingested proteins. Furthermore, we ob- served that alanine and glutamine metabolisms rely both on GDH and are interdependent, regulating amino acid availability for gluconeogenesis during starving period.

2.1.2 Introduction

Our daily protein turnover is subjected to three interconnected fluxes of amino acids: di- etary protein intake, de novo synthesis, and release of amino acids by proteolysis. The availability of energy substrates, in particular glucose, influences the net protein utiliza- tion and the catabolism of amino acids. Upon starvation or prolonged physical exercise, proteolysis in skeletal muscles promotes transport of amino acids, mainly as alanine and glutamine, from the peripheral tissues to the liver. This favors gluconeogenesis to main- tain hepatic glucose production and euglycemia once glycogen stores are exhausted, with the accompanying consequence of increased production of ammonium (NH⁺₄) and the ac- cumulation of its ammonium cation NH⁺₄ [Schutz, 2011]. The latter situation can also be prompted by the ingestion of high protein diet, which stimulates catabolism and oxida- tion of amino acids that gives rise to substantial load of NH⁺₄ [Jackson, 1999]. Hyperam- monemia may be toxic for the brain as it alters neurotransmission and neurotransmitter recycling, an effect witnessed by liver failures such as hepatic encephalopathy [Haussinger and Schliess, 2008].

Safe disposal of ammonium is carried out by dedicated hepatic metabolism, namely the formation of urea and the synthesis of glutamine, each molecule carrying two non- toxic nitrogen. Accordingly, the liver plays a crucial role in the adaptation to whole-body protein turnover. Regarding the zonation of these liver detoxifying pathways, there is an exclusive distribution of urea cycle enzymes in the periportal hepatocyte subpopulation,

while glutamine synthetase (GS) is mostly present in the perivenous part. Glutamate dehydrogenase (GDH) plays a key role in each of these ammonium disposal systems and is homogenously distributed throughout the liver [Haussinger et al., 1992; Boon et al., 1999]. Zones with urea cycle and GS activities are anatomically aligned one after the other. Sequentially, ureagenic periportal hepatocytes are first supplied with blood, then are the glutamine-synthesizing cells. This reflects the functional organization with peri- portal low-affinity but high capacity urea system followed by perivenous high-affinity and low-capacity glutamine synthesis [Haussinger, 1990b]. As a consequence, most of the blood ammonium from the portal circulation is metabolized in the periportal zone into urea for kidney excretion, the remaining being scavenged by fine tuning perivenous hepatocytes for glutamine synthesis [Qvartskhavaet al., 2015b].

The urea cycle should be under tight control to prevent the loss of valuable amino acids when their availability is scarce; in particular when dietary protein uptake is low. Upon a high protein diet, extensive amino acid catabolism mobilizes the urea cycle as it represents the main way of excretion of their toxic byproduct NH⁺₄. Due to the responsiveness of this system, daily dietary variations can be counteracted and the potentially toxic high circulating ammonium avoided [Payne and Morris, 1969]. Beside urea, the liver can generate glutamine as a complementary molecular sink for ammonium disposal. Blood glutamine concentrations vary according to the different dietary protein intake, although indirectly through the action of the kidneys which use glutamine to maintain acid-base homeostasis [Qvartskhavaet al., 2015b; Matthews and Campbell, 1992].

The metabolism of amino acids involves dehydrogenase and aminotransferases where glutamate and α-ketoglutarate are reaction partners. The transfer of an amino group toα-ketoglutarate produces glutamate which in turn can be used by GDH or aspartate aminotransferase (ASAT). Detoxification of ammonium to urea involves deamination and transamination. GDH deaminating reaction provides ammonium and ASAT transami- nates oxaloacetate to produce aspartate, both products being necessary for urea genera- tion. Importantly, GDH and aminotransferases are catalyzing reversible reactions, which enables hepatic nitrogen metabolism to tailor substrate concentrations to urea cycle capac- ity. GDH is an enzyme acting at the interface of carbohydrate and amino acid metabolism, thus bridging protein to glucose pathways [Karacaet al., 2018b]. The bidirectional reac- tion catalyzed by GDH allows either the synthesis of glutamate or conversely its oxidation with the concomitant generation of ammonium [Brosnanet al., 1996; Cooperet al., 1988].

Which of these fluxes is predominant is still a matter of debate for hepatic GDH. Adding to the complexity of the enzymatic control, GDH is allosterically regulated by ADP (ac- tivator) and GTP (inhibitor), as witnessed by disorders resulting from mutations in the