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Implications of arginine deficiency for growth and organ maturation. Studies on hair, muscle, brain and lymphoid organ maturation de Jonge, W.J.

Publication date 2001

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Citation for published version (APA): de Jonge, W. J. (2001). Implications of arginine deficiency for growth and organ maturation. Studies on hair, muscle, brain and lymphoid organ maturation.

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Download date:05 Oct 2021 ChapterChapter I

ArginineArginine biosynthesis and metabolism

WouterWouter J de Jonge, Maaike J Bruins, 2Nicolaas E Deutz,2Peter B Soeters, 11 Wouter H hamers

'Departmentt of Anatomy and Embryology,3 Department of Biochemistry, Academicc Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, department of Surgery,, Maastricht University (AZL), The Netherlands.

5 5 ChapterChapter I

Contents: Contents:

1-Biosynthesiss of argin in e 9 1.11 Intestinal citrulline biosynthesis 1.22 Arginine biosynthesis 1.33 Hepatic arginine metabolism 1.44 Ontogeny of arginine metabolism

2-Dietaryy demand of arginine 16 2.11 Dispensable and indispensable amino acids 2.22 the homeostasis of creatine reflects a delicate arginine balance 2.33 Arginine demands in growing mammals 2.42.4 Species differences in arginine requirement and biosynthesis

3-Inbornn errors of metabolism leading to congenital arginine deficiency 19 3.11 ornithine aminotransferase 3.22 ornithine transcarbamoylase 3.33 argininosuccinate synthetase

4-Metabolismm of arginine 4.11 Intestinal utilization and catabolism of glutamine and arginine 22 4.22 Arginase I and II 4.33 Agmatine 4.44 Proline 4.55 Poly amines 4.66 Nitric oxide

5-- Regulation of metabolism by arginine 28 5.11 Effect of arginine on protein synthesis and degradation 5.22 Endocrine activity arginine

6 6 6-Guanidinoo compounds 6.11 The ornithine guanidine bi-cycle 6.22 Guanidino compounds formed after transamidination 6.33 Formation of GSA by reactive oxygen species 6.44 Pathological effects

7-Argininee transporters 7.11 Amino acid transporters 7.22 System y + 7.33 Regulation of arginine transporters

8-Argininee as an immunonutrient 8.11 Arginine and immune function 8.22 Possible mechanisms of action

9-Scopee of the thesis ChapterChapter I

AbbreviationsAbbreviations and enzymes with enzyme codes used'.

A-I I [ECC 3.5.3.1] :: hepatic arginase A-n n [ECC 3.5.3.1] :: kidney-type arginase AGAT T [ECC 2.1.4.1] :: arginine:glycine amidinotransferase AAT T [ECC 2.6.1.1.] :: aspartate aminotransferase ASL L [ECC 4.3.2.1] :: argininosuccinate lyase ASS S [ECC 6.3.4.5] :: argininosuccinate synthetase CPS S [ECC 6.3.4.16] :: carbamoylphosphate synthetase GMT T [ECC 2.1.1.2] :: s-adenosylmethionine:guanidinoacetate methyltransferase e OAT T [ECC 2.6.1.13] :: ornithine aminotransferase ODC C [ECC 4.1.1.17] :: ornithine decarboxylase OTC C [ECC 2.1.3.3] :: ornithine transcarbamoylase P5C C [ECC n.a.] :: pyrroline-5-carboxylate synthetase PDG G [ECC 3.5.1.2.] :: phosphate-dependent glutaminase PO O [ECC n.a.] :: proline oxidase SI I [ECC 3.2.1.48-

ECC = enzyme code, n.a.= not assigned Introduction Introduction

1-Biosynthesis1-Biosynthesis ofarginine

1.1-Intestinal1.1-Intestinal citrulline biosynthesis Thee organ perfusion studies by Windmueller and Spaeth *'2 established that de novo synthesiss of citrulline, as well as ornithine and proline, takes place in the small intestinee (Fig. 1). Citrulline synthesis requires NH3, CO2, and ornithine. The NH3 is derivedd from glutamine via the action of glutaminase. Together with bicarbonate, NH33 can form carbamoylphosphate by the action of carbamoylphosphate synthetase (CPS-1;; EC 6.3.4.16), an enzyme that requires N-acetylglutamate for activity. The carbamoyll group is transferred to ornithine to form citrulline in a reaction catalyzedd by ornithine transcarbamoylase (OTC; EC 2.1.3.3). In addition to glutamate,, proline can function as a precursor for the intestinal ornithine synthesis (Fig.. 1) 3, via the formation of P5C by proline oxidase, and the subsequent non- enzymaticc conversion into L-glutamyl-y-semialdehyde 4. Thee enzymes involved in citrulline synthesis, phosphate-dependent glutaminase,, OAT, and aspartate aminotransferase are found in a variety of tissues 55''66,, but CPS I, OTC, and N-acetyl glutamate synthetase expression are restricted to thee liver and intestinal mucosa 7. P5C synthetase, which catalyzes the multistep conversionn of glutamate via glutamyl-y-semialdehyde into P5C 4, is present almost exclusivelyy in the small intestinal mucosa, with only trace amounts in other tissues 4.. P5C is, in turn, converted into ornithine by OAT. For this reason, the intestine is thee only organ that has the capacity of net production of ornithine. In (most) other organs,, including liver, OAT converts ornithine into glutamate. Human patients sufferingg from a deficiency in P5C synthetase develop hyperammonemia and hypoornithinemiaa 8. In addition, these patients have decreased proline levels, implyingg that P5C-S may also have a P5C-R like activity. The essential role of the intestinee to provide citrulline for arginine biosynthesis elsewhere, is also demonstratedd by the arginine deficiency that results from inhibition of intestinal

9 9 ChapterChapter I citrullinee synthesis, as a result of inhibition or deficiency of OTC ' , OAT ' , or byy massive resection of the small bowel . Inn intestinal tissue, arterial and luminal glutamine is the main metabolicc contributor of intestinal citrulline biosynthesis 2. The magnitude of the rolee of proline in citrulline synthesis is disputed. In pigs, there is no arterial uptake off proline by the gut 14, suggesting that only dietary proline is metabolized into citrulline.. Indeed, a prominent role for proline is suggested by the observation that, att least in pigs, proline is among the most abundant amino acids in the sow's milk

14 4

protein n<4 ~ ~ Glutamine e - purines 11 -glutaminase 10-AAT 10-AAT CX-KGG <4r Glutamate e asparanginn oxaloacetate 2-P5C-S2-P5C-S NHj ATp^ C02 9-PO 9-PO proline e - P5C A— Glu-sem m 4-CPS 4-CPS \\ 3-OAT W W CP P OrnVV 5-OTC

urea a if f ornithine e Cit t cycle e 7-ASL7-ASL 6-ASS —— AS

Fig.. 1: Scheme of the relation between glutamine and the ornithine cycle. Enzymes that catalyze the reactionss are: 1- phosphate-dependent glutaminase [EC 3.5.1.2.], 2- pyrroline-5-carboxylate synthetase [EC n.a.],, 3- ornithine aminotransferase [EC 2.6.1.13], 4- carbamoyl-phosphate synthetase [EC 6.3.4.16], 5- ornithinee transcarbamoylase [EC 2.1.3.3], 6- argininosuccinate synthetase [EC 6.3.4.5], 7- argininosuccinate lyasee [EC 4.3.2.1], 8- hepatic arginase [EC 3.5.3.1]/ - kidney-type arginase [EC 3.5.3.1], 9- proline oxidase [EC n.a.].. 10- aspartate aminotransferase [EC 2.6.1.1]. Enzymes 6, 7 and 8 are cytosolic, 1-5 and 9 are mitochondrial,, and enzyme 10 is both cytosolic and mitochondrial.

Thoughh proline oxidase (PO) is reported to be present mainly in liver and kidney 6'15,, other groups have detected a relatively high level of PO activity in the intestinal

10 0 Introduction Introduction mucosaa of pig and rat16'17. This difference has been attributed to instability of the enzymee during homogenization of, specifically, mucosal tissue6.

1.2-A1.2-A rgin ine biosyn thesis 1.2.1-Renal1.2.1-Renal arginine biosynthesis. InIn the adult kidney, arginine is made endogenously by converting citrulline into arginine,, mainly in the renal proximal tubules l'18, via the cytosolic enzymes ASS andd ASL. The kidney accounts for 60 % of arginine synthesis from circulating citrullinee 19 (see section 2.2.2). Although the kidney expresses CPS-I, (unpublished observation),, as well as glutaminase and glutamate dehydrogenase (GDH)20'21 in the preweaningg period, the adult kidney lacks the capacity for carbamoylphosphate synthesis. . Thee citrulline that is metabolized into arginine by the kidneys derives primarilyy from the intestine x'21. The amount of citrulline taken up for arginine synthesiss has been shown to be proportional to the amount released by the intestine U3.. Furthermore, a positive correlation between renal citrulline uptake and arginine productionn has been shown in humans 24, sheep 25 and rats 26. The capacity of the kidneyy to synthesize arginine exceeds the capacity of the intestine to synthesize citrullinee several-fold, indicating that renal arginine synthesis depends on intestinal citrullinee synthesis 2?. In addition, the renal arginine flux is highly regulated: renal ASSS and ASL mRNA as well as activity are upregulated during a high protein diet28, 29 9 uponn starvation , or upon an acute decrease in circulating arginine levels under pathologicall conditions30. Nextt to the cytosolic arginine-synthesizing enzymes, the kidney expresses A-III (EC 3.5.3.1)31, but the expression of arginase, ASS and ASL is segregated in differentt parts of the nephron, arginine synthesis is restricted to the upstream portionss of proximal convoluted tubules, whereas arginase activity is present mainly inn the cortical and outer medullary portions of straight proximal tubules. Hence, theree is little or no co-expression of these enzymes in the same cell32, allowing for netnet renal arginine biosynthesis from the citrulline reabsorbed from glomerular

11 1 ChapterChapter I filtratefiltrate in proximal convoluted tubuli cells, while in straight tubuli, arginase-derived ureaa passively diffuses into the luminal fluid entering Henle's loops32.

1.2.2-Extra-renal1.2.2-Extra-renal arginine biosynthesis ASSS mRNA is expressed in the neurons of the myenteric plexus of the adult small intestinee 33, suggesting the existence of a neuronal arginine-citrulline cycle for regenerationn of arginine for nitric oxide synthesis. Such a cycle has been described inn several other nitric oxide producing cell-types, like endothelial cells ' , macrophagess 36, and neurons 37"39. The picture therefore emerged that citrulline synthesiss is confined to the small intestinal enterocytes, whereas arginine biosynthesiss occurs in many tissues, the kidney being the most prominent. Presumably,, the co-locali2ation of ASS and ASL with NOS serves to assure that cellularr NO synthesis becomes independent of circulating arginine. Thus, ASS, ASL,, and nNOS immune reactivity (or NADPH-diaphorase activity) have been shownn to colocalize in the central nervous system of rat 37'38 and pig 39. Whether ASSS and ASL indeed colocalize with nNOS in the intestinal neurons and whether thiss renders these neurons independent of extracellular arginine through intra- neuronall recycling of L-citrulline to L-arginine, is not clear. In vitro, inhibitory neuromuscularr transmission in murine proximal colon was stimulated by arginine, butt not by citrulline alone (0.1-2 mM), which in principle indicates that enteric nervess were not able to convert citrulline into arginine 40. In contrast, when nitrergicc transmission was blocked by the NOS antagonist L-NAME, sustained neuronall exposure to citrulline (0.2 mM) reversed the effects of L-NAME ' , revealingg the capacity of enteric nerves to recycle L-citrulline to L-arginine to sustainn NO synthesis. Most likely, enteric neurons thus do not depend on extracellularr arginine for generation of NO. Measurement of the inhibitory componentt of the intestinal enteric neuron system, evoked by NANC neurons, in organn bath experiments using intestinal tissue from our arginine-depleted transgenicc F/A mice 42, may provide evidence for the (in)dependence of enteric neuronss on extracellular arginine availability.

12 2 Introduction Introduction

Similarr to neurons, macrophages, activated by LPS or IFNy, have been shownn to express ASS, ASL 43, and A-I and A-II 44. Despite the upregulation of ornithinee cycle enzymes required for regeneration of arginine from citrulline, macrophagess remain dependent on extracellular arginine 45, as is shown by the upregulationn of their arginine transport capacity (via the enhanced expression of CAT-22 46) in conjunction with iNOS induction 47'48. The upregulation of either A-I (A-III is not upregulated) or iNOS may be subject to the nature of the inflammatory response:: the upregulation of A-I may be directed by Th2-derived anti-inflammatory cytokines,, whereas Thl-associated pro-inflammatory cytokines induce iNOS expressionn . In section 4, the potential role of macrophage A-I induction in counteractingg the macrophage NO output is further discussed.

l.S-Hepaticl.S-Hepatic arginine metabolism Thee liver does not participate in whole-body arginine synthesis, despite very high levelss of ASS and ASL. This is because the very high arginase content of hepatocytess prevents the net production of arginine, which emphasizes the importancee of the liver for urea production (ammonia detoxification) rather than forr arginine biosynthesis. Moreover, the liver lacks expression of the high affinity cationicc amino acid transporter (CAT-1) 50,51, which is required for efficiënt hepaticc im- or export of arginine from the circulation. In concert with the argument thatt the liver does not contribute arginine to the circulation, patients with inherited ornithinee cycle deficiencies, who need supplementation of arginine, continue to do soo after transplantation of the liver52'53.

1.4-ontogeny1.4-ontogeny of arginine metabolism Inn rapidly growing suckling rats, endogenous arginine biosynthesis is crucial to compensatee for the insufficient supply of arginine via the milk 54. During the sucklingg period, the kidney has a limited capability for the synthesis of arginine 27. Evidencee is accumulating that, in mammals, the intestine rather than the kidney playss a major role in arginine biosynthesis in this period. Thus, the enterocytes of

13 3 ChapterChapter I

1U7 55 56 thee small intestine express CPS, AGA synthetase, OTC, ASS and ASL ' - 5 i.e. thee enzymes required for arginine production from glutamine 3, but do not express arginasee 33. Similarly, enzymes that convert glutamine and proline to ornithine, glutaminase,, P5CS and OAT, are all present in concentrations well above adult levelss in the period before weaning 57"60, whereas the activity of pyrroline-5- carboxylatee reductase (P5CR), the enzyme that can divert pyrroline-5-carboxylate towardss proline, shows a reciprocal pattern 56. The intestine appears to play a similarr role in human neonates as well, as destruction of the enterocytes in necrotizingg enterocolitis also results in a selective decrease in circulating arginine 61.. In mammals, ASS and ASL, disappear from the enterocytes in the peri weaning period,, concurrent with the appearance of endogenous arginase . After weaning, thee role of the gut therefore becomes confined to citrulline biosynthesis. Thee ontogeny of arginine metabolism in intestinal epithelium is associated withh its maturation, as expression of ornithine cycle enzymes changes during migrationn along the crypt-villus axis. From the formation of villi onwards, glutaminasee 62, OAT, CPS and OTC mRNA are concentrated near the base of the villus,, whereas ASS and ASL mRNA are only present near the top 33'42. We have suggestedd that this baso-apical difference in gene-expression relates to the time afterr stem-cell division, and migration of the enterocytes along the crypt-villus axis 33.. The enterocytes near the top of the villus are "older" than those at the base, implyingg that the presence of OAT, CPS and OTC protein in all villus enterocytes, includingg those at the top of the villus, is due to the half-life of these proteins that weree synthesised when the enterocytes were still localized at the base of the villus. Becausee their mRNAs are only present in the apical enterocytes, ASS and ASL apparentlyy become expressed only several days after enterocyte cell division and/or migrationn along the villus axis. The transit time of the enterocytes between the cryptt and the top of the villus, which decreases from 11-14 days in the first postnatall week, to 2-3 days after weaning 63,64, may thus explain the difference betweenn the pre- and post weaning expression pattern 33. In contrast with this hypothesis,, however, a decrease in enterocyte migration rate, through the transgenic

14 4 Introduction Introduction overexpressionn of E-cadherin, is not accompanied by a change in distribution of the terminall differentiation marker liver-type FABP along the crypt-villus axis, suggestingg that differentiation, at least for liver-type FABP, is largely cell nonautonomous65. . Together,, the observed expression pattern of ornithine cycle enzymes suggestss the existence of a metabolic compartimentation of citrulline synthesis in thee enterocytes at the base of the villus and arginine synthesis in the top of villus enterocytes.. Such a two-compartment system along the villus axis may enable the gutt to simultaneously meet the needs for the synthesis of citrulline and arginine duringg the suckling period. Further examination of the expression of arginine transporters,, or measurement of arginine and citrulline concentration in the two compartmentss should confirm this hypothesis.

15 5 ChapterChapter I

2-Dietary2-Dietary demand ofarginine

2.1-Dispensable2.1-Dispensable and indispensable amino acids Proteinn requirements have long been assessed by the sum of requirementss of the essential amino acids and the nitrogen from non-essential aminoo acids 66. By preparing diets containing mixtures of amino acids as the only sourcess containing dietary nitrogen, the quantitative requirement for each amino acidd was determined. In rats, it was found that of the 20 amino acids, that are incorporatedd into protein, 10 could not be excluded from the diet: valine, leucine, isoleucine,, methionine, threonine, lysine, phenylalanine, tryptophan, histidine, and argininee 67. Excluding any one of these essential amino acids, except arginine, resultedd in weight loss, and eventually death. However, although exclusion of argininee led to decreased weight gain, but allowed the animals to survive . The endogenouss synthesis of arginine was apparently adequate for survival, but not for optimall growth. The classification of arginine as being a dispensable or indispensablee amino acid is therefore a matter of definition, as arginine can be synthesizedd endogenously, but in limited quantities only. Irrespective of definitions, thee limited endogenous biosynthetic capacity is most obvious under conditions of increasedd demand for- or catabolism of arginine 67. Indeed, arginine supplementationn has been shown to ameliorate recovery and reduce hospital state underr catabolic conditions, such as sepsis or trauma68'69, also reviewed in " .

2.2-The2.2-The homeostasis of creatine reflects a delicate arginine balance Thee highest level of creatine is found in muscle, Sertoli cells 73, and brain 74',, where it plays an essential role in the energy metabolism 74'75. The biosynthesis off creatine first comprises the formation of guanidino acetic acid (GAA) from L- argininee and glycine by L-arginine-glycine amidinotransferase (AGAT EC 2.1.4.1., Fig.. 2), followed by the transfer of a methyl group by S- adenosylmethionine:guanidinoacetatee methyltransferase (GMT) 75. The delicate balancee of the dietary supply and endogenous biosynthesis ofarginine with its daily

16 6 Introduction Introduction expendituree is nicely illustrated by the direct relation between the dietary intake of argininee and the body pool size of its product creatine 76'77. A young adult male excretess on average of 23 mg (0.2 mmol) of creatinine per kg body weight per day. Thus,, 0.2 mmol arginine per kg body weight is required for creatine synthesis 66. Thee recommended daily allowance for protein is 0.8 gram protein per kg of body weightt , supplying 0.25 mmol of arginine per kg of body weight. Creatine synthesiss alone, therefore, requires 80% of the daily intake of arginine. This tight balancee may well explain why the creatine pool size is influenced significantly by changess in the quantities of arginine, glycine or creatine in the diet76'78.

Orn n OTC OTC

ornithine e cycle e

O/OH O/OH MG G

Figuree 1: Metabolic routes of creatine synthesis from arginine. Enzymes that catalyze the indicated reactionss are given in italics. AGAT transamidinates the guanidino group of arginine to glycine, but also too the other substrates indicated. Proposed transamidination substrates: glycine (Gly), (5-aminopropionic acidd (GPA), y-aminobutyric acid (GABA), 5-aminovaleric acid (6-AVA), and products: (3- guanidinopropionicc acid (GPA), a-guanidinobutyric acid (GBA), and 5-guanidinovaleric acid (GVA). Productss formed by reactive oxygen (see text): guanidinosuccinic acid (GSA), methylguanidine (MG) and guanidinee (G).GSA and MG are most probably formed from argininosuccinate (AS) and CTN, respectively,, via a reaction with a reactive oxygen species (see also section 6).

23-Arginine23-Arginine demands in growing mammals

17 7 ChapterChapter I

Daviss et al compared the supply of amino acids via the milk and their accumulationn in body proteins of rat to estimate the degree to which each amino acidd in the milk is utilized for protein accretion. The essential amino acids were all utilizedd for protein synthesis with efficiencies of around 75 %, indicating that only 25%% of amino acids are degraded in the suckling period 54. This finding corroboratess an earlier finding that excretion of urea in this period is very small79, seesee also chapter IV. Similar findings were reported for other species like pig, calf, sheep,, chicken and human 80,8\ However, 170 and 270% of ingested arginine and glycine,, respectively, i.e. the precursors of creatine, was found in the lean body mass,, indicating that they were actively synthesized.

2.4-Species2.4-Species differences in arginine requirement and biosynthesis Ass described above, it has been shown that dietary arginine is required for optimal growthh of species like rodents and pigs, but for obligate carnivorous species like catss and mink, it is an essential amino acid 28, due to the virtual absence of P5C synthetasee and OAT in the intestine of these carnivores 82"85(see also 3.1). The resultingg deficiency in ornithine biosynthetic capacity renders their ornithine availabilityy directly dependent on dietary arginine. The result of this can be devastating:: cats develop hyperammonemia and show clinical symptoms of ammoniaa toxicity within 2 hours after consumption of an amino acids containing meall without arginine 86'87, whereas non-carnivorous species show only minimal signss of intoxication when fed arginine-free diets 88. Since ornithine and citrulline occurr only in very low concentrations in ordinary dietary proteins, feline species dependd on dietary arginine 67'87 to spike the ornithine cycle and maintain high capacityy for ammonia detoxification. Thus, in carnivorous species, dietary arginine supplyy can be regarded critical for maintaining the urea cycle capacity for detoxifyingg ammonia

18 8 Introduction Introduction

3-inborn3-inborn errors of metabolism leading to congenital arginine deficiency deficiency

3.13.1 Ornithine aminotransferase Thee small intestinal mucosa is the only site of synthesis of pyrroline-5- carboxylatee (P5C) from glutamate or prolinee 4'28 (Fig. 1). P5C is a precursor for the non-enzymaticc conversion to glutamate-y-semialdehyde, which, in turn, is the substratee of OAT for ornithine formation. Carnivorous species, like the cat, have onlyy 18% (per gram of mucosa), and 5% (per kilogram body weight) of intestinal P5CC activity compared with rat. This limitation in the de novo synthesis of ornithine formss the metabolic basis for the severe hyperammonemia found in cats fed an arginine-deficientt diet90. Patientss that lack OAT 91'92 develop hyperornithemia and gyrate atrophy (GA),, which is characterized by night blindness and diminished peripheral vision in thee first and second decades of life, followed by progression to total blindness. In GAA patients, hyperornithemia is associated with a reduction in the formation of creatinee 9\ via inhibition of AGAT activity by the excess ornithine 94. This creatine deficiencyy has been proposed to account for pathophysiology of the disease 95, as thee mass of skeletal muscle tissue in GA patients is only 24% of that of controls. Unexpectedly,, arginine was found to be an essential amino acid in young growing patientss with this inborn error 93. Mice, in which the OAT gene was genetically targeted,, can serve as an animal model for GA 12. In young mice with this deficiency,, hypo-ornithemia (56 uM, 50% of normal) and -argininemia (26 uM, lesss than 10% of normal), and an impaired growth rate are seen, demonstrating the requirementt for ornithine biosynthesis from pyrroline-5-carboxylate at this age 12. Interestingly,, OAT-deficient mice develop hyperornithemia (>lmM) as adults, elegantlyy demonstrating that the OAT enzyme is directed towards ornithine synthesiss in the neonatal small intestine and liver, but is reversed towards P5C

19 9 ChapterChapter I synthesiss in many adult tissues. A similar phenomenon seems to occur in human patientss as they grow older91.

3.23.2 Ornithine transcarbamoylase Thee human deficiency of OTC, the gene being located on the X- chromosome,, was first described in 1962 96. It is regarded as the most common ornithinee cycle deficiency in man 10. Affected male infants generally die in the first feww weeks after birth, due to acute hyperammonemic coma, though some chronic casess are known97. Well studied murine models of OTC deficiency are the sparse- furfur (spf) and sparse-fur with abnormal skin and hair (spf-ash) mice. These mutant micee suffer from congenital hyperammonemia and hypoargininemia and can serve ass a useful model to study the neurochemical consequences of congenital urea cyclee disorders 97, including hyperammonemic encephalopathy. Spf-ash mice are characterizedd by retarded body- and fur growth, and an excitable hyper-reactive temperamentt 10'98. Biochemically, these mice are characterized by a significantly elevatedd orotic acid excretion, elevated serum ammonia, glutamine and tryptophan levelss l0", and a decrease in all other amino acid levels ". Especially the ornithine cyclee intermediates arginine, ornithine and citrulline are decreased to 20-50% of normal". . Inn children, encephalopathy as a result of chronic hyperammonemia often seenn in OTC deficiency is associated with changes in cerebral neurotransmitter systemss 10°. Similarly, Spf mice display behavioral abnormalities 98 and brain damagee l0!, which can be attributed to alterations in a number of neurotransmitter systems,, (NO 102, ATP, and glutamate 103), ammonia 103, or the tryptophan-derived excitotoxinn quinolinic acid 104. Moreover, Spf mice have an elevation in the levels off neurotoxic guanidino compounds, such as GSA 105. Recently, we described behaviorall aberrations, similar to those in Spf mutants, in our mouse model of argininee deficiency (F/A mice) 42. We showed that this model is more selective in itss amino acid deficiency than Spf mutants, as F/A mice have normal levels of ammonia,, urea, and all amino acids except arginine. Although, the formation of GSA

20 0 Introduction Introduction iss increased in both F/A and Spf mice, this compound is not increased in the brain off F/A mice, whereas it is in Spf-ash mice I06. Hence, a comparison of the two modelss suggests that the deficiency of arginine, or its direct metabolites (NO, agmatinee or creatine), is the determining factor in the development of neuromotor deficitss in both models. This conclusion would also justify the arginine treatment of OTC-deficientt hyperammonemic patients52.

3.33.3 Argininosuccinate synthetase Citrullinemiaa is an autosomal recessive disorder, caused by a deficiency of argininosuccinatee synthase (ASS). It is characterized by elevated levels of blood citrulline,, ammonia, and orotic aciduria 107. Patients suffer from a disturbance of consciousnesss and coma, and most die with cerebral edema within a few years of onsetI08.. In man, ASS deficiency can manifest as a neonatal (CTLN1), or an adult- onsett form (CTLN2) and as an asymptomatic form 52109. CTLN1 is associated withh mutations in the ASS gene, leading to due to ASS enzyme defects, whereas CTLN22 is a form of the disease that is defined by a decrease in ASS protein, but withh normal kinetic properties no. The protein being effected in CTLN2 may be a calcium-dependentt mitochondrial transporter with a role in urea cycle function 108. Thee phenotype of ASS-deficient mutant mice closely resembles that of humans whoo lack the ASS enzyme, 107. Homozygous mutant animals develop high levels of bloodd citrulline, become hyperammonemic, and die within one or two days after birth. .

21 1 ChapterChapter I

4-Metabolism4-Metabolism ofarginine

4.1-Intestinal4.1-Intestinal utilization ofglutamine and arginine Thee small intestine of the adult rat extracts 25-33% of arterial glutamine in aa single pass, which accounts for 30% of whole body glutamine utilization I7'1U. Untill the pioneering work of Windmueller on glutamine utilization by rat jejunum 2, itt was tacitly assumed that all dietary amino acids absorbed by the small intestinal mucosaa entered the portal circulation intact and became available for extra- intestinall tissues n. However, intraluminally delivered glutamine is utilized extensivelyy by the small intestinal mucosa: 66% of intraluminal glutamine is metabolizedd in a single pass by rat jejunum ' . Thus, most of the glutamine is metabolizedd in the small intestinal epithelium and does not reach the portal circulationn n. Subsequently, it was shown that glutamine, and not glucose, is the mainn metabolic fuel of enterocytes. Extraction ofglutamine from the circulation is directlyy related to blood glutamine level, and in conditions associated with a decreasedd circulatory glutamine level, such as starvation, glutamine utilization in thee intestine declines, whereas, on the other hand, the hepatic glutamine uptake increasess m. Inn adult rats, 40% of the luminal arginine, absorbed by the intestinal mucosa,, is catabolized in a single pass, and the remaining 60% enters the intestinal venouss blood intact17'113. A similar percentage was found for humans l14'115. These numberss indicate that substantial amounts of dietary arginine are not available for extra-intestinall tissues. For pig enterocytes, it was shown that 93, 4, and 1% of argininee was metabolized to ornithine, citrulline and CO2 respectively 55. In adult pigg enterocytes, the majority of the arginine that is metabolized to ornithine (56%), iss subsequently metabolized to proline. However, in contrast to post-weaning pigs, prolinee is an essential amino acid in neonatal pigs . This can be explained by the lackk of arginase in the neonatal pig intestine.

22 2 Introduction Introduction

4.2-Arginase4.2-Arginase I and II 4.2.1-Tissue4.2.1-Tissue distribution Arginasee is the most widespread of urea cycle enzyme in terms of tissue distributionn ,16. In mammals, the presence of at least two different arginase genes hass been established 117"119. in man, hepatic arginase I (A-I) is primarily found in liver,, but also in extra-hepatic tissues such as erythrocytes 120 and enterocytes 42. Studiess in man and rats have demonstrated that liver and kidney-type arginase (A-II) differr from one another in both immunogenic and electrophoretic properties 121122. Arginasee II is more widespread and found primarily in kidney, adult intestine, stomach,, brain, mammary gland, pancreas, epididymis, lung and skin n6. A-I and A-II mayy be coexpressed in the rodent kidney 123, small intestinal enterocytes 42, rat aorticc endothelial cells and murine macrophages U6124. The expression of A-II is highlyy species-dependent: the A-II content of small intestine and mammaryy gland of thee rat is a factor 20 higher than that in man or dog 125'126.

4.2.2-Enzyme4.2.2-Enzyme properties Ureotelicc mammals excrete the ammonia that is formed during the catabolismm of amino acids as urea. Arginine regulates urea synthesis by allostericallyy activating the synthesis of N-acetyl glutamate, the essential co-factor off CPS-1 t27. The arginase reaction is highly exergonic with a change in free energy off -23 kcal/mol, making the reaction irreversible 128. Arginase has by far the highestt specific activity of the ornithine cycle enzymes (a Vmax of more than 4000 umol/min/mgg ' ), but the high Km of 1-15 raM causes it to work at much lower velocityy in vivo. Its activity strongly depends on the presence of its cofactor Mn2+ 130.. Reflecting arginase's high capacity, , tissue arginase activities and tissue argininee content are inversely related: liver, with the highest arginase content, has thee lowest arginine content, while kidney, muscle and spleen have only 1 % of liver arginasee activity, but have a 5 to 10 fold higher arginine content (Fig. 3) 131. In additionn to L-arginine, other substrates for arginase are : L-canavanine, L-

23 3 ChapterChapter I homoarginine,, L-argininic acid, and agmatine, but with 7-10 fold higher Y^. D- argininee and GABA are not substrates , indicating that the suitability of alternate substratess for liver arginase is determined by the presence of a guanidino group, the lengthh of the amino acid carbon skeleton and the substituents around C-oc. Lysine inhibitss arginase 132, and functions as an arginine antagonist in mammals 133. As a result,, the metabolic effects of feeding an arginine-deficient diet resemble the effectt of feeding a diet containing a high lysine relative to arginine content.

4.2.3-Subcellular4.2.3-Subcellular localization A-II and A-II have a different subcellular localization: A-I is found in the cytosol , thoughh a substantial fraction of A-I (10 % of total activity) is associated with microsomess and mitochondria 135. A-II is strictly associated with in the mitochondriaa 136. It has been suggested that the the mitochondrial localization of A- III facilitates the synthesis of glutamate and proline from ornithine 1I6, given the intramitochondriall localization of ornithine aminotransferase.

4.2.4-Regulation4.2.4-Regulation of arginase expression Activatedd murine macrophages metabolize arginine by two alternative pathways involvingg either the inducible enzyme NO synthase (iNOS) or arginase. Macrophagess contain both A-I and A-II, with the A-II form predominating. Activationn of macrophages, by LPS or ylFN, induces a concurrent induction of iNOSS and A-I 137 138. In addition, arginine synthesizing enzymes ASS and ASL have beenn shown to be co-induced with iNOS , implying that activated macrophages cann regenerate arginine from citrulline and are independent of extracellular arginine forr NO synthesis. However, cellular arginine import is elevated in macrophages whenn NOS expression is activated 47, via enhanced expression of CAT-2 139, indicatingg that activated macrophages require extracellular arginine for sustained substratee supply for the generation of NO.

24 4 Introduction Introduction

27.88 4

t t 0.3 3

0.4 4 * * 0.2 2 arginasee activity arginine e (U/g) ) (umoll / g) 0.2 2 -- 0.1 D D D D

liverr kidney sPleen muscle Fig.. 3: relation between arginase activity and arginine extractable content in several tissues. 140 0 Adaptedd from Gopalakrishna, 1979 . Open boxes: arginase activity; closed boxes: arginine content.

4.3-Agmatine. 4.3-Agmatine. Agmatinee is a metabolite of arginine, formed via arginine decarboxylase (ADC).. Agmatine and ADC activity have been described to be widespread in brain, liver,, kidney, intestine, adrenal glands and macrophages ,, but the function of agmatinee is not well understood. Agmatine can be hydrolyzed by agmatine urohydrolasee (agmatinase; EC 3.5.3.11) to putrescine, and hence agmatine is a metabolicc precursor for the biosynthesis of higher polyamines. On the other hand, agmatinee may suppress cell proliferation by inhibiting ODC activity and subsequentt polyamine biosynthesis 143. In addition, agmatine may play a role in celll signaling by controlling NO production by inhibiting NO synthases 144. Recently,, agmatine has been identified as an neurotransmitter in the brain, that bindss the N-methyl-D-aspartate (NMDA) subclass of glutamate receptors in hippocampall neurons . Modulation of the receptor was shown to be mediated by thee interaction between the guanidino group and the neuronal channel pore. Though

25 5 ChapterChapter I thee authors claimed this treat to be selective for agmatine 145, other (neurotoxic) guanidinoo compounds i$ee Chapter 6), like guanidinosuccinic acid (GSA) and methylguanidinee (MG) have been shown to share similar properties146ï47.

4.4-Protine. 4.4-Protine. Ornithinee can be converted by the enzyme ornithine aminotransferase (OAT)) and proline oxidase via proline-5-carboxylate into proline. All these reactionss occur in the mitochondrion6. Proline may stimulate collagen synthesis in hydroxylatedd form 148. This may offer an explanation for arginine's ameliorating effectt on the wound healing process and cell proliferation

4.5-Poly'amines. 4.5-Poly'amines. Ornithinee also serves as precursor in polyamine synthesis and is decarboxylatedd by the rate-limiting enzyme ornithine decarboxylase (ODC; EC 4.1.1.17)) to putrescine, the first polyamine in the biosynthetic pathway that can furtherr be converted into spermine and spermidine. Polyamine synthesis has been correlatedd with increased arginase and ODC activity in the kidney 15° and the small intestinee 151. Polyamines are known to exert regulatory properties in cell proliferationn 143,152 and, in that way, are involved in tissue growth and wound repair.

4.6-Nitric4.6-Nitric oxide. Another,, quantitatively minor, pathway in the urea cycle is the NO synthase (NOS)) pathway in which the free-radical NO and citrulline are stoichiometrically formedd by the action of NOS (EC 1.14.13.39) I53. Three distinct isoforms of NOS aree recognized, representing three different gene products l54. Two of these isoenzymes,, defined as cNOS, are expressed constitutively and are calcium- dependent,, whereas one isoform, termed inducible NOS (iNOS or NOS II), is only synthesizedd de novo after stimulation and is non-calcium dependent !55156. One cNOSS subtype, named endothelial NOS (eNOS or NOS III), is mostly membrane- boundd and is present in several cells, mostly endothelial cells 157158. The other

26 6 Introduction Introduction cNOSS subtype is cytosolic and mostly present in neuronal tissue and therefore definedd as neuronal NOS (nNOS or NOS I). The iNOS isoform is cytosolic can be 155 156159 expressedd in a great variety of cells including macrophages and epithelia - i Thee cNOS enzyme is permanently present in cells and is (transiently) activated by temporaryy rises in free Ca2+. The iNOS isoform is not present at basal levels but can bee induced in response to inflammatory cytokines and endotoxin, including LPS 157.

27 7 ChapterChapter I

5-- Regulation of metabolism by arginine

5.15.1 Effect of arginine on protein synthesis and degradation Duringg disease and injury the organism responds by changing the rates in thee synthesis and/or degradation, i.e. turnover of proteins. Supplementation of intravenouss or enteral arginine is associated with alterations in protein metabolism .. These effects may, at least in part, be mediated by the arginine-mediated hormonee release such as insulin and growth hormone (GH), which are known to producee anabolic effects by inhibiting the loss of body protein and stimulating aminoo acid transport into the cells 161 {see 5.2). The rate of translation of specific mRNAss determines the rate of protein synthesis, whereas post-translational modificationn of proteins affects protein function. Arginyl-tRNA is not only an immediatee precursor for protein biosynthesis, but it is also involved in the post- translationall conjugation of arginine with N-termini of proteins bearing N-terminal aspartatee or glutamate, thereby targeting these proteins for degradation via the ubiquitin-dependentt proteolytic pathway I62. Arginine may thus be involved in modulationn of protein breakdown by post-translational arginylation 163.

5.25.2 Endocrine activity arginine Argininee exerts a secretagogue effect by stimulating the secretion of the pituitary,, pancreatic, and adrenal hormones. The pituitary GH has been shown to 164165 increasee in response to intravenous administration of arginine in humans ; butt also after oral administration l66,167. Arginine was also found to exert the releasee of prolactin, both after intravenous 168 and oral 167 administration. Furthermore,, arginine administration stimulates the secretion of several 169170 171 in pancreaticc hormones like insulin ? glucagon pancreatic polypeptide , andd the GH-inhibiting hormone somatostatin m. This secretagogue effect of argininee has been shown to act via mechanisms dependent, as well as independent off the production of nitric oxide78174. Several recent reports show that the effects off arginine constituting the induction of vasodilatation, inhibition of platelet

28 8 Introduction Introduction aggregationn and blood viscosity are mediated, in part, by arginine-induced endogenouss released insulin 175176, Besides these pancreatic hormonal responses, argininee was also found to stimulate the secretion of catecholamines ,75177 by the adrenall gland.

29 9 ChapterChapter I

6-Guanidino6-Guanidino compounds

6.1-6.1- the ornithine-guanidine bi-cycle Soo far, approximately 120 naturally occurring guanidino compounds have beenn described in plants and tissues 178 (for structural formulas: see Chapter VI, appendixx II). Previously, Natelson and Sherwin proposed the existence of a guanidinoo cycle 179, which, next to the ornithine cycle, would be important in nitrogenn metabolism. Though the intermediates have been detected in bacteria, the existencee of this cycle has never been proven in mammals. This guanidine cycle was purportedd to be an alternative pathway to ensure the synthesis of creatine phosphate, neededd for muscular contraction. In mammals, which do not possess urease, urea cann be oxidized by the cytochrome P-450 system to give hydroxyurea Hydroxyureaa can either enter the ornithine cycle via the formation of carbamoylphosphate,, or can be hydrolyzed to give hydroxylamine and enter the guanidinee cycle. Guanidinoacetic acid (GAA), the precursor for creatine (CT), can bee formed from glycine and arginine (via the ornithine cycle), or from glycine and canavaninee (via the guanidine cycle) 18°. According to Natelson, the two cycles wouldd be operative in parallel, and were therefore specified as the 'ornithine- guanidinee bi-cycle' 181 (Fig. 4). The existence of such a bi-cycle however, has never beenn shown in mammals.

6.2-6.2- guanidino compounds formed after transamidination. AA major route of formation of guanidino compounds from arginine is the transferr of the amidino group from arginine to glycine, yielding ornithine and GAA, thee immediate precursor for creatine (CT) and creatinine (CTN). A proposed schemee of reactions is given in Fig 2. The reaction, catalyzed by the enzyme AGAT (ECC 2.1.4.1), is reversible l82, and catalyzes the rate-determining step in creatine synthesiss 18°. AGAT accepts a wide variety of substrates as amidine acceptors: in additionn to the physiological substrate oc-amino acetic acid (glycine), a- aminoproprionicc acid (- a-alanine), a-aminobutyric acid or a-aminovaleric acid can

30 0 Introduction Introduction

functionn as substrate, yielding guanidinopropionic acid (GPA), guanidinobutyric acidd (GBA), and guanidinovaleric acid (GVA), respectively178,180,18 2 2(Fig.2) . .

Ure e Ornn OTC ^ Cit ureido-Hser Can n

ornithinee cycle y / guanidine cycle Cit t

Cav v

Fig.. 4: Proposed interaction of the ornithine cycle and the guanidine cycle via the "ornithine-guanidine bi-cycle"" of Natelson et at. Excess urea can be re-utilized via this cycle. For details see text). Guanidine cyclee substrates and products: hydroxyamine (OH-amine), homoserine (Hser), ureido homoserine (ureido- Hser),, canavanino succinate (CavSA), cnavanine (Cav), canaline (Ca). Intermediates in creatine synthesis: argininee (Arg), guanidinoacetate (GAA), creatine (CT), glycine (Gly).

Inn murine models of arginine deficiency, either as a result of OTC-deficiency (spf andd spf-ash mice) , or of A-I overexpression 42, GPA, GBA, and GAA are decreased,, demonstrating that availability of arginine as amidine donor determines thee formation of these GCs.

6.3-formation6.3-formation ofGSA by reactive oxygen species

31 1 ChapterChapter I

Thee formation of guanidino compounds under several pathological conditions is demonstrated,, and some GCs, such as the neurotoxic guanidinosuccinic acid (GSA), guanidinee (G), and methylguanidine (MG), have been reported to add to the (neuro)pathologyy of several metabolic diseases. Elevated levels of the guanidine derivativess MG as well as GSA, have been detected in the urine and plasma of patientss with uremia, and both compounds were shown to be toxic ' ' . The exactt mechanism of GSA synthesis remains to be established. Several alternative biochemicall routes of GSA formation have been previously postulated. According too this proposed guanidine cycle pathway, GSA can be formed from canavaninosuccinate.. As already stated, the existence of a guanidine cycle in mammalss has never been demonstrated. Perhaps a more plausible route of GSA synthesiss was proposed by Cohen et aln4: GSA may be produced due to a shift in activityy of AGAT to use aspartate rather than glycine as amidine donor. Another proposedd enzymatic pathway of GSA synthesis is based on the cleavage of argininosuccinatee by ASL to carbamoyl-asparatate and ornithine, instead of arginine andd fumarate, and subsequent formation of GSA. This reaction may be stimulated by urea,, which is known to inhibit ASL activity l85, thus favoring AS accumulation. Indeed,, addition of urea to the medium dose-dependently increases GSA synthesis inn isolated rat hepatocytes. In addition, urea levels correlate positively with GSA andd MG concentration in patients with renal failure and after partial nephrectomyy in rodents 187188. On the other hand, the decreased GSA synthesis in

11 OQ humann patients with hyperargininemia as a result of arginase deficiency cannot easilyy be explained by this hypothesis. However,, evidence is accumulating that the conversion of AS in GSA and betainee may be non-enzymatic, via the reaction of argininosuccinic acid with the actionn of an oxygen radical species. Such a conversion has already been shown to occurr in vitro, in isolated rat hepatocytes 183. In a similar series of experiments, OTCC inhibition by DL-norvaline inhibited GSA synthesis in isolated hepatocytes, suggestingg that the urea cycle enzymes catalyze intermediate reactions in the GSA syntheticc pathway 190. Following urea injection, hepatic GSA levels also increased

32 2 Introduction Introduction inn vivo, but there was little change in hepatic arginine 190, indicating that GSA synthesiss is stimulated by urea, and is directly dependent on arginine availability. Thee latter is corroborated by the increased GSA synthesis in arginine-deflcient transgenicc mice Similarly,, MG has been shown to be generated after the reaction of an oxygenn radical with CTN 191192, in uremia MG may arise from the degradation of CTN-byy the gut flora I93. The concentration of MG varies with the GSA concentrationn in patients with renal insufficiency. Together, the toxic GCs MG and GSAA most probably are generated by the cleavage of CTN or AS respectively, by reactivee oxygen. Such a mechanism would provide the rational for the use of active oxygenn scavengers for treatment of uremia.

6.4-Pathological6.4-Pathological effects Thee clinical syndrome of uremia is due to the failure of not only the excretoryy but also the metabolic, regulatory and endocrine functions of the kidney. InIn addition to elevations in CTN and urea, acute and chronic renal failure is recognizedd by a state of hypercitrullinemia 194. Citrulline proved to be a more sensitivee indicator than urea or creatine, as citrulline levels correlated with the degreee of nephrectomy, and hypercitrullinemia developed in the range of 10% to 33%% nephrectomy without any changes in urea and creatine 194. Thus, hypercitrullinemiaa is a specific marker of normal function of the proximal tubule, whereass an impaired creatine and urea clearance apparently can be compensated. A numberr of guanidino derivatives are also considered to be important indicators of renall failure 193. GSA positively correlates with plasma urea 186 and indicates the severityy of renal failure ,95,196. MG also appears in plasma after renal failure 197. GSAA and MG are recognized as candidate markers, which reflect the pathological stagee of nephritis 147198'199. The enhanced production of certain GCs, in particular GSAA and MG, has been associated with the etiology of the uremic syndrome. Their 184 196200 201 increasedd synthesis has been reported in renal insufficiency - > ) m partiallyy nephrectomized rats and mice ,87, during endotoxemia-induced sepsis 202,

33 3 ChapterChapter I

andd in patients with hypothyroidism . Furthermore, an increased GSA concentrationn is found during under hyperammonemic conditions due to consumptionn of an arginine-free diet in carnivorous species 204, or under normal ammoniaa concentrations in mice models of arginine deficiency 42. Uremiaa has been shown to be associated with neurological disorders 205" ,, and a proportion of patients with renal failure suffers from central and peripherall nervous system impairments 206. Mildly uremic mice display acute behaviorall deficits, which may relate to analogous symptoms in uremic patients sufferingg from uremic encephalopathy208. The role of GSA in uremic neurotoxicity andd coma is still controversial and needs further investigation, but GSA, G, and MG havee been shown to be (neuro)toxic in vitro 209. Moreover, GSA has been shown to causee convulsions after intracerebroventricular injection in mice 147. The elevated levelss of GSA and MG may well play a role in the neurological consequences of uremiaa via activation of NMDA receptors147-210. Mice, suffering from low levels of argininee and elevated level of circulating GSA and MG displayed behavioral deficits,, such as hyperactivity as well as several more specific deficits in neuromotorr abilities and coordinated movements 42'98.

34 4 Introduction Introduction

7-Arginine7-Arginine transporters

7.1-Amino7.1-Amino acid transporters Fivee systems are known to mediate transport of cationic amino acids over thee cell membrane: system y+, y*L, b+, b*0, and B^. These transport systems can be distinguishedd by their affinity for cationic amino acids, their dependence on sodium,, and their capacity to share transport with zwitterionic amino acids 2U. Systemss b+, b^, and B*0, first discovered in rat blastocysts 212, are found in brush borderr membranes, and have a broad specificity for zwitterionic and dibasic amino acids.. Systems b+ (cationic amino acids-preferring) and b40 (cationic and neutral aminoo acids-preferring) are sodium-independent, and B^ (cationic and neutral aminoo acids-preferring) is sodium-dependent. Transport of arginine, ornithine, and lysinee across plasma membranes is mostly performed via the sodium-independent aminoo acid-transport system, formerly designated y+ 213'214.

7.2-System7.2-System y+ Thee major mammalian cationic amino acid-transport system is the nearly ubiquitouss system y+, which facilitates Na+-independent arginine, lysine, and ornithinee transport driven by membrane potential 2l5. Expression in oocytes revealedd the ecotropic murine leukemia virus receptor, now named CAT-1 216, as a CATT . Since, four homologous human and rodent genes make up the family of cationicc amino acid transporters (CATs) that encode system y+, CAT-1, 2A, and a splicee variant CAT-2B, CAT-3 and CAT-4214. CAT-11 expression is nearly ubiquitous and produces a single protein. Underr basal conditions, CAT-1 is absent from hepatocytes 215, probably to prevent unnecessaryy transport and metabolism of arginine by the hepatic arginase in the hepatocytes.. However, CAT1 expression is present in regenerating liver, and in the casess when hepatic cationic amino acid transport is needed, such as following feeding,, cellular growth and illness.

35 5 ChapterChapter I

AA full length cDNA clone from a previously identified murine T-lymphoma celll line (Tea, T early activation gene) showed significant homology (61%, 2I8) withh CAT-1 and amino acid-transport capacity in oocytes2I9. Two splice variants of thiss gene were identified and termed CAT-2A and -B. mCAT-2 expression is highly tissue-specificc and is expressed using at least two widely separated promoters 51. CAT-2AA is expressed in the liver and is regarded as the low affinity variant, whereas CAT-2BB is the high affinity variant, expressed in T-cells. CAT2A is highly expressedd in liver, skeletal muscle, especially after trauma, and skin, while CAT2B expressionn is high in brain, lung, testis, uterus, and activated, but not resting T-cells (expressionn was found in T-cell thymoma cells, but was low in thymocytes 215). CATT 2A and 2B proteins are 97% identical, as they differ only one 41 amino acid domain215'219. . Recently,, two new members of the CAT family have been cloned; a brain- 220 0 specificc isoform, which again has strong similarity with CAT-1, termed CAT-3 , andd CAT4 (previously termed HCAT3). The latter gene is highly expressed in skeletall muscle, intestine, kidney, and placenta and in eye and in retinal pigmented epithelium.. CAT4 is homologous to the amino acid permease CD98 light chain 221. AA defect in CAT4 expression at the basolateral membrane of epithelial cells in kidneyy and intestine has been associated with the development of lysinuric protein intolerance,, a rare autosomal recessive disease, characterized by poor feeding, vomiting,, diarrhea, and episodes of hyperammonemic coma222223.

7.3-Regulation7.3-Regulation ofarginine transporters CATT I and CAT2 proteins expressed in Xenopus oocytes exhibit high- affinity-loww capacity cationic amino acid transport activities 224. There is a steric constraintt for arginine binding to the CAT-1 transporter as a-methylation completelyy abrogates arginine binding 217. Similarly, the affinity for D-arginine is 200 times less than for the L-arginine 225. On the contrary, L-homoarginine is specificallyy transported with high affinity, making it a useful probe to study CAT-1 activity,, as physiological concentrations of L-homoarginine are low. The kinetic

36 6 Introduction Introduction

constantss Vmax (maximal transport rate when excess substrate is present) and Km (substratee concentration at which the transport rate is half maximal) for arginine uptakee depend on the CAT isoform. In hepatocytes in the basal state, the y+ system

(CAT2A)) is barely detectable having a Km value of 2-5 mM and a Vmax of 3.3 nmol" 11 1225 • mg of protein"' • min" i.e. a 10-70 fold higher apparent Km and Vmax than the CAT11 and 2B (100-150 \M 214). The high Km value of CAT2A for arginine (way abovee the normal plasma concentration) underscores the minimal rate of arginine uptakee by hepatocytes under normal physiological conditions 225. The precise cellularr localization of arginine transporters may account for the "arginine paradox",, the dependence of endothelial NO synthesis on extracellular arginine availability,, while the intracellular arginine concentrations (0.1-1.0 mM) greatly exceedd the Km of eNOS for arginine (2-10 uM). The apparent Km for arginine of NOO synthesis by intact endothelial cells is approximately 75-150 JIM 6, which is in thee range of normal physiological plasma concentrations, and of the Km values of thee arginine transport systems.

Glucocorticoidss and insulin induce expression of the CAT1 gene in liver cellss through induction of transcription and stabilization of its mRNA 50. The CAT1 genee is subject to adaptive regulation by arginine availability 'm vitro, as arginine depletionn increases CAT1 mRNA stability, causing an increase in CAT1 protein, and transportt capacity 226. CAT2A is, in contrast to CAT1 and 2B, hardly trans- stimulatedd 227. Therefore, the 41 amino acid domain which lacks in the protein sequencee of CAT2A is thought to be responsible for substrate recognition and possiblyy the mechanism of substrate translocation215,219. Quiescentt lymphocytes from spleen, lymph nodes, and Peyer's patches constitutivelyy express CAT2B, although these cells exhibit little transport of argininee via the systems y or y+L 214-215. Upon mitogenic activation, however, transcriptss of CAT2B rapidly accumulate215, underscoring a role for arginine in this T-celll activation process 228. In addition, system y+ can be induced by inflammatory cytokiness in hepatocytes 229 and macrophages 46.

37 7 ChapterChapter I

Otherr cationic amino acids and positively charged analogues are effective inhibitorss of arginine uptake by system y+. For example, arginine uptake can be competitivelyy inhibited by lysine, ornithine, canavanine, and certain NOS inhibitors, suchh as N-monomethyl-L-arginine and N-iminoethyl-L-ornithine 6. N-nitro-L- argininee and N-nitro-L-arginine methyl ester, or aminoguanidine, on the other hand, havee no effect on arginine uptake " .

38 8 Introduction Introduction

8-Arginine8-Arginine as immunonutrient.

8.18.1 Arginine and immune f unction Onlyy limited information is presently available as to how nutrients can influencee immune function or the development of lymphoid organs. An intriguing featuree of the nutritional benefit of arginine is, next to its function as a building blockk for protein synthesis, its irnmunosupportive effect, especially under catabolic conditionss 233,234. Arginine was already identified as an immunonutrient more than 200 years ago, in studies on wound healing ' ' . Since, arginine has often been indicatedd to influence immune system responses 72, either via the increased productionn of polyamines237, effects on tumor growth 238, by ameliorating survival duringg sepsis239, or directly on T-cell gene expression 228. Based on these studies, argininee has widely been added to postoperative supplemental formulas, like Impact®® (Novartis), at doses as high as 100 gr per kg formula 240, but the actual molecularr basis of the beneficial effect of arginine on lymphocyte biology has remainedd unclear through the years.

8.28.2 Potential mechanism of action ADP-ribosylationADP-ribosylation of arginine residues Inn the transgenic arginine-deficient mice described in Chapter V, the developmentt of the lymphoid system is affected. This finding may hint towards an importantt aspect of arginine's irnmunosupportive effect. The influence of the argininee deficiency seems to be restricted mainly to the early development of B- cellss in the bone marrow, as we found no defective T-cell maturation in the thymus,, or reduction of T-cell number in peripheral lymphoid organs. Despite theirr hampered maturation, transgenic B-cells residing in the spleen were able to proliferatee normally upon B-cell specific in vitro stimulation. We are currently testingg the proliferative ability of splenic T cells, but preliminary data already

39 9 ChapterChapter I indicatee an impaired production of IFNy, and possibly IL4 and 6, upon CD3/CD28 inin vitro stimulation, corroborating other reports228. InIn addition to a temporary hampered B cell maturation, the F/A transgenic micee suffer from a temporary inhibition of muscle development. An attractive hypothesiss on how arginine may influence both lymphocyte responses and muscle growthh is a possible enhancement of ADP-ribosylation of arginine residues in lymphocytee membrane proteins. Mono-ADP-ribosylation is a posttranslational modificationn of proteins in which the ADP-ribose moiety of NAD+ is transferred to thee guanidino group of arginine on a target molecule 241. This reaction is catalyzed byy mono-ADP ribosyltransferases (ART), which are exo-enzymes imbedded in the celll membrane. ART is abundantly expressed in myocytes, as well as immune cells. AA membrane protein that was identified as a substrate for ART was Ct7-integrin 242. Mice,, lacking this integral suffer from muscular dystrophy, which starts five days afterr birth243, a phenotype similar to the arginine-deficient F/A-mice.

Thee involvement of ART in immune response regulation is already indicatedd by the observation that strains of rats, which lack one of the ART enzymes (ART-1),, develop autoimmunity 244. ART-1 and 2, isoforms of the murine enzyme, whichh are active on CD4+ and CD8+ T-lymphocytes, and may be functional in downregulatingg their activation state 245. ART-1 and 2 have been shown to specificallyy ribosylate members of the integrin family of adhesion molecules, like lymphocyte-functionn associated protein-1 (LFA-1) 246, or T-cell membrane proteinss important in eliciting T-cell mediated immune responses, like CD27, CD28,, CD43, CD44 and CD45R 242. ADP-ribosylation of T-lymphocytes is known too attenuate T-cell proliferation, cytotoxicity, and cytokine excretion , probably becausee T-cell receptors fail to associate in a contiguous and functional receptor clusterr after stimulation242. B-cell responses are not affected by ADP-ribosylation 245.. Hence, the observed inactivity of T-lymphocytes (preliminary data), but intact B-celll response upon stimulation in our F/A-2 transgenic mice perfectly matches thee functional consequences of an over-ribosylation of T-cells. Such an over- ribosylationn during hypo-argininemia could be due to the fact that ARTs not only

40 0 Introduction Introduction

ADP-ribosylatee arginines in the backbone of proteins, but also free arginine. Thus, argininee and agmatine are the naturally competitive inhibitors of ADP-ribosylation off cell-surface molecules, and a decrease in circulating arginine, as seen in our F/A transgenics,, then results in an enhanced ribosylation of cell surface proteins. However,, the involvement of ADP-ribosylation in the arginine deficiency phenotypee remains to be demonstrated.

41 1 ChapterChapter I

9-Scope9-Scope of the thesis

Thee amino acid arginine is more than a building block for the synthesis of protein; it servess important physiological functions, such as the detoxification of ammonia, andd it is a precursor for the synthesis of nitric oxide (Science's molecule of the yearr in 1990), agmatine, creatine and polyamines. Furthermore, it serves as a secretagoguee for glucagon and growth hormone, and evidence is accumulating that itit has intriguing capacities in promoting lymphocyte function. Lactating mammals havee been shown to accumulate large amounts of arginine, compared to mother's milk,, implying endogenous arginine biosynthetic capacity. This thesis aims to establishh the specific functions of arginine for neonatal development. Inn Chapter II, we identified the suckling small intestine as an organ with argininee biosynthetic capacity, after studying the spatial distribution of ornithine cyclee enzymes along the rat small intestinal villus axis. The enterocytes of neonatal andd suckling rats have the capacity to synthesize arginine, but this capacity is shown too be lost after weaning. Furthermore, we found a compartimentation of the citrullinee and arginine synthesis over the villus axis: the basal enterocytes seem to synthesizee citrulline, and the apical enterocytes arginine. A possible role of the changess in turnover rate of enterocytes during development in the establishment of thiss metabolic compartimentation is discussed. Wee investigated the significance of this intestinal arginine mrtabolism for neonatall development further in a murine model of chronic arginine deficiency. To thiss end, two lines of transgenic mice which express different levels of arginase I in theirr enterocytes were generated, in order to establish lines of mice with a graded severityy of arginine deficiency. In Chapter III, both lines were analyzed, and indeed thiss approach led to a, surprisingly selective, reduction in circulating arginine concentration,, further underscoring the importance of enterocytes in maintaining argininee homeostasis. We describe the development of a pronounced phenotype in thee arginine-deficient mice, which corresponds with the degree of arginine

42 2 Introduction Introduction deficiencyy and which includes the retardation of hair- and muscle growth, and an impairedd development of the lymphoid tissue, in particular Peyer's patches. Ass mentioned earlier, arginine serves as a precursor for the synthesis of creatinee and other guanidino compounds. In Chapter IV, the metabolic effects of a deficiencyy of arginine, in particular of arginine's guanidino group, are considered. Decreasess in the concentration of arginine lead to corresponding decreases in its transaminidationn products and, hence, creatine synthesis. However, hypoargininemicc conditions also induce the accumulation of certain neurotoxic guanidinoo compounds, associated with the development of psychomotor deficits, a phenomenonn which parallels the enigmatic guanidino compound disturbances seen inn patients with renal failure or hyperammonia. These data hint to a role for an enhancedd oxidative stress under these conditions. Anotherr feature of arginine is its purported immunosupportive effect, which has neverr been explained, despite more than 20 years of research on this matter. As describedd in Chapter HI, hypoargininemia affects the development of lymphoid organs,, reflected in the virtual absence of Peyer's patches. In Chapter V, this featuree is further explored. Arginine deficiency leads to reduced B-cell numbers in thee peripheral lymphoid organs, but T cells numbers are unaffected. Further researchh into the cause of this reduction led to the bone marrow, where it was unexpectedlyy revealed that arginine deficiency impaired early B-cell maturation at thee transition from the pro- to pre- B cell stage

43 3 ChapterChapter I

References References

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