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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.

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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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ChapterChapter IV

Effectt of arginine deficiency on circulating and tissue

aminoo acids and guanidino compounds, and on

behaviorall development

WouterWouter J de Jonge, Bart Marescau, Rudi D 'Hooge, Peter P De Deyn,

MarcellaMarcella M Hallemeesch, Nicolaas EP Deutz, Jan M Ruijter, Wouter H

Lamers Lamers

Departmentt of Anatomy & Embryology Academic Medical Center, University of

jnsterdam,, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands, 2Department <

Neurology,, Laboratory of Neurochemistry and Behavior, Born-Bunge Foundation,

0,, Antwerp, Belgium, 3Departu

University,, The Netherlands.

Universityy of Antwerp (U1A), Antwerp, Belgium, department of Surgery, Maastricht

111 1

ChapterChapter IV

Abstract t Argininee is an intermediate of the ornithine cycle and serves as a precursor for the

synthesiss of nitric oxide, creatine, agmatine, and proteins. It is considered to be a

conditionallyy essential amino acid, because endogenous synthesis only barely

meetss daily requirements. In rapidly growing suckling rats, endogenous arginine

biosynthesiss is crucial to compensate for the insufficient supply of arginine via the

milk.. Evidence is accumulating that the intestine rather than the kidney plays a

majorr role in arginine metabolism in this period. Accordingly, ectopic expression

off hepatic arginase in murine enterocytes by genetic modification induces a

selectivee arginine deficiency. The ensuing phenotype, the severity of which

correlatess with the level of transgene expression in the enterocytes, could be

reversedd with arginine supplementation. We now report that these transgenic mice

continuee to suffer from an arginine deficiency after the arginine biosynthetic

enzymess disappear from the enterocytes. Post-weaning catch-up growth in

arginine-deficientt mice is characterized by increased levels of all amino acids

exceptt arginine. Furthermore, amino acid concentrations, including arginine, are

lowerr in adult male than in adult female transgenes. Decreases in the concentration

off plasma and tissue arginine lead to corresponding decreases in most metabolites

off arginine. However, the accumulation of the toxic guanidino compounds

guanidinosuccinicc acid and methylguanidine corresponds with arginine deficiency,

possiblyy reflecting a higher oxidative stress under hypoargininemic conditions.

Hypoargininemiaa is also associated with disturbed neuromotor behaviour, although

brainn levels of toxic guanidino compounds and ammonia are normal.

112 112

Abbreviations Abbreviations

BiochemicalBiochemical implcations ofarginine deficiency

CPSS [EC 6.3.4.16]

OTCC [EC 2.1.3.3]

ASSS [EC 6.3.4.5]

ASLL [EC 4.3.2.1]

A-II [EC 3.5.3.1]

A-ÜÜ [EC 3.5.3.1]

AGATT [EC 2.1.4.1]

ND D

Harg g

ArgA A

GAA A

CT T

cm cm MG G

G G

GABA A

y-GBA A

P-GPA A

5-GVA A

oc-K-5-GVA A

GSA A

a-NAA A

carbamoylphosphatee synthetase

ornithinee transcarbamoylase

argininosuccinatee synthetase

argininosuccinatee lyase

hepaticc arginase

non-hepaticc arginase

L-arginine-glycinee transamidinase

neonatall day, days after birth

homoarginine e

argininicc acid

guanidinoaceticc acid

creatine e

creatinine e

methylguanidine e

guanidine e

y-aminobutyricc acid

y-guanidinobutyricc acid

p-guanidinopropionicc acid

8-guanidinovalericc acid

a-keto-8-guanidinovalericc acid

guanidinosuccinicc acid

a-N-acetylarginine e

113 3

ChapterChapter IV

Introductio n n

Argininee is an intermediate of the ornithine cycle and serves as a precursor for the

synthesiss of nitric oxide (NO), creatine, agmatine, and proteins (Wu and Morris,

1998).. Arginine is not considered to be an essential amino acid. In adults, a major

sourcee of arginine biosynthesis from citrulline is the renal proximal convoluted

tubulee (Featherston et al., 1973; Levillain et al., 1992). Citrulline, in turn, is

synthesizedd in the small intestine from glutamine and proline (Windmueller and

Spaeth,, 1981). Although arginine is not considered to be an essential amino acid in

adultt humans, its endogenous biosynthetic capacity only amounts to approx. 20%

off daily expenditure (Visek, 1986). Hence, its bioavailability may become

insufficientt under conditions of increased demand, such as growth (Visek, 1986)

andd tissue repair (Thornton et al., 1997), or as a result of decreased dietary supply

(dee Lorgeril, 1998). For this reason, arginine is regarded as a conditionally

essentiall amino acid.

Inn rapidly growing suckling rats, endogenous arginine biosynthesis is crucial to

compensatee for the insufficient supply of arginine via the milk (Davis et al., 1993).

Evidencee is accumulating that the intestine rather than the kidney plays a major

rolee in arginine biosynthesis in the suckling period. Thus, the enterocytes of the

smalll intestine express the enzymes required for arginine production from

glutaminee and proline (Wu, 1997) and do not express arginase (De Jonge et al.,

1998).. Furthermore, we have recently shown that overexpression of hepatic

arginasee in enterocytes induces an arginine deficiency (De Jonge et al, submitted).

Thee intestine appears to play a similar role in human neonates as well, as

destructionn of the enterocytes in necrotizing enterocolitis also results in a

selectivee decrease in circulating arginine (Zamora et al., 1997). In mice and rats,

argininosuccinatee synthetase and argininosuccinate lyase, the enzymes that

synthesizee arginine from citrulline, disappear from the enterocytes in the

postweaningg period, concurrent with the appearance of endogenous arginase (De

Jongee et al., 1998), so that the role of the gut becomes confined to citrulline

biosynthesis. .

114 4


Thee reduction in circulating arginine concentration in transgenic mice that express

differentt levels of arginase I in their enterocytes ("F/A" transgenes) depends on

thee expression level of transgenic arginase. The degree of arginine deficiency

correlates,, in turn, with the degree of retardation of hair- and muscle growth, and

thee development of the lymphoid tissue (De Jonge et al, submitted). Expression of

arginasee in all enterocytes is necessary to elicit arginine deficiency. Phenotypic

abnormalitiess are reversed by daily injections of arginine, but not creatine.

Becausee the phenotypic symptoms largely disappear after weaning, the question

arosee whether circulating levels of arginine in relation to the other important

aminoo acids normalize in our transgenic mice when intestinal arginine

biosynthesiss ceases after weaning.

Ornn QIC A-I/A-U A-I/A-U

lysine e A A

argA A a-NAA A

a-K-5-GVA A

Gly y P-alanine e

GABA A

55 AVA

P-GPA A Y-GBA A 5-GVA A

ornithine e cycle e

O/OH' O/OH'

Figuree 1: Metabolic routes ofarginine. Enzymes that catalyze the indicated reactions are given in italics. Arginasee accepts arginine as well as homoarginine as a substrate. Similarly, AGAT transamidinates the guanidinoo group ofarginine to glycine, but also to the other substrates indicated. The a-amino group of argininee can be transaminated to a-K-8-GVA, andacetylated to a-NAA. GSA and MG are most probably formedd from argininosuccinate (AS) and CTN, respectively, via a reaction with a reactive oxygen species.

115 5

ChapterChapter IV

Wee also investigated the accumulation of guanidino compounds, the

metabolitess of arginine that retain the guanidinium group. A schematic

representationn of the relation of arginine with the ornithine cycle and its guanidino

metabolitess is given in Fig. 1. Homoarginine, lysine, and homocitrulline (not

shown)) are homologues of arginine, ornithine, and citrulline, respectively, and as

suchh are alternative intermediates of the ornithine cycle. A major metabolic route

off the guanidino group of arginine is transamidination to glycine to yield

guanidinoaceticc acid (GAA), and subsequently creatine (CT) and creatinine (CTN).

Inn addition to the physiological substrate glycine (oc-aminoacetic acid), its

homologuess p-alanine (p-aminoproprionic acid), y-aminobutyric acid, and 8-

aminovalericc acid can function as substrates of the enzyme AG AT, yielding P-

guanidinopropionicc acid (p-GPA), y-guanidinobutyric acid (y-GBA), and 8-

guanidinovalericc acid (S-GVA), respectively (Fritsche et al., 1999; Marescau et al.,

1992b;; Wyss and Kaddurah-Daouk, 2000). Furthermore, the oc-amino group of

argininee can be transaminated and acetylated, yielding oc-keto-8-guanidinovaleric

acidd (a-K-S-GVA) and a-N-acetylarginine (a-NAA), respectively. Hydrogenation

off a-K-S-GVA produces argininic acid (ArgA) (Robin and Marescau, 1985).

Methylguanidinee (MG) forms after the reaction of an oxygen radical with CTN

(Nakamuraa et al., 1991). Similarly, guanidinosuccinic acid (GSA) may be the

productt of the reaction of argininosuccinic acid and the action of an oxygen

radicall species (Aoyagi et al., 1999). Some guanidino compounds, in particular a-

NAA,, GSA and MG, are toxic and play a role in the pathology of renal (Tomida et

al.,, 2000) and liver (Marescau et a l, 1995) failure, in particular the metabolic

(Horowitzz et al., 1970) and neurological (da Silva et al., 1999; De Deyn et al.,

2000;; D'Hooge et al., 1996) consequences of these diseases. The neurotoxicity of

guanidinoo compounds appears to be more pronounced in the growing than in the

adultt animal (D'Hooge et al., 1992; D'Hooge et al., 1994). The concentration of

guanidinoo compounds was therefore determined in plasma and several tissues of

10-dayy old homozygous arginine-deficient transgenic mice, as well as in wild-type

116 6


controls.. At this age, intestinal biosynthetic capacity for arginine is maximal (De

Jongee et al., 1998) and the hypoargininemic phenotype of the transgenic mice

mostt pronounced (De Jonge et al, submitted). The relation between circulating

argininee concentration and the formation of guanidino compounds was investigated

byy arginine supplementation. Because of the neurotoxic potential of some

guanidinoo compounds, like GSA and MG, and the possible long-term

consequencess of alterations of GAB A and glycine levels on neurotransmission, we

alsoo determined the consequences of arginine deficiency on neuromotor

development. .

117 7

ChapterChapter IV

Methods s

Animals.Animals. Mice were kept under environmentally controlled conditions (lights on

att 8;00 a.m., off at 8;00 p.m.; water and rodent chow ad lib; 20-22 ° C, 55%

humidity).. Animal experiments were done in accordance with the guidelines of the

locall Animal Research Committee. Litters discovered in the morning were

assignedd ND 0. The animals were weaned at three weeks of age. For arginine

injections,, nest size was adjusted to 7 pups. Pups received a subcutaneous

injectionn of 5 mmol/kg of arginine-HCl (150 mM), twice daily (9;00 a.m. and 6;00

p.m.)) from postnatal day 5 onward. Controls were injected with saline (0.9 %

sodiumm chloride).

GenerationGeneration of transgenics. Intestinal fatty-acid binding protein (FABPi)/arginase

II transgenic mice (F/A) were generated in the FVB-strain. Generation of the

transgenicss is described in detail elsewhere (De Jonge et al, submitted). Briefly,

arginasee I (A-I) was specifically expressed in enterocytes by coupling the A-I

structurall gene to the FABPi promoter/enhancer element. Two lines, F/A-l and

F/A-2,, which express A-I in all enterocytes, but which differ 2-fold in the level of

A-II expression (F/A-2 = 2 * F/A-l), were analyzed.

TissueTissue and blood sampling. Pups were separated from their mother and kept at 37° C

forr one hour prior to sacrifice. After decapitation, blood was collected into heparin-

containingg tubes and centrifuged at 2,000 x g for 5 minutes at 4° C. Fifty uL of plasma

wass added to 4 mg of lyophilized sulphosalicylic-acid, mixed, frozen in liquid nitrogen

andd stored at -70° C. Tissue samples were collected, flushed in ice-cold phosphate-

bufferedd saline (PBS), rapidly frozen in liquid nitrogen and stored at -70° C until

analysis. .

DeterminationDetermination of amino acid and guanidino compound concentrations. Jejunum

andd plasma amino acids were determined by fully automated HPLC as described

118 8


(vann Eijk et al., 1993). Norvaline was used as an internal standard. For analysis of

guanidinoo compounds, plasma was deproteinized with an equal volume of 20 %

trichloroaceticc acid, followed by centrifugation at 16,000 x g at 4 °C. The

supernatantt was diluted and injected into an LC5001 amino acid analyzer

(Biotronic,, Maital, Germany), adapted for guanidino compound determination. For

guanidinoo compound determination in tissue, approximately 40 mg tissue was

homogenizedd (Tissue Tearor®, model 985-370 type 2, Biospec Products,

Bartesville,, USA) in 350 ul of ice-cold water. Thirty ul was taken for urea

determination.. The tissue homogenizer was washed immediately with 350 ul 30 %

trichloroaceticc acid, the wash fluid was added to the first pool and the total was

vortexed.. The acetic protein complexes were precipitated by centrifugation at

208000 x g at 4 °C. Diluted supernatant was injected. Guanidino compounds were

separatedd on a cation-exchange column using sodium citrate buffers and detected

withh the ninhydrin fluorescence method (Marescau et al., 1986). Urea nitrogen

wass determined with diacetylmonoxime (Ceriotti, 1971). Blood ammonia

concentrationn was determined using the using the ammonia test kit (Menarini

Diagnostics,, Florence, Italy), according to the manufacturer's instructions.

BehavioralBehavioral tests. Mice of 3 months of age were used. Each animal was put in a

separatee cage (16x22 cm2) between 3 infrared photobeams connected to a

microprocessorr counter. Cage-activity was recorded between 16:00 p.m. and 8:00

a.m.,, and expressed as total number of beam crossings during the recording period.

Openn field activity was recorded in animals on a reversed dark-light schedule

duringg the dark phase of the cycle, and their movements in a brightly lit area were

trackedd for 10 minutes using a computerized video tracking system (San Diego

Instruments,, USA). Exploratory activity was additionally assessed in a dark/light

transitionn box consisting of a large illuminated (45x75 cm2) and a smaller (10x75

cm2),, dark compartment. A dividing wall allowed transition between compartments

throughh four evenly spaced 4-cm holes. Animals on reversed dark-light cycle were

119 9

ChapterChapter IV

placedd in the box for 10 min (starting from the dark compartment) during the dark

phasee of the light cycle, and exploration of the illuminated area was registered

usingg two photobeams (at 1 and 7 cm distance to the dividing wall) and a

microprocessor-basedd counter.

Forr the wire suspension test, animals were put with their front paws on a

tautt steel wire (0.6 mm diameter), 46 cm above tabletop, and were to remain

suspendedd for 120 s using their front paws only. Latency of the first slip and

numberr of slips within the 120-sec test period were recorded. In the rotarod test,

animalss received a 1-min training trial at 4 rpm followed by four testing trials at

10-minn intervals on an accelerating rotarod (Ugo Basile, Varese, Italy). Each

testingg trial consisted of a 5-min session during which the rod accelerated linearly

fromm 4 to 40 rpm. The time the animals were able to stay on the rod was recorded

automatically.. Finally, gait characteristics were recorded using a runway apparatus.

Withh their hind paws wetted with ink, the animals walked on a strip of paper down a

brightlyy lit corridor (40 cm long, 4.5 cm wide) towards a dark goal box.

Recordingss were made in duplicate, and maximal distances between prints of left

andd right paws were measured from the tracks.

Passivee avoidance learning was tested in a two-compartment step-through

box.. Animals on reversed dark-light cycle were put in the small (5x9 cm2) brightly

litt compartment of the box. After 5 sec, the sliding door was opened, leading to

thee larger (20x30 cm2), dark compartment. Upon entrance into the dark

compartment,, the door was closed and animals received a slight electric foot

shockk (0.3 mA, 1 sec). Twenty-four hrs later, the procedure was repeated, and

step-throughh latency was recorded up to 300 sec.

StatisticalStatistical analyses. Biochemical data on amino acids and guanidino compounds

weree tested with a repeated measure analysis of variance (ANOVA) per compound

group.. Because of significant interactions between compound, genotype and age, a

two-wayy ANOVA (amino acids; factors age and genotype) or a one-way ANOVA

(guanidinoo compounds; factor genotype) was carried out per compound. In case of

120 0


aa significant effect of genotype or genotype-age interaction in these ANOVAs,

Dunnett'ss multiple comparison test between genotypes was applied. Results were

consideredd significantly different if pO.01.

Forr the amino acids with a significant age effect, a mean amino acid level

wass calculated by removing the multiplicative variation between amino acids (J.M.

Ruijterr et al, submitted). To this end, the correction factors for removal of

variationn were deduced from a matrix of between amino acid ratios. The mean

aminoo acid level of adult wild type mice was set at 100. Thus, in the resulting mean

aminoo acid all included amino acids carry equal weight.

121 1

ChapterChapter IV

Results s

LevelsLevels of circulating amino acids in wild-type and transgenic mice during

postnatalpostnatal development.

F/A-ll and F/A-2 are transgenic mouse lines that were genetically modified to

expresss hepatic arginase (A-I) in their enterocytes in order to annul their capacity

too synthesize arginine (De Jonge et al, submitted). The level of hepatic arginase in

thee enterocytes of transgenic line F/A-l is 50% of that of line F/A-2.

plasma a

Tablee 1: statistical analysis (two-way ANOVA)) of changes in amino acid levelss in WT, F/A-l and F/A-2 postnatall and adult mice.Significant effectss (pO.01) of genotype (G), age (A),, or genotype-age interaction (I) are givenn in each column. * Indicates genotypee effects which depend solely onn age group 5-10 weeks.

ala a ars s Clt t glu u gly y met t orn n ser r tau u thr r gin n his s lys s trp p asn n il e e leu u

#

val l

genotype e _ _

G G _ _ _ _ G G _ _ G* * G* * _ _ G* * _ _ _ _ _ _ G G ----_ _ G* * --

age e

A A ----

A A --

A A A A A A A A A A _ _

A A A A A A A A A A _ _

A A A A

genotype-age e interaction n

_ _ ----_ _ I I --I I ----_ _ _ _ _ _ ------------

--

Too evaluate whether the apparent normalization of body and fur growth after

weaningg was associated with the normalization of circulating arginine levels,

circulatingg amino acid concentrations, including arginine, were determined as a

functionn of age. In suckling wild-type mice, the concentration of plasma arginine

iss 250 uM and gradually declines to 150-170 uM in adult male and female animals

(Fig.. 2A). In homozygous suckling and weanling F/A-l mice, plasma arginine is

reducedd to 80-125 uM, whereas in homozygous F/A-2 suckling and weanling

animals,, plasma arginine averages only 70 uM.

122 2


30 0 AA arginine (|xM)

WTT F/A-l F/A-2 20 0

10 0

BB ornithine (|iM)

WTT F/A-l F/A-2

0" "

CC glycine (pM) WTT F/A-l F/A-2

12 2

— ii 1 1 — i i 1 1 — i 1 i 1 i 1 f

11 3 7 ad weeks s

DD tryptophan (jxM) WTT F/A-l F/A-2

122 -

88

44

11 3 7 ad weeks s

££ citrulline (|iM) -WTT F/A-l F/A-21

JJ 1 3 7 ad

weeks s

Figuree 2: Plasma amino acids as a function of age. Thee plasma concentration (uM) of arginine (panel A), ornithine (panel B), glycine (panel C), tryptophan (panell D), citrulline (panel E), and the mean of all other amino acids that decline with age (panel F) in wild-type,, homozygous F/A-l and F/A-2 mice in the preweaning period (1-2 weeks of age), in the periweaning periodd (3-4 weeks), in the adolescent period (5-10 weeks), and in the adult period (>10 weeks). Arginine is significantlyy decreased, whereas glycine, tryptophan, and ornithine plasma concentrations are increased inn the F/A transgenics. Note the higher amino acid levels in the adolescent period of F/A-2 mice and the

123 3

ChapterChapter IV

higherr arginine levels in adult female (open symbols) compared to male (closed symbols) F/A transgenic mice.. Panel F represents the means of plasma concentrations of the amino acids that significantly decline withh age (asparagine, glutamate, histidine, lysine, ornithine, serine, isoleucine, methionine, valine, threoninee and tyrosine). Since the concentrations were corrected for multiplicative variation (see materials andd methods), all amino acids weigh equally in this mean. No difference between sexes was found in sucklingg and adolescent mice. Asterisks indicate significant differences between male and female (P < 0.01). .

Noo sex differences were found in suckling and weanling animals. In adult female

F/A-ll and -2 mice, arginine concentration increases to 130-150 uM, whereas in

adultt transgenic males, it remains depressed (P < 0.01) at 110 and 80 uM in F/A-

11 and F/A-2 mice, respectively (Fig. 2A).

Inn the preweaning period, arginine deficiency is associated with an

increasedd concentration of glycine and ornithine (De Jonge et al, submitted).

Tablee 1 shows the effects of genotype and age, as well as the interaction between

genotypee and age on circulating amino acid concentrations in wild-type, F/A-l and

F/A-22 mice from birth to adulthood. As shown in Fig. 2A, the decline in circulating

argininee concentration depends strongly on genetic background (p<0.0005). In

additionn to ornithine (Fig. 2B) and glycine (Fig. 2C), circulating tryptophan (Fig.

2D),, serine, threonine and phenylalanine concentrations are increased in

transgenics.. The significance of the genotype-related difference in ornithine,

serine,, threonine and phenylalanine (Table I) depends solely on the increase in

plasmaa amino-acid concentration in 5-10 weeks-old F/A-2 mice, as exclusion of

thiss age group from the ANOVA abolishes significant differences between wild-

type,, F/A-l, and F/A-2 mice (Table I, genotype effects indicated by asterisks).

Sincee the increased plasma concentration in 5-10 weeks-old F/A-2 mice is not

limitedd to the amino acids mentioned (Fig. 2F and next paragraph), we felt justified

too consider only tryptophan levels as being affected by the F/A genotype.

Thee Table further shows a significant age effect in the plasma levels of

alanine,, glutamate, methionine, ornithine, serine, taurine, threonine, histidine,

lysine,, tryptophan, asparagine, isoleucine, tyrosine, and valine, which is a decline

withh age in all mice (Fig. 2F), whereas the levels of citrulline (Fig. 2E), glutamine,

andd leucine do not change significantly in concentration during postnatal

124 4


developmentt (Table I). In wild-type mice, arginine, glycine and ornithine also

exhibitt an age-dependent decline, indicating that their circulating level is

determinedd by both genotype and age. Fig. 2F shows the age-dependent decline of

thee mean concentration of the amino acids with a significant age effect. Two

featuress are remarkable. First, the circulating concentration of these amino acids

iss significantly higher in fully-grown, adult transgenic females than in males, but

nott in wild-type control animals, showing that this gender difference is caused by

thee genotype, i.e. is the result of low circulating arginine concentrations earlier in

life.. Second, the circulating concentration of these amino acids is significantly

higherr in adolescent (5-10 weeks old) F/A-2 transgenic mice than in either wild-

typee or F/A-l transgenic mice. The relatively high level of amino acids in

adolescentt F/A-2 mice coincides with the phase of catch-up growth from the

growthh retardation that they incur in the first 3 weeks of life (De Jonge et al,

submitted). .

GuanidinoGuanidino compounds under arginine deficiency

Arginine,Arginine, homoarginine and urea (Fig.SA)

Argininee concentration of transgenic mice was significantly decreased in all

organss analyzed. The effect was most pronounced in muscle, in which arginine

levelss decreased approx. 8-fold, and least in liver. No differences in urinary

argininee concentration were observed. Despite the 2-fold difference in intestinal

arginasee expression and an associated aggravation of the phenotype (De Jonge et

alaltt submitted), no striking differences in tissue and plasma arginine concentration

betweenn lines F/A-l and F/A-2 were observed in this series of assays (cf. Fig. 2 A).

Argininee deficiency was accompanied by an even more pronounced decrease in the

argininee homologue homoarginine. Unlike arginine, the decreased homoarginine

levelss resulted in a decline in urinary excretion. Furthermore, homoarginine levels

inn most organs were more affected in line F/A-2 than F/A-l. Significant increases

inn tissue urea concentration were only found in muscle and jejunum, that is, the

125 5

ChapterChapter IV

organn with the most pronounced drop in arginine concentration (muscle) and the

organn with transgenic arginase activity (jejunum). Although urea levels tended to

bee higher in F/A-2 than F/A-l and WT mice, plasma and urinary urea was not

significantlyy increased in the transgenic animals.

Arg Arg

|jMM or brain umol/kgg 20

(mMM 20 mmol/kg) )

brain n muscle e liver r kidney y je j j urine e inj/10 0

plasmaa plasma urine inj j

Figuree 3A: Tissue, plasma and urinar y concentration of guanidino compounds. Concentrationn of guanidino compounds in wild-type (black bars), F/A-l (gray bars), and F/A-2 (white bars)) mice at ND 10. Panel A: arginine (Arg), homoarginine (Harg) and urea. Tissue concentrations are givenn in umol/kg, plasma and urinary concentrations in uM, except for urea concentrations, which are in mmol/kgg or mM. <DL is below detection limit. Values are means SEM, with n = 6-21. Asterisks indicate significantt difference (p<0.05) from wild-type means using a one-way ANOVA.

126 6

BiochemicalBiochemical implcations of arginine deficiency

80 0

40 0

uMM or umoll / kg 2

brainn muscle liver kidney jej plasma plasma urine/10 urine injj inj/10

3-GPA A

II * * <DLL .In.fcj - <DLL <DL

brainn muscle liver kidney jej

Y-GBA A

plasmaa plasma urine urine injj inj

<DL L

brainn muscle liver kidney jej plasma plasma urine urine injj inj

Figuree 3B: Tissue, plasma and urinar y concentration of guanidino compounds. panell B: the transamidination products, guanidinoacetic acid (GAA), P-guanidinopropionic acid (p-GPA), andd a-guanidinobutyric acid (y-GBA).

OtherOther products of arginine metabolism (Fig. 3B and C).

Argininee deficiency causes significant decreases of the transamidination products

GAA,, P-GPA, and y-GBA in most tissues (Fig. 3B). However, plasma GAA level

wass not affected. Tissue and urinary concentrations exceed plasma concentrations,

indicatingg active transport.

127 7

ChapterChapter IV

400 0

200 0

0 0

brainn muscle liver kidney

CTNN T

jejj plasma plasma urine urine injj inj

Li i <DLL <DL <DL

(jiMM or brain muscle liver kidney umol/kg g

20 0 a-K-8-GVA A

10 0

jejj plasma plasma urine/10 urine injj inj/10



Figuree 3C: Tissue, plasma and urinar y concentration of guanidino compounds. panell C: the GAA products creatine (CT) and creatinine (CTN), the transamination product a-amino,8-guanidinovalericc acid (a-K-8-GVA) and its hydrogenation product argininic acid (ArgA).

128 8

BiochemicalBiochemical implcations of arginine deficiency

GSA A

* *

I I * *

* * -fc c

* *

brainn muscle

MG G

<DLL <DL

liver r

<DL L

kidney y

<DL L

jej j

<DL L

plasmaa f

.. <DL ,.

I I

Jm m (uMM or brain muscle liver kidney jej umol/kg g

4 4

plasmaa plasma urine urine injj inj

<DLL <DL <DL <DL üüt. . 15 5


(X-NAA A

-- In —ii <DL U I

<DLL <DL <DL

brainn muscle liver kidney jej plasma plasma urine urine injj inj/10

Figuree 3D: Tissue, plasma and urinar y concentration of guanidino compounds. panell D: guanidinosuccinic acid (GSA), methylguanidine (MG) and guanidine (G), and the transacetylationn product a-N-acetylarginine (a-NAA).

129 9

ChapterChapter IV

Thee changes in transaminidation products are similar in F/A-l and F/A-2 mice.

Tissuee creatine (CT) concentration in hypoargininemic animals follows the

changess in GAA concentration, though in contrast to GAA, plasma and urinary CT

levelss are decreased in the transgenic animals (Fig. 3C). No effect of arginine

deficiencyy was observed for the CT product CTN. The transamination product a-

K-8-GVAA and its hydrogenation product ArgA are decreased in tissues and plasma,

butt not in urine (Fig. 3C). Generally, the decreases in a-K-8-GVA, and argA were

equall in F/A-2 and F/A-l mice.

TheThe formation ofGSA, MG, G, anda-NAA (Fig. 3D)

GSAA levels were increased in liver, kidney, jejunum and plasma of the transgenic

mice.. Furthermore, its excretion into urine was substantially increased. MG, which

wass only detectable in urine, was increased three-fold in F/A-2 mice. G, which was

additionallyy detectable in jejunum and plasma, was increased only in the urine of

thee transgenic mice. Importantly, clear differences in the urinary excretion were

observedd between line F/A-l and F/A-2, levels being the highest in the most

affectedd line. In transgenic brain or muscle, GSA levels were unaltered, although

brainn arginine concentration is reduced to 40% and arginine in muscle to less that

15%% of that of wild-type animals. In those tissues in which the a-amino

acetylationn product a-NAA was detectable, it was decreased in both transgenic

lines,, whereas its excretion into urine was increased.

EffectEffect of arginine treatment

Inn order to demonstrate that the alterations in guanidino compounds in the

transgenicss are caused by arginine deficiency, we treated transgenic mice with

twicee daily subcutaneous injections of arginine, starting at ND5 . Mice were

sacrificedd at ND10, 6 hours after the last arginine injection. At sacrifice, plasma

argininee concentration was 560, 170 and 90 uM in wild-type, homozygous F/A-l

andd F/A-2 mice, respectively, reflecting the influence of arginase activity in the

130 0


transgenicc intestine. The difference in plasma urea, GAA, CTN, a-K-8-GVA,

ArgA,, GSA and G between F/A-2 animals on the one hand and the wild-type and

F/A-ll animals on the other, shows that urea production mirrored circulating

argininee concentration. In a few cases (CTN, a-K-S-GVA and G), the plasma

guanidinoo compound concentration in F/A-l animals was also significantly lower

thann that in wild-type animals. Similar differences were found for J3-GPA, y-GBA

andd CT, but these numbers did not reach significance. Urinary CT excretion

paralleledd the corresponding circulating compounds. The urinary data also reveal

thatt the bulk of the injected arginine is metabolized by arginase and excreted as

ureaa in both control and transgenic mice. Whereas the urinary excretion of most

compoundss increased or remained constant upon arginine supplementation, that of

GSAA decreased in the transgenic mice. In summary, the observed response of

plasmaa and urinary guanidino compound concentration to arginine supplementation

indicatess that the altered concentrations of guanidino compounds that are found in

F/AA mice, reflect the decreased availability of arginine. Because of the rapid

establishmentt of the equilibriums (< 6 hours), plasma guanidino compound

concentrationss can be interpreted as fluxes.

BehavioralBehavioral alterations in adult F/A transgenics

Cerebrall arginine concentrations are decreased to 50% and 40% in F/A-l and F/A-2

transgenics,, respectively. These decreased levels of arginine may limit cerebral NO

synthesis,, as has been shown for endothelial cells (Arnal et al., 1995) and activated

macrophagess (Norris et al., 1995), and decreased NO synthesis has been reported to

impairr long-term potentiation (Son et al., 1996). Furthermore, in both transgenic lines

thee cerebral content of creatine is decreased to 70%, while the concentration of the

purportedd neurotoxins GSA and ammonia (WT: 39 11, F/A-l 40 11, F/A-2 44 14

uM),, are not altered. In view of this potential modification of metabolism in the brain

off F/A transgenic mice, it was decided to evaluate its possible consequences for brain

function. .

131 1

ChapterChapter IV

WÜd-typee F/A-l F/A-2

CageCage activity beamm crossings

OpenOpen field activity pathh length (cm) comerr entries (cm)

WireWire suspension latencyy first slip (s) slips/1200 s

RotarodRotarod (trial 4) timee on the rod (s)

GaitGait test maximall span (cm)

PassivePassive avoidance learning step-throughh latency (s)

8,4000 700

3,9000 300 722 4.0

104 11 0.22 1

2711 14

6.44 0.2

3000 0.0

10,0000 *

4,4000 0 866 6.0

555 14* 1.00 2

2322 19

6.22 0.2

2388 32*

12,2000 1300*

5,5000 0 * 1177 *

422 12*

1622 20*

5.33 *

1866 *

Tablee 2. Behavioral assessment in wild-type and transgenic groups. Valuess are mean SEM, n=10-l 1. Asterisks indicate significant difference from wild-type using Dunnett's multiplee comparison test after significant between-genotype effect (ANOVA).

Tablee 2 shows that both F/A transgenic lines were impaired in several behavioral tests,

andd that the F/A-2 group generally deviated more from wild-type than the F/A-l group.

Cagee and open field activity were increased in the F/A transgenic mice compared to

controls.. In the open field tests, ambulatory activity measures like path length and

cornerr entries, differed significantly between the groups, whereas exploratory activity

measures,, like entries and dwell in center did not. Accordingly, transitions between the

compartmentss of the dark-light box were not significantly different between the groups.

Together,, these tests demonstrated general hyperactivity in the transgenic mice, with

thee F/A-2 line being most severely affected. This hyperactivity could not be reduced to

aa qualitative activity difference (e.g., increased exploration). Several additional, more

specificc neuromotor abilities were tested. Wire suspension (grip strength and

endurance)) and rotarod performance (equilibrium and coordination) showed significant

decreasess in F/A transgenics. Analysis of their gaits revealed that F/A mice walked with

shorterr paces than controls. Finally, passive avoidance learning was impaired since the

132 2


step-throughh latency was decreased in F/A-l and F/A-2 transgenic mice compared to

controls. .

133 3

ChapterChapter IV

Discussion n

ArginineArginine deficiency and amino acids

Endogenouss arginine biosynthesis is a typical example of interorgan metabolic

cooperation.. The intestine is special in that it has the unique capacity to synthesize

citrulline,, whereas many other tissues express argininosuccinate synthetase and

argininosuccinatee lyase to metabolize the citrulline originating from the intestine

orr from local NO synthesis to arginine. In the adult mammal, 60% of arginine

formationn from citrulline occurs in the kidney (Yu et al., 1996). However, the

intestinee rather than the kidney appears to play a major role in arginine metabolism

inn the suckling period (Herzfeld and Raper, 1976; Hurwitz and Kretchmer, 1986).

Accordingly,, the F/A transgenic mice, which express different levels of arginase in

theirr enterocytes, suffer from a graded reduction in circulating arginine

concentration,, in particular during the suckling period. The functional significance

off the intestine in arginine metabolism is not yet fully understood, but the degree

too which hair and muscle growth (De Jonge et al, submitted), and B-cell

maturationn (De Jonge et al, submitted) are disturbed in F/A transgenic mice, is

directlyy related to the level of arginase that accumulates in the enterocytes. We

noww report that circulating arginine levels in F/A transgenes remain low after the

capacityy to synthesize arginine in the gut ceases to exist, probably due to the

continuedd breakdown of circulating arginine by intestinal arginase. The accelerated

disappearancee of injected arginine in the transgenic mice shows that the capacity

too catabolize circulating arginine already exists in suckling F/A mice. For this

reason,, we do not know at present to what extent overexpression of arginase in the

enterocytess depresses circulating arginine by interfering with local biosynthesis

orr by increasing catabolism of circulating arginine. We also do not know why

circulatingg arginine levels are lower in male than in female transgenic mice.

Argininee belongs to the group of amino acids, of which the circulating

concentrationn declines concurrent with the declining growth rate in the course of

postnatall development. The expression of arginase in enterocytes is sufficient to

completelyy abolish the high circulating arginine levels in the suckling period,

134 4


suggestingg that the observed phenotype was a direct consequence of arginine

deficiency.. The mechanism by which low arginine concentrations prevent hair and

musclee growth, remains to be solved. Because extremely arginine-rich proteins

suchh as trichohyalin, are present in normal amounts in F/A-2 mice (De Jonge et al,

submitted),, the most obvious explanation, that is the precursor function ofarginine

forr protein synthesis cannot account for the phenotype of hypoargininemia.

Argininee is also known as a secretagogue of growth-promoting hormones, such as

growthh hormone (Barbul et al., 1983) and insulin (Palmer et al., 1975), via

mechanismss dependent, as well as independent of the production of nitric oxide

fromfrom arginine (Junand Wennmalm, 1994). However, animals of the F/A-2 line

showw catch-up growth after weaning, even though the concentration of circulating

argininee remains low. The association of this delayed growth spurt with the 50%

increasedd concentration of virtually all amino acids, is probably not a coincidence.

Together,, these data suggest that the ratio between arginine and the other members

off the age-dependent group of amino acids determines whether or not growth is

inhibited.. This hypothesis is supported by the difference in growth behavior of

F/A-l,, which grow normally, and F/A-2 mice, which are severely affected, even

thoughh the additional decrease of arginine levels is modest. We are presently

followingg these and other leads.

Anotherr intriguing observation is the rise in circulating arginine levels in

adultt F/A females, but not in males. The effect is more pronounced in the F/A-2

thann in the F/A-l line. Since the intestinal arginase expression is similar in both

sexess and since arginine biosynthesis by e.g. the kidneys can be adaptively

increasedd (Prins et al., 1999), this finding suggests that females are able to mount

aa more effective adaptive increase in arginine biosynthesis than males.

Alternatively,, the requirements for arginine may be higher in males than in

females,, e.g. for creatine synthesis, because creatine accumulates to very high

levelss in testis and muscle ((Moore, 2000); see also next paragraph). Since this

genderr difference is also observed for the other age-dependent amino acids in the

135 5

ChapterChapter IV

F/AA transgenics, the cause may also be more general, e.g. a lower rate of amino

acidd catabolism in female arginine-deficient animals. Irrespective, this gender

differencee may well be of clinical importance, since a common disease such as

atherosclerosis,, has been hypothesized to represent an arginine-deficiency disease

(Cooke,, 1998) and since males are more prone to develop atherosclerosis than

femaless (Clarkson et al., 1985). In view of the atheroprotective effect of estrogens

(Elhagee et al., 2000), future studies should also address the interaction between

estrogenss and arginine metabolism.

Thee instant hydrolysis of newly synthesized and circulating arginine by

transgenicc arginase in the enterocytes of suckling F/A mice leads to a drop of

circulatingg and tissue arginine levels. As described in Chapter III, arginine

deficiencyy was associated with an increase in the levels of ornithine and glycine,

whenn only the preweaning period is considered. The inclusion of the postweaning

andd adult mice in the current analyses reveals genotype-dependent increases in the

levelss of ornithine, glycine, serine, tryptophan, threonine and phenylalanine. The

40%% increase in circulating glycine may well be due to the diminished synthesis of

creatine.. Creatine plays an essential role in the energy metabolism of muscle,

nervee and testis (Walker, 1979), and accounts for a sizable portion of arginine

catabolismm (Visek, 1986). Moreover, GAA levels in brain, liver and kidney reach

maximumm levels in the suckling period (Watanabe et al., 1985), underscoring the

importancee of arginine metabolism for CT synthesis in this period. The diminished

synthesiss of creatine is due to the decreased concentration of available arginine

andd not to feedback inhibition of the transaminidase reaction by creatine or

ornithine.. Conceivably, ornithine concentration could be increased as a result of

thee increased flux through arginase, but increased ornithine levels were only

observedd in intestine, where creatine levels are not altered and transaminidase

activityy is virtually absent (McGuire et al., 1986). The genotype-related increases

inn serine, threonine and phenylalanine were found to be solely due to their

increasedd level at 5-10 weeks concentrations in F/A-2 animals. Since all amino

acidss except arginine were elevated at this time (Fig 2F), we do not attach too

136 6


muchh importance to it. At present, we have no explanation for the increase in

circulatingg tryptophan. Interestingly though, a similar increase in tryptophan was

foundd in brain of OTC deficient .sp/mice (Bachmann and Colombo, 1984).

Thee absence of an increased urea concentration in tissue, plasma or urine

iss at first glance surprising in view of the overexpression of arginase in the

enterocytess and the ensuing consumption of body arginine content. However, as

alsoo shown by the extremely low urinary urea content, amino acid catabolism in

thee suckling period is very low (Blommaart et al., 1993). Hence, dietary and

endogenouslyy synthesized arginine largely ends up in protein or creatine. Loss via

thee creatine/creatinine pathway virtually stops in the F/A mice. Arginine content in

rodentt protein is 3.5-4% (Davis et al., 1993), so that arginine degradation in the

intestinee only represents a tiny fraction of the total substrate for urea synthesis.

ArginineArginine deficiency and guanidino compounds

Thee decreased arginine availability in F/A mice decreased the concentration of

guanidinoo compounds that form via the transaminidation pathway, but caused an

accumulationn of GSA, MG and G. Strictly speaking, our data do not allow

conclusionss about fluxes. However, we observed that 6 hours after an arginine

injection,, the concentration ofarginine and the arginine metabolites urea, GAA, p-

GPA,, Y-GBA, CTN, cc-K-8-GVA, ArgA, GSA, and G were still substantially

increasedd in plasma of wild-type mice, but were similar to the corresponding

controll animals in F/A-2 mice, with animals from the F/A-l line taking an

intermediatee position. The plasma levels of these compounds therefore

correspondd with the actual availability ofarginine instead of with the administered

argininee load, in other words, are a parameter for flux. The exception is

homoarginine,, which is not a metabolite of arginine. This conclusion implies that

thee decline in arginine concentration, and not the accumulation of transaminidation

productss (McGuire et al., 1984; Sipila, 1980), determines the decrease in flux

throughh transamidinase.

137 7

ChapterChapter IV

Thee increase in tissue, plasma and urinary GSA levels and urinary G levels

correspondss with the increased severity of arginine deficiency. The higher

concentrationn of GSA and G in the urine of transgenic mice upon arginine loading

confirmss that the formation of these compounds is enhanced under arginine-

deficientt conditions. Increased a-NAA, GSA, MG and G levels are also observed

inn renal failure (Cohen et al., 1968; De Deyn et al., 1986; Marescau et al., 1997),

subtotall nephrectomy (Al Banchaabouchi et al., 1998; Levillain et al., 1995),

endotoxinn treatment (Deshmukh et al, 1997), and after consumption of an

arginine-freee diet by strict carnivores (Deshmukh and Rusk, 1989). As in the

diseasedd states mentioned, the contribution of GSA and/or MG and G to the

developmentt of the arginine-deficient phenotype remains to be established.

Itt has been proposed that GSA is a transamidination product of arginine, when

aspartate,, instead of glycine, is the amidine donor (Cohen et al., 1968). However,

sincee the flux through transaminidase is slowed down due to arginine deficiency in

F/AA mice, this explanation is not favored. GSA concentrations positively correlate

withh plasma urea (Marescau et al., 1992a). Urea, in turn, inhibits argininosuccinate

lyasee (Menyhart and Grof, 1977), suggesting that argininosuccinate may

accumulatee locally. The hypothesis that GSA, as well as MG, form upon

interactionn of argininosuccinate and creatine with free radicals (Aoyagi et al.,

1996)) is therefore more attractive, the more since it has been shown that free

radicalss form in the neonatal intestine (Musemeche et al., 1993), and probably

elsewheree under catabolic conditions.

ArginineArginine deficiency and behavioral deficits

F/AA transgenic mice displayed hyperactivity as well as several more specific

deficitss in coordinated neuromotor abilities. Since passive avoidance learning was

alsoo impaired in these animals, although the motor requirements of this task are

minimal,, these deficits are probably not caused by muscular problems. Behavioral

deficits,, comparable with the ones seen in the F/A mice, have been associated with

increasedd levels of urea and CTN in nephrectomized mice (Al Banchaabouchi et

138 8


al.,, 1999). Uremic toxins, in particular GSA, affect cerebral neurotransmitter

systemss and are thought to cause the psychomotor deficits seen in uremic

encephalopathyy (D'Hooge et al., 1996). Behavioral alterations, similar to those

seenn in F/A mice, were found in 5p/-mutant mice (Batshaw et al., 1995). Unlike

F/AA mice, these mice suffer from increased ammonia levels. However, brain

ammonia,, urea, CTN and GSA concentrations are unchanged in F/A mice, virtually

excludingg these factors as causes of their neuromotor deficits. Similarly, GSA

levelss were not elevated in brain of spf mice (Batshaw et al., 1995). However, we

cannott presently exclude that the slightly elevated circulating levels of tryptophan,

aa precursor for cerebral production of serotonin and quinolinate, a known

excitotoxinn (Batshaw et al., 1993), are responsible for the observed neuromotor

deficitss in F/A mice. Likewise, elevated brain tryptophan levels in spf mice

(Bachmannn and Colombo, 1984) have been associated with a two-fold increase in

brainn quinolinic acid, neuropathology (Robinson et al, 1995), and impaired

passivee avoidance (Batshaw et al., 1995).

Ass arginine is required for NO synthesis in neurons, the low level of

circulatingg arginine in the F/A mice may be limiting for NO synthesis. Limitation

off cerebral NO synthesis has been shown in spf mice, which also suffer from

argininee deficiency (Ratnakumari et al., 1996). A hampered NO synthesis impairs

synapticc plasticity, motor coordination and memory functions (O'Dell et al., 1994;

Sonn et al., 1996). Neuronal NOS-deficient mice show behavioral alterations, but

unlikee F/A transgenics, wire suspension, open field activity, pole equilibrium and

severall other neuromotor tests were not altered in these animals (Nelson et al.,

1995).. Furthermore, mice lacking nNOS, especially males, were reported to be

extremelyy aggressive, but we did not observe any signs of this aggressive behavior

inn F/A mice.

139 9

ChapterChapter IV

Conclusion Conclusion

F/AA transgenic mice suffer from a life-long deficiency in circulating arginine and

lastingg behavioral deficits. Despite a low circulating arginine level, F/A-2

transgenicss show a catch-up growth after weaning, which is temporally related to

increasedd circulating levels of all other amino acids. Arginine levels in adult F/A

femaless are higher than in adult F/A males, possible as a result of the extra

requirementt for CT synthesis in muscle and testis. Low levels of arginine lead to a

decreasee in the flux through transaminidase and accumulation of GSA and G,

probablyy as a result of a higher oxidative stress under hypoargininemic conditions.

WdJJ and MH were supported by a grant from the Dutch Foundation for Scientific Research (NWO, grant numberr 902-23-028); BM and PDD were funded by the Fund for Scientific Research Belgium (FWO, grant numberr 6.00.27.97); RD is a postdoctoral FWO fellow.

140 0


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