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Department of Anatomy & Embryology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands;
*
Department of Neurology, Laboratory of Neurochemistry and Behavior, Born-Bunge Foundation, University of Antwerp (UIA), Antwerp, Belgium; and
Department of Surgery, Maastricht University, The Netherlands
2To whom correspondence should be addressed. E-mail: W.H.Lamers{at}AMC.UVA.NL.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • hypoargininemia • transgenes • intestine • guanidinosuccinic acid • methylguanidine • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Arginine is not considered to be an
essential amino acid for adults on the basis of nitrogen balance. In
adults, a major site of arginine biosynthesis from citrulline is the
renal proximal convoluted tubule (2
,3)
. Citrulline, in
turn, is synthesized in the small intestine from glutamine and proline
(4
,5)
. The endogenous arginine biosynthetic capacity
amounts to only 
20% of daily expenditure (6)
. Hence,
its bioavailability may become insufficient under conditions of
increased demand, such as growth (6)
and tissue repair
(7)
, or as a result of decreased dietary supply
(8)
. For this reason, arginine is regarded as a
conditionally essential amino acid.
In rapidly growing suckling neonates, endogenous arginine biosynthesis
is crucial to compensate for the insufficient supply of arginine via
the milk (9
,10)
. Evidence is accumulating that the
intestine rather than the kidney plays a major role in arginine
biosynthesis in the suckling period. Thus, the enterocytes of the small
intestine express the enzymes required for arginine production from
glutamine and proline (5)
and do not express arginase
(11)
. Furthermore, we showed recently that overexpression
of hepatic arginase in enterocytes induces arginine deficiency
(12)
. The intestine appears to play a similar role in
human neonates as well because destruction of the enterocytes in
necrotizing enterocolitis also results in a selective decrease in
circulating arginine (13)
. In mice and rats,
argininosuccinate synthetase
(ASS)3
and argininosuccinate lyase (ASL), the enzymes that synthesize arginine
from citrulline, disappear from the enterocytes in the postweaning
period, concurrent with the appearance of endogenous arginase
(11)
, so that the role of the gut becomes confined to
citrulline biosynthesis.
The reduction in circulating arginine concentration in transgenic mice
that express different levels of arginase I in their enterocytes
("F/A" transgenes) depends on the expression level of transgenic
arginase. The degree of arginine deficiency correlates, in turn, with
the degree of retardation of hair and muscle growth, and the
development of the lymphoid tissue (12)
. Expression of
arginase in all enterocytes is necessary to elicit arginine deficiency.
Phenotypic abnormalities are reversed by daily injections of arginine,
but not creatine. Because the phenotypic symptoms largely disappear
after weaning, the question arose whether circulating levels of
arginine in relation to the other important amino acids are normalized
in our transgenic mice when intestinal arginine biosynthesis from
citrulline ceases after weaning.
We also investigated the accumulation of guanidino compounds, the
metabolites of arginine that retain the guanidinium group. A schematic
representation of the relation of arginine with the ornithine cycle and
its guanidino metabolites is given in Figure 1
. Homoarginine (Harg), lysine and homocitrulline (not shown) are
homologues of arginine, ornithine and citrulline, respectively, and as
such are alternative intermediates of the ornithine cycle. A major
metabolic route of the guanidino group of arginine is transamidination
to glycine to yield guanidinoacetic acid (GAA), and subsequently
creatine (CT) and creatinine (CTN). In addition to the physiologic
substrate glycine (
-aminoacetic acid), its homologues, ß-alanine
(ß-aminoproprionic acid), 
-aminobutyric acid (GABA) and
-aminovaleric acid, can function as substrates of the enzyme
L-arginine-glycine transamidinase (AGAT), yielding
ß-guanidinopropionic acid (ß-GPA), -guanidinobutyric acid
(-GBA) and -guanidinovaleric acid (-GVA), respectively
(14
–16)
. Furthermore, the -amino group of arginine can
be transaminated and acetylated, yielding -keto--guanidinovaleric
acid (-K--GVA) and -N-acetylarginine (-NAA),
respectively. Hydrogenation of -K--GVA produces argininic
acid (ArgA) (17)
. Methylguanidine (MG) forms after the
reaction of an oxygen radical with CTN (18)
. Similarly,
guanidinosuccinic acid (GSA) may be the product of the reaction of
argininosuccinic acid and the action of an oxygen radical species
(19)
. Some guanidino compounds, in particular -NAA, GSA
and MG, are toxic and play a role in the pathology of renal
(20)
and liver (21)
failure, in particular
the metabolic (22)
and neurological (23
–25)
consequences of these diseases. The neurotoxicity of guanidino
compounds appears to be more pronounced in growing than in adult
animals (26
,27)
. The concentration of guanidino compounds
was therefore determined in plasma and several tissues of 10-d-old
homozygous arginine-deficient transgenic mice, as well as in
wild-type controls. At this age, intestinal biosynthetic capacity
for arginine is maximal (11)
and the hypoargininemic
phenotype of the transgenic mice most pronounced (12)
. The
relation between circulating arginine concentration and the formation
of guanidino compounds was investigated by arginine supplementation.
Because of the neurotoxic potential of some guanidino compounds such as
GSA and MG, and the possible long-term consequences of alterations
of GABA and glycine levels on neurotransmission, we also determined the
consequences of arginine deficiency on neuromotor development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Transgenic mice of the FVB-strain were bred at the local animal facility. Mice were kept under environmentally controlled conditions (lights on at 0800 h, off at 2000h;water and food were consumed ad libitum;20–22°C,55% humidity). Diets consisted of the following: crude protein, 24.8%; crude fat, 6.6%; crude fiber, 3.6%; and minerals, 4.5% (rodent AM-II diet, Hope Farms, Woerden, The Netherlands). Animal experiments were done in accordance with the guidelines of the local Animal Research Committee. Litters discovered in the morning were assigned neonatal day (ND) 0. The mice were weaned at 3 wk of age. For arginine injections, nest size was adjusted to 7 pups. Pups received a subcutaneous injection of 5 mmol/kg of arginine-HCl (150 mmol/L) twice daily (0900 h and 1800 h) from ND 5 onward. Mice were killed at ND 10, 6 h after the last arginine injection. Controls were injected with saline (150 mmol/L NaCl). For the neuromotor activity tests, 3- to 5-mo-old mice were used.
Generation of transgenic mice.
Intestinal fatty acid binding protein (FABPi)/arginase I transgenic
mice (F/A) were generated in the FVB-strain. Generation of the
transgenic mice is described in detail elsewhere (12)
.
Briefly, hepatic arginase I (A-I) was specifically expressed in
enterocytes by coupling the A-I structural gene to the FABPi
promoter/enhancer element. Two lines, F/A-1 and F/A-2, expressed
A-I in all enterocytes. The level of A-I in the enterocytes of
transgenic line F/A-1 is 50% of that of line F/A-2.
Tissue and blood sampling.
Pups were separated from their dams and kept at 37°C for 1 h before killing. After decapitation, blood was collected into heparin-containing tubes and centrifuged at 2000 x g for 5 min at 4°C. Plasma (50 µL) was added to 4 mg of lyophilized sulfosalicylic acid, mixed, frozen in liquid nitrogen and stored at -70°C. Tissue samples were collected, flushed in ice-cold PBS, rapidly frozen in liquid nitrogen and stored at -70°C until analysis.
Determination of amino acid and guanidino compound concentrations.
Jejunum and plasma amino acids were determined by fully automated HPLC
as described (28)
. Norvaline was used as an internal
standard. For analysis of guanidino compounds, plasma was deproteinized
with an equal volume of 20% trichloroacetic acid, followed by
centrifugation at 16,000 x g at 4°C. The
supernatant was diluted and injected into an LC5001 amino acid analyzer
(Biotronic, Maital, Germany), adapted for guanidino compound
determination. For guanidino compound determination in tissue, 40 mg
tissue was homogenized (Tissue Tearor, model 985–370 type 2, Biospec
Products, Bartesville, OK) in 350 µL of ice-cold
water; 30 µL was taken for urea determination. The
tissue homogenizer was washed immediately with 350 µL
of 30% trichloroacetic acid. The wash fluid was added to the first
pool and the total was mixed with a vortex mixer. The acetic protein
complexes were precipitated by centrifugation at 20,800 x g at 4°C. Diluted supernatant was injected. Guanidino
compounds were separated on a cation-exchange column using sodium
citrate buffers and detected with the ninhydrin fluorescence method
(29)
. Urea nitrogen was determined with diacetylmonoxime
(30)
. Blood ammonia was determined using the Blood Ammonia
Checker (Menarini Diagnostics, Florence, Italy), according to the
manufacturer’s instructions.
Behavioral tests.
Each mouse was put in a separate cage (16 x 22 cm2) crossed by three infrared photobeams connected to a microprocessor counter. Cage activity was recorded between 1600 and 0800 h, and expressed as total number of beam crossings during the recording period. Open field activity was recorded in mice on a reversed dark-light schedule during the dark phase of the cycle, and their movements in a brightly lit area were tracked for 10 min using a computerized video tracking system (San Diego Instruments, San Diego, CA). Exploratory activity was additionally assessed in a dark/light transition box consisting of a large illuminated (45 x 75 cm2) and a smaller (10 x 75 cm2), dark compartment. A dividing wall allowed transition between compartments through four evenly spaced 4-cm holes. Mice on a reversed dark-light cycle were placed in the box for 10 min (starting from the dark compartment) during the dark phase of the light cycle, and exploration of the illuminated area was registered using two photobeams (1 and 7 cm from the dividing wall) and a microprocessor-based counter.
For the wire suspension test, mice were put with their front paws on a taut steel wire (0.6 mm diameter), 46 cm above tabletop, and were to remain suspended for 120 s using their front paws only. Latency of the first slip and number of slips within the 120-s test period were recorded. In the rotarod test, mice received a 1-min training trial at 4 rpm followed by four testing trials at 10-min intervals on an accelerating rotarod (Ugo Basile, Varese, Italy). Each testing trial consisted of a 5-min session during which the rod accelerated linearly from 4 to 40 rpm The time the mice were able to stay on the rod was recorded automatically. Finally, gait characteristics were recorded using a runway apparatus. With their hind paws wetted with ink, the mice walked on a strip of paper down a brightly lit corridor (40 cm long, 4.5 cm wide) toward a dark goal box. Recordings were made in duplicate, and maximal distances between prints of left and right paws were measured from the tracks.
Passive avoidance learning was tested in a two-compartment step-through box. Mice on a reversed dark-light cycle were put in the small (5 x 9 cm2) brightly lit compartment of the box. After 5 s, the sliding door was opened, leading to the larger (20 x 30 cm2), dark compartment. Upon entrance into the dark compartment, the door was closed and mice received a slight electric foot shock (0.3 mA, 1 s). Twenty-four hours later, the procedure was repeated, and step-through latency was recorded up to 300 s.
Statistical analyses.
Biochemical data on amino acids and guanidino compounds were tested with a repeated-measures ANOVA, with guanidino compounds and amino acids as repeated factor, and genotype and age as grouping factors. Because of significant interactions among compound, genotype and age, a two-way ANOVA (amino acids; factors age and genotype) or a one-way ANOVA (guanidino compounds; factor genotype, and behavioral data, factor genotype) was carried out per compound. In case of a significant effect of genotype or genotype-age interaction in these ANOVA, Dunnett’s multiple comparison test between genotypes was applied. Significant differences between male and female amino acid levels, as well as behavioral data, were determined using Dunnett’s multiple comparison test after one-way ANOVA. Except for the analyses of guanidino compounds (P < 0.05), differences were considered significant if P < 0.01.
For the amino acids with a significant age effect, a mean amino acid level was calculated by removing the multiplicative variation between amino acids. To this end, the correction factors for removal of variation were deduced from a matrix of between amino acid ratios. The mean amino acid level of adult wild-type mice was set at 100. Thus, in the resulting mean amino acid, all induced amino acids carry equal weight.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In wild-type and transgenic mice, circulating amino acid
concentrations, excluding proline and cysteine, were determined as a
function of age. In suckling wild-type mice, the concentration of
plasma arginine was 250 µmol/L and gradually declined to
150–170 µmol/L in adult males and females (Fig. 2A
). In homozygous suckling and weanling F/A-1 mice, plasma arginine was
reduced to 80–125 µmol/L, whereas in homozygous F/A-2
suckling and weanling mice, plasma arginine was only 70
µmol/L. In adult female F/A-1 and -2 mice, arginine
concentration increased to 130–150 µmol/L, whereas in
adult transgenic males, it remained depressed at 110 and 80
µmol/L in F/A-1 and F/A-2 mice, respectively (Fig. 2
A; P < 0.01).
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shows the effects of genotype and age, as well as the interaction of
both variables on circulating amino acid concentration in
wild-type, F/A-1 and F/A-2 mice from birth to adulthood. As shown
in Figure 2
A, the decline in circulating arginine
concentration depended strongly on genetic background (P
< 0.0005). In addition to ornithine (Fig. 2
B) and
glycine (Fig. 2
C), circulating tryptophan (Fig. 2
D), serine, threonine and phenylalanine concentrations were
increased in transgenic mice. This genotype-related difference in
ornithine, serine, threonine and phenylalanine (Table 1)
was solely
dependent on the increase in plasma amino acid concentration in 5- to
10-wk-old F/A-2 mice because exclusion of this age group in the ANOVA
abolished the significant differences between wild-type, F/A-1 and
F/A-2 mice (Table 1)
. Because the increased plasma concentrations in 5-
to 10-wk-old F/A-2 mice were not limited to the amino acids mentioned
(Fig. 2
F and next paragraph), we considered only tryptophan
levels, in addition to arginine and glycine, to be affected by the F/A
genotype.
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, whereas the levels of citrulline (Fig. 2
E),
glutamine and leucine did not change (Table 1)
. In wild-type mice,
arginine, glycine and ornithine also exhibited age-dependent
declines, indicating that their circulating levels are determined by
both genotype and age. Two features of the age-dependent decline in
the concentration of the amino acids (Fig. 2
F) were
remarkable. First, the circulating concentrations of these amino acids
were significantly higher in fully grown, adult transgenic females than
in males, but not in wild-type control mice, showing that this
gender difference was caused by the genotype, i.e., is the result of
low circulating arginine concentrations earlier in life. Second, the
circulating concentration of these amino acids was significantly higher
in adolescent (5- to 10-wk old) F/A-2 transgenic mice than in either
wild-type or F/A-1 transgenic mice. The relatively high level of
amino acids in adolescent F/A-2 mice coincided with the phase of
catch-up growth from the growth retardation that they incurred in
the first 3 wk of life (12)
. Guanidino compounds under arginine deficiency
Arginine, Harg and urea.
Arginine concentration of transgenic mice was significantly lower than
in wild-type mice in all organs analyzed (Fig. 3A
; P < 0.05). The effect was most pronounced in muscle,
in which arginine levels were decreased to only 10–15% of
wild-type mice, and least in liver. No differences in urinary
arginine concentration were observed. Despite the difference in
intestinal arginase expression and an associated aggravation of the
phenotype (12)
, no striking differences in tissue and
plasma arginine concentration were found between lines F/A-1 and F/A-2
(cf. Fig. 2
A). Arginine deficiency in F/A-1 and -2
transgenic mice was accompanied by an even more pronounced deficiency
in the arginine homologue, Harg, compared with wild-type mice.
Unlike arginine, the decreased Harg levels resulted in a decline in
urinary excretion. Furthermore, Harg levels in most organs were more
affected in line F/A-2 than F/A-1. Significant increases in tissue urea
concentration were found only in muscle and jejunum, that is, the organ
with the most pronounced drop in arginine concentration (muscle) and
the organ with transgenic arginase activity (jejunum). Plasma
(P = 0.157) and urinary (P = 0.226)
urea levels tended to be higher in F/A-2 than F/A-1 and wild-type
mice.
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-GBA in most tissues (Fig. 3
B;
P < 0.05). However, plasma GAA level was not affected.
Tissue and urinary concentrations exceeded plasma concentrations,
indicating active transport. The changes in transaminidation products
were similar in F/A-1 and F/A-2 mice. Tissue CT concentration in
hypoargininemic mice followed the changes in GAA concentration
although, in contrast to GAA, plasma and urinary CT levels were lower
in the transgenic mice (Fig. 3
C). No effect of arginine
deficiency was observed for the CT product CTN. The transamination
product -K--GVA and its hydrogenation product, ArgA, were lower
in tissues and plasma, but not in urine (P < 0.05;
Fig. 3
C). Generally, the decreases in -K--GVA, and
ArgA were similar in F/A-2 and F/A-1 mice.
The formation of GSA, MG, guanidine (G) and -NAA.
GSA levels were increased in liver, kidney, jejunum and plasma of the
transgenic mice (P < 0.05; Fig. 3
D).
Furthermore, its excretion into urine was substantially increased
(P < 0.05). MG, which was detectable only in urine,
was increased twofold in F/A-2 mice, whereas G, which was also
detectable in jejunum and plasma, was increased only in the urine of
the transgenic mice (P < 0.05). In brain and muscle of
transgenic mice, GSA levels were unaltered, although brain arginine
concentration was reduced to 40% and that in muscle to <15% of that
of wild-type mice. In those tissues in which the -amino
acetylation product, -NAA, was detectable, it was decreased in both
transgenic lines, whereas its excretion into urine was increased
(P < 0.05).
Effect of arginine treatment
To demonstrate that the alterations in guanidino compounds in the
transgenic mice were caused by arginine deficiency, we treated
transgenic mice with arginine during the neonatal period. At the time
of killing at ND 10, plasma arginine concentration was 560, 170 and 90
µmol/L in wild-type, homozygous F/A-1 and F/A-2 mice,
respectively, reflecting the influence of arginase activity in the
transgenic intestine. The difference in plasma urea, GAA, CTN,
-K--GVA, ArgA, GSA and G between F/A-2 mice on the one hand and
the wild-type and F/A-1 mice on the other, showed that urea
production mirrored circulating arginine concentration. In a few cases
(CT, CTN, -K--GVA and G), the plasma guanidino concentration in
F/A-1 mice was also significantly lower than that in wild-type mice
(P < 0.05). Similar trends were found for ß-GPA
(P = 0.256) and -GBA (P = 0.091).
Urinary CT excretion paralleled that of the corresponding circulating
compounds. The urinary data also revealed that the bulk of the injected
arginine was metabolized by arginase and excreted as urea in both
control and transgenic mice. The urinary excretion of most compounds
increased or remained constant upon arginine supplementation, whereas
that of GSA was significantly lower in the transgenic mice (Fig. 3
D, upper panel). In summary, the observed
response of plasma and urinary guanidino compound concentration to
arginine supplementation indicated that the altered concentrations of
guanidino compounds that were found in F/A mice reflected the decreased
availability of arginine. Because of the rapid establishment of the
equilibriums (<6 h), plasma guanidino compound concentrations can be
interpreted as fluxes.
Behavioral alterations in adult F/A transgenic mice
Cerebral arginine concentrations were decreased to 50 and 40% in
F/A-1 and F/A-2 transgenic mice, respectively. Furthermore, in both
transgenic lines, the cerebral content of CT was decreased to 70%,
whereas the concentration of the purported neurotoxins GSA (Fig. 3
D), and ammonia (wild-type, 39 ± 11; F/A-1, 40
± 11; F/A-2, 44 ± 14 µmol/L plasma) were not
altered. In view of this potential modification of metabolism in the
brain of F/A transgenic mice, we decided to evaluate its possible
consequences for brain function.
Both F/A transgenic lines were impaired in several neuromotor tests and
the F/A-2 mice deviated in more tests from wild-type mice than did
the F/A-1 group (Table 2
). Cage and open field activity were increased in the F/A transgenic
mice compared with controls (P < 0.05). In the open
field tests, ambulatory activity measures such as path length and
corner entries differed significantly between F/A-2 and wild-type
mice (P < 0.05), whereas exploratory activity measures
such as entries and dwell in center did not (data not shown).
Accordingly, transitions between the compartments of the dark-light
box were not significantly different among the groups (not shown).
Together, these tests demonstrated general hyperactivity in the
transgenic mice, especially in the F/A-2 line. This hyperactivity could
not be reduced to a qualitative activity difference (e.g., increased
exploration). Several additional, more specific neuromotor abilities
were tested. Wire suspension (grip strength and endurance) and rotarod
performance (equilibrium and coordination) were significantly decreased
in F/A transgenic mice (P < 0.05). F/A mice walked
with shorter paces than controls (P < 0.05). Finally,
passive avoidance learning was impaired because the step-through
latency was less in F/A-1 and F/A-2 transgenic mice than in controls
(P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Endogenous 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 ASS and ASL to metabolize the citrulline originating
from the intestine or from local NO synthesis to arginine. In adult
mammals, 60% of arginine formation from citrulline occurs in the
kidney (31)
. However, the intestine rather than the kidney
appears to play a major role in arginine metabolism during the suckling
period (32
,33)
. Accordingly, the F/A transgenic mice,
which express different levels of arginase in their enterocytes, suffer
from a graded reduction in circulating arginine concentration, in
particular during the suckling period. The functional importance of the
intestine in arginine metabolism is not yet fully understood, but the
degree to which hair and muscle growth (12)
, and
B-cell maturation (12)
are disturbed in F/A transgenic
mice is directly related to the level of arginase that accumulates in
the enterocytes. We now report that circulating arginine levels in F/A
transgenic mice remain low after the capacity to synthesize arginine in
the gut ceases to exist, probably due to the continued breakdown of
circulating arginine by intestinal arginase. The accelerated
disappearance of injected arginine in transgenic mice shows that the
capacity to 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 enterocytes depresses circulating
arginine by interfering with local biosynthesis or by increasing
catabolism of circulating arginine. We also do not know at present why
circulating arginine levels are lower in male than in female transgenic
mice.
Arginine belongs to the group of amino acids whose circulating
concentration declines concurrently with the declining growth rate in
the course of postnatal development. The expression of arginase in
enterocytes of the F/A transgenic mice is sufficient to completely
abolish the high circulating arginine levels in the suckling period,
suggesting that the observed phenotype was a direct consequence of
arginine deficiency. The mechanism by which low arginine concentrations
prevent hair and muscle growth remains to be determined. Because the
abundance of extremely arginine-rich proteins, such as
trichohyalin, is similar in wild-type and F/A-2 mice
(12)
, the most obvious explanation for the decreased
growth rate of the F/A-2 transgenic mice, that is, the incomplete
charging of arginyl-tRNA for protein synthesis, does not seem to
account for the phenotype of hypoargininemia. Arginine is also known as
a secretagogue of growth-promoting hormones, such as growth hormone
(34)
and insulin (35)
, via mechanisms
dependent as well as independent of the production of nitric oxide from
arginine (36)
. However, mice of the F/A-2 line show
catch-up growth after weaning, even though the concentration of
circulating arginine remains low. The coincidence of this delayed
growth spurt (12)
with the temporarily increased
concentration of virtually all amino acids in the F/A-2 line relative
to the F/A-1 and wild-type mice, is probably not a coincidence.
Together, these data suggest that the ratio between arginine and the
other members of the age-dependent group of amino acids determines
whether growth will be inhibited. This hypothesis is supported by the
difference in growth behavior of F/A-1 mice, which grow normally, and
F/A-2 mice, which are severely affected, even though the additional
decrease of arginine levels is modest. We are presently following these
and other theories.
No sex difference in the plasma level of arginine was observed in
wild-type mice. However, an intriguing observation was the rise in
plasma arginine levels in adult F/A females, but not in males, because
intestinal arginase expression was similar in both sexes. The effect
was more pronounced in the F/A-2 than in the F/A-1 line (Fig. 2)
. It
has been shown that arginine biosynthesis by, for example, the kidneys
can be adaptively increased in response to low arginine levels
(37)
. The current finding suggests that F/A females are
able to mount such an adaptive increase in arginine biosynthesis more
effectively than F/A 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 levels in testis
and muscle [(38)
; see also next paragraph]. Because this
gender difference is also observed for the other age-dependent
amino acids in the F/A transgenic mice, the cause may also be more
general, e.g., a lower rate of amino acid catabolism in female
arginine-deficient mice. This gender difference may well be of
clinical importance because a common disease such as atherosclerosis
may represent an arginine-deficiency disease (39)
and
because males are more prone to develop atherosclerosis than females
(40)
. In view of the atheroprotective effect of estrogens
(41)
, future studies should also address the interaction
between estrogens and arginine metabolism.
The instant hydrolysis of newly synthesized and plasma arginine by
transgenic arginase in the enterocytes of suckling F/A mice causes a
drop of plasma and tissue arginine levels. The current analyses of
amino acid levels in wild-type and transgenic mice reveals
genotype-dependent increases in the levels 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, nerve and testis (42)
and accounts for a sizable
portion of arginine catabolism (6)
. Moreover, GAA levels
in brain, liver and kidney reach maximum levels in the suckling period
(43)
, underscoring the importance of arginine metabolism
for CT synthesis in this period. The diminished synthesis of CT is due
to the decreased concentration of available arginine and not to
feedback inhibition of the transaminidase reaction by CT or ornithine.
Conceivably, ornithine concentration could be increased as a result of
the increased flux through arginase, but increased ornithine levels
were observed only in intestine, where CT levels are not altered and
transaminidase activity is virtually absent (44)
. The
genotype-related increases in serine, threonine and phenylalanine
were found to be due solely to their increased level at 5–10 wk in
F/A-2 mice. However, all amino acids measured except arginine were
elevated at this time (Fig. 2
F). At present, we have no
explanation for the increase in circulating tryptophan. Interestingly
though, a similar increase in tryptophan was found in brain of
ornithine transcarbamoylase–deficient spf mice
(45)
.
The absence of an increased urea concentration in tissue (except
muscle), plasma or urine is at first glance surprising in view of the
overexpression of arginase in the enterocytes and the ensuing
consumption of body arginine content. However, as also shown by the
extremely low urinary urea content, amino acid catabolism in the
suckling period is very low (46)
. Hence, dietary and
endogenously synthesized arginine serve largely to produce protein or
creatine. Loss via the creatine/creatinine pathway virtually stops in
the F/A mice. Arginine content in preweaning rat protein is 8.5%
(9)
, so that arginine degradation in the intestine
represents only a tiny fraction of the total substrate for urea
synthesis.
Arginine deficiency and guanidino compounds
The decreased arginine availability in F/A mice decreased the
concentration of guanidino compounds that form via the transaminidation
pathway, but caused an accumulation of GSA, MG and G. Strictly
speaking, our data do not allow conclusions about fluxes. However, we
observed that 6 h after an arginine injection, the concentrations
of arginine and the arginine metabolites, urea, GAA, ß-GPA, -GBA,
CTN, -K--GVA, ArgA, GSA and G, were still substantially increased
in plasma of wild-type mice, but were similar to the corresponding
control mice in F/A-2 mice, with mice from the F/A-1 line intermediate.
The plasma levels of these compounds therefore correspond to the actual
availability of arginine rather than to the arginine load administered;
in other words, they are a proxy for flux. The exception is Harg, which
is not a metabolite of arginine. This conclusion implies that the
decline in arginine concentration, and not the accumulation of
transaminidation products (47
,48)
, determines the decrease
in flux through transamidinase.
The increase in tissue, plasma and urinary GSA levels and urinary G
levels corresponds to the increased severity of arginine deficiency.
The higher concentration of GSA and G in the urine of transgenic mice
after arginine loading confirms that the formation of these compounds
is enhanced under arginine-deficient conditions. Increased -NAA,
GSA, MG and G levels are also observed in renal failure
(49
–51)
, subtotal nephrectomy (52
,53)
,
endotoxin treatment (54)
and after consumption of an
arginine-free diet by strict carnivores (55)
. As in
the diseased states mentioned, the contribution of GSA and/or MG and G
to the development of the arginine-deficient phenotype in F/A mice
remains to be established.
It has been proposed that GSA is a transamidination product of
arginine, when aspartate, instead of glycine, is the amidine donor
(49)
. However, because the flux through transaminidase is
slowed down due to arginine deficiency in F/A mice, this explanation is
not favored. GSA concentrations positively correlate with plasma urea
(56)
. Urea, in turn, inhibits ASL (57)
,
suggesting that ASS may accumulate locally. The hypothesis that GSA as
well as MG form upon interaction of ASS and CT with free radicals
(58)
is therefore more attractive, particularly because it
has been shown that free radicals form in the neonatal intestine
(59)
and probably elsewhere under catabolic conditions.
Arginine deficiency and behavioral deficits
F/A transgenic mice displayed hyperactivity as well as several
more specific deficits in coordinated neuromotor abilities. Because
passive avoidance learning was also impaired in these mice, although
the motor requirements of this task were minimal, these deficits were
probably not caused by muscular problems. Behavioral deficits,
comparable to the ones seen in the F/A mice, have been associated with
increased levels of urea and CTN in nephrectomized mice
(60)
. Uremic toxins, in particular GSA, affect cerebral
neurotransmitter systems and are thought to cause the psychomotor
deficits seen in uremic encephalopathy (23)
. Behavioral
alterations, similar to those seen in F/A mice, were found in
spf-mutant mice (61)
. Unlike F/A mice, these
mice suffer from increased ammonia levels. However, brain ammonia,
urea, CTN and GSA concentrations were unaffected in F/A mice, virtually
excluding these factors as causes of their neuromotor deficits.
Similarly, GSA levels were not elevated in brain of spf mice
(61)
. However, we cannot presently exclude that the
slightly elevated circulating levels of tryptophan, a precursor for
cerebral production of serotonin and quinolinate, an excitotoxin
(62)
, were responsible for the observed neuromotor
deficits in F/A mice. Similarly, elevated brain tryptophan levels in
spf mice (45)
have been associated with a
100% increase in brain quinolinic acid, neuropathology
(63)
and impaired passive avoidance (61)
.
Because arginine is required for NO synthesis in neurons, the low level
of circulating and cerebral arginine in the F/A mice may be limiting
for NO synthesis. Limitation of cerebral NO synthesis has been shown in
spf mice, which also suffer from arginine deficiency
(64)
. Limited NO synthesis impairs synaptic plasticity,
motor coordination and memory functions (65
,66)
. Neuronal
nitric oxide synthase (nNOS)-deficient mice show behavioral
alterations, but unlike F/A transgenic mice, wire suspension, open
field activity, pole equilibrium and several other neuromotor tests
were not altered in these mice (67)
. Furthermore, mice
lacking nNOS, especially males, were reported to be extremely
aggressive, but we did not observe any signs of this aggressive
behavior in F/A mice.
F/A transgenic mice suffer from a life-long deficiency in
circulating arginine and lasting behavioral deficits. Despite a low
circulating arginine level, F/A-2 transgenic mice show catch-up
growth after weaning (12)
, which is temporally related to
increased circulating levels of all other amino acids measured.
Arginine levels in adult F/A females are higher than in adult F/A
males, possibly as a result of the extra requirement for CT synthesis
in muscle and testis. Low levels of arginine lead to a decrease in the
flux through transaminidase and accumulation of GSA and G, probably as
a result of a higher oxidative stress under hypoargininemic conditions.
|
FOOTNOTES |
|---|
3 Abbreviations used: A-I (EC 3.5.3.1),
hepatic arginase; AGAT (EC 2.1.4.1), L-arginine-glycine
transamidinase; ArgA; argininic acid; ASL (EC 4.3.2.1),
argininosuccinate lyase; ASS (EC 6.3.4.5), argininosuccinate
synthetase; CT, creatine; CTN, creatinine; F/A, FABPi/arginase I
transgenic mice; FABPi, fatty acid binding protein; G, guanidine; GAA,
guanidinoacetic acid; GABA, -aminobutyric acid; -GBA,
-guanidinobutyric acid; ß-GPA, ß-guanidinopropionic acid; GSA,
guanidinosuccinic acid; -GVA, -guanidinovaleric acid;
-K--GVA, -keto--guanidinovaleric acid; MG, methylguanidine;
-NAA, -N-acetylarginine; ND, neonatal day, days
after birth; nNOS, neuronal nitric oxide synthase.
Manuscript received January 22, 2001. Initial review completed March 28, 2001. Revision accepted July 20, 2001.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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