Download Defective intestinal amino acid absorption in Ace2 null mice

Am J Physiol Gastrointest Liver Physiol 303: G686 –G695, 2012.
First published July 11, 2012; doi:10.1152/ajpgi.00140.2012.
Defective intestinal amino acid absorption in Ace2 null mice
Dustin Singer,1 Simone M. R. Camargo,1 Tamara Ramadan,1 Matthias Schäfer,2 Luca Mariotta,1
Brigitte Herzog,1 Katja Huggel,1 David Wolfer,3 Sabine Werner,2 Josef M. Penninger,4 and
François Verrey1
1
Institute of Physiology and Zürich Center for Integrative Human Physiology, University of Zürich, Zurich, Switzerland;
Institute of Cell Biology, ETH Zurich, Switzerland; 3Institute of Anatomy, University of Zurich and Institute of Human
Movement Sciences, ETH Zurich, Switzerland; and 4Institute for Molecular Biotechnology of the Austrian Academy of
Sciences, Vienna, Austria
2
Submitted 4 April 2012; accepted in final form 9 July 2012
Hartnup disorder; L-tryptophan; niacin; angiotensin converting enzyme 2
B0AT1 (SLC6A19) cotransports a
broad range of neutral amino acids with Na⫹ across the apical
membrane of small intestine and kidney proximal tubule epithelial cells (5, 8, 10, 18, 28). B0AT1 was recently shown to
need specific partner proteins for expression at the plasma
membrane, in particular, Ace2 in small intestine (11, 20) and
transmembrane protein 27 (Tmem27; collectrin) in the kidney
proximal tubule (15, 22). A general B0AT1 knockout mouse
THE AMINO ACID TRANSPORTER
Address for reprint requests and other correspondence: F. Verrey, Institute of
Physiology, Univ. of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland (e-mail: [email protected]).
G686
model has been described recently, showing that this transporter is essential for normal body weight gain and optimal
growth control (7). The kidney-specific lack of B0AT1 was
reported in mice lacking partner protein Tmem27 (15, 22).
These mice were shown to display a massive aminoaciduria
caused by the lack of B0AT1 and of other Na⫹-dependent
amino acid transporters, and their neutral amino acid serum
levels appeared to be decreased. On the other hand, the lack of
Ace2 has been shown to lead to a lack of B0AT1 protein
expression specifically in the intestine and, compared with
Tmem27, Ace2 was shown to differentially interact with some
B0AT1 Hartnup mutants (11). However, the study of Ace2 has
as yet mostly focused on its role in the renin angiotensin
system and not on its role as B0AT1 partner protein. For
example, Ace2 was shown to decrease angiotensin II levels and
thus functionally counteract the effects of the angiotensin
converting enzyme. Old (ⱖ 6 mo) ace2 null mice were shown
to develop a variety of effects attributed to an increased level
of angiotensin II, in particular, decreased cardiac contractility
(14), oxidative stress, and inflammation in heart (26) as well as
kidney glomerulosclerosis (25). It has also been suggested that
Ace2 may play a role in energy homeostasis, as ace2 null mice
were shown to display a selective decrease in first-phase
insulin secretion in response to glucose and a progressive
impairment of glucose tolerance (24). At the level of the small
intestine, where Ace2 is highly expressed at the luminal brushborder membrane (17), its role has not been studied besides
assessing the fact that it is required for the expression of
B0AT1. Additionally we have shown in a recent collaborative
study that ace2 null mice display an increased susceptibility to
intestinal inflammation induced by epithelial damage (data not
shown). A possible role of intestinal Ace2 within a luminal
angiotensin system has been suggested based on the recent
observation that angiotensin II downregulates sodium-dependent glucose uptake in ex vivo small intestine rings, presumably via luminal AT1 receptors (36).
Hartnup disorder (OMIM 234500) is an autosomal recessive
impairment of epithelial neutral amino acid transport in kidney
proximal tubule and small intestine that is, in most cases,
caused by mutations of the SLC6A19 gene (18, 31). The
consistent characteristic of Hartnup cases is an increased urinary excretion of neutral amino acids due to impaired renal
transport (3, 21). This is not always accompanied by an
intestinal transport defect (29, 32). Whereas most cases remain
asymptomatic apart from the aminoaciduria, clinical symptoms
reminiscent of pellagra appear in some cases, in particular,
photosensitive skin rash, diarrhea, cerebellar ataxia, and psychotic behavior. These unusual symptoms have been suggested
0193-1857/12 Copyright © 2012 the American Physiological Society
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Singer D, Camargo SMR, Ramadan T, Schäfer M, Mariotta L,
Herzog B, Huggel K, Wolfer D, Werner S, Penninger JM, Verrey
F. Defective intestinal amino acid absorption in Ace2 null mice. Am
J Physiol Gastrointest Liver Physiol 303: G686 –G695, 2012. First
published July 11, 2012; doi:10.1152/ajpgi.00140.2012.—Mutations
in the main intestinal and kidney luminal neutral amino acid transporter B0AT1 (Slc6a19) lead to Hartnup disorder, a condition that is
characterized by neutral aminoaciduria and in some cases pellagralike symptoms. These latter symptoms caused by low-niacin are
thought to result from defective intestinal absorption of its precursor
L-tryptophan. Since Ace2 is necessary for intestinal B0AT1 expression, we tested the impact of intestinal B0AT1 absence in ace2 null
mice. Their weight gain following weaning was decreased, and
Na⫹-dependent uptake of B0AT1 substrates measured in everted
intestinal rings was defective. Additionally, high-affinity Na⫹-dependent transport of L-proline, presumably via SIT1 (Slc6a20), was
absent, whereas glucose uptake via SGLT1 (Slc5a1) was not affected.
Measurements of small intestine luminal amino acid content following gavage showed that more L-tryptophan than other B0AT1 substrates reach the ileum in wild-type mice, which is in line with its
known lower apparent affinity. In ace2 null mice, the absorption
defect was confirmed by a severalfold increase of L-tryptophan and of
other neutral amino acids reaching the ileum lumen. Furthermore,
plasma and muscle levels of glycine and L-tryptophan were significantly decreased in ace2 null mice, with other neutral amino acids
displaying a similar trend. A low-protein/low-niacin diet challenge led
to differential changes in plasma amino acid levels in both wild-type
and ace2 null mice, but only in ace2 null mice to a stop in weight gain.
Despite the combination of low-niacin with a low-protein diet, plasma
niacin concentrations remained normal in ace2 null mice and no
pellagra symptoms, such as photosensitive skin rash or ataxia, were
observed. In summary, mice lacking Ace2-dependent intestinal amino
acid transport display no total niacin deficiency nor clear pellagra
symptoms, even under a low-protein and low-niacin diet, despite
gross amino acid homeostasis alterations.
INTESTINAL AMINO ACID ABSORPTION
MATERIAL AND METHODS
Animals. The ace2 wild-type (WT) and knockout mice were housed
in standard conditions and fed a standard diet for 8 wk. They were
then either kept on a standard diet or switched to a LP/LN diet for up
to 85 additional days. Generation of the knockout mice was described
elsewhere (14). All procedures for mice handling were according to
the Swiss Animal Welfare laws and approved by the Kantonales
Veterinäramt Zürich.
Growth curve. Pups from ace2⫹/⫺ ⫻ ace2⫹/y breeding weights
were recorded starting 1 day after birth. Pups were identified by
marking the body with a Securiline alcohol-resistant lab marker
(Precision Dynamics, San Fernando, CA). The tail was similarly
marked when fur appeared on the body. At day 21, pups were
weaned from the mother, separated by sex, and biopsies were
sampled for genotyping. WT and knockout mouse body weight was
averaged separately for each litter (4 litters) and compared by a
paired t-test.
Metabolic cage experiments. Animals were adapted to metabolic
cages (Tecniplast, Buguggiate, Italy) for 3 days before data collection,
where they had free access to corresponding diets and drinking water.
Daily food/water intake, urine/feces output, and body weights were
measured. Urinary pH was measured using a pH microelectrode
(model 691 pH-meter; Metrohm). Urinary creatinine was measured by
the Jaffe method (30). Urinary and plasma urea were measured using
the diacetyl monoxime method (37). Urinary electrolytes (Na⫹, K⫹,
Ca2⫹, Mg2⫹, Cl⫺, SO2⫺
4 ) were measured by ion chromatography
(Metrohm ion chromatograph; Herisau, Switzerland).
Blood was collected by decapitation and 1 ␮l of heparin⫺Na⫹
25,000 IE/5 ml (B. Braun, Melsungen, Germany) was added. Plasma
was collected after centrifugation at 6,000 g and 4°C. Organs were
harvested after a NaCl 0.9% perfusion through the heart. Plasma total
niacin (nicotinic acid and nicotinamid acid) was measured using
ID-Vit niacin assay (Immundiagnostik, Bensheim, Germany).
Diet treatments. After 8 wk, mice were either fed a standard diet
[normal protein (NP): 20% casein/30 mg/kg niacin (AIN93G) KlibaNafag, Kaiseraugst, Switzerland] or a low-protein/low-niacin diet
(LP/LN; 7% casein to 1.2 mg/kg niacin). Experiments were performed
after 75 to 85 days of diet treatment.
Amino acid measurements. Ice-cold methanol deproteinization of
the plasma was performed as described elsewhere (2). Liver was
homogenized and deproteinized in cold 10% sulfosalicylic acid (wt/
vol ⫽ 1:3) on ice. Supernatant was collected after two 15,000 g
centrifugation steps for 15 min at 4°C. Deproteinized samples or
mouse urine collected over 24 h were then derivatized using AccQ
Tag (Waters, Milford) and analyzed on an Acquity UPLC (Waters)
according to the manufacturer’s instructions by the Functional
Genomics Center Zurich (FGCZ) (13).
Proximal small intestine ring uptake. Uptake of radiolabeled amino
acids and glucose was performed as previously described (11) on
proximal small intestine (first two-thirds) segments. Briefly, everted
small intestine rings were incubated in bubbling (Oxycarbon) KrebsTris buffer (pH 7.4) containing either 1 mM glycine (0.01 ␮Ci
14
C-Gly/ml), 1 mM L-tryptophan (0.1 ␮Ci 3H-L-Trp/ml), 200 ␮M
L-proline (0.1 ␮Ci 3H-L-Pro/ml), for 5 min, or 5 mM D-glucose (0.1
␮Ci 3H-D-Gluc/ml) for 2 min at 37°C.
Intestine rings were dried at 55°C o/n on cellulose (Sartorius AG,
Goettingen, Germany) and weighed. The rings were then lysed in 0.75
N NaOH for 6 h, neutralized with 10 N HCl, and the radioactivity was
determined by liquid scintillation. Na⫹ was replaced by N-methyl-Dglucamine in the condition without Na⫹ (⫺Na⫹). Amino acid transport was expressed relative to dry tissue weight and related to the
average of the ⫺Na⫹ condition. Selected groups were compared by
repeated-measures one-way ANOVA, followed by Bonferroni’s posttest (⫹Na⫹ vs. ⫺Na⫹ for each genotype and ⫹Na⫹ ace2⫹/y vs.
⫹Na⫹ ace2⫺/y).
Intestinal luminal amino acid measurements following gavage. WT
and ace2⫺/y mice were starved for 18 h in metabolic cages to empty
their intestinal lumen. Seven h after light onset, during the inactive
phase, animals received by gavage a mixture of all proteinogenic
amino acids dissolved in PBS 1 ⫻ pH 7.4 at a final concentration
10-fold higher than the plasma concentration in WT animals. The
solution was supplemented with 7 ␮Ci/ml 3H-and 1.5 ␮Ci/ml 14Cradiolabeled amino acid. After 1 h, animals were anesthetized with
isofluorane and killed by cervical dislocation. The blood was collected
and the gastrointestinal system, from stomach to rectum collected.
The segments, namely stomach, small intestine (divided in 4 segments), caecum, and colon were washed with 1 ml PBS 1⫻ pH 7.4 at
room temperature. Flushed intestinal content was digested with 1 ml
Solvable (PerkinElmer) overnight at 50°C and distained with 30%
H2O2 (200 ␮l). The content was mixed with 15-ml cocktail scintillation Ultima Gold (PerkinElmer) and the amount of added radiolabeled
tracer 3H-tryptophan, 14C-glycine or 14C-isoleucine measured. The
concentration of the amino acids was calculated in picomols, and the
results expressed as picomol per milligram of wet segment weight.
For UPLC measurements of luminal amino acids, the content was
centrifuged at 16,000 g for 5 min at 4°C using a table-top centrifuge.
The supernatant was deproteinized and measured as described above
in the amino acid measurements section.
RotaRod test. The RotaRod consists of a rotating drum with an
accelerating (day 1, 6 to 60 rpm) or fixed speed (day 2, average speed
reached on day 1) [model 47600; Ugo Basile, Comerio, Italy; (33)].
The time at which the animal drops off the drum is measured
(maximum testing time: 300 s). Five trials were performed on each
day.
UVB irradiation. Mice were anesthetized by intraperitoneal injection of ketamine/xylazine and subsequently shaved. Mice were then
irradiated with 100 mJ/cm2 UVB using a Medisun FH-54 lamp
(Schulze and Böhm, Huerth, Germany), equipped with six UVBTL/12 bulbs (9 W each; Philips, Amsterdam, The Netherlands), which
emit UVB light in the range of 280 to 315 nm with a peak emission
at 312 to 315 nm. Forty-eight hours later, the irradiated mice were
killed, the skin was fixed in 4% paraformaldehyde and stained with
hematoxylin and eosin.
Statistics. Data are presented as means ⫾ SE. Analyses were done
by running the GraphPad Prism 4.0 software (GraphPad).
RESULTS
Defect in amino acid homeostasis and growth of ace2 null
mice. In view of the defective intestinal amino acid absorption
that we had described in ace2 null mice (11) and of the growth
defect observed in mice entirely lacking B0AT1 (7), we fol-
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to be the consequence of inappropriate niacin (vitamin B3,
nicotinic acid) levels caused by decreased intestinal uptake of
its precursor L-tryptophan (Trp). In normal conditions, Trp
only accounts for the synthesis of a small part of the required
niacin, the major part being directly acquired from the diet. It
is thus hypothesized that in Hartnup disorder, low Trp absorption might lead to niacin deficiency and thus to pellagra-like
symptoms only in combination with environmental influences
such as poor diet, diarrhea, or other factors (29). This hypothesis is supported by the fact that these symptoms are successfully treated with oral niacin supplementation, but no experimental evidence has yet provided a direct confirmation of this
hypothesis (21).
The present study therefore aims at clarifying the impact of
intestinal B0AT1 absence in ace2 null mice on amino acid
handling in vivo, especially in regard to Trp and the pellagralike symptoms associated with Hartnup disorder.
G687
G688
INTESTINAL AMINO ACID ABSORPTION
30
*
Body weight (g)
*
*
20
*
*
10
*
ace2 +/y
ace2 -/y
0
0
10
40
50
Fig. 1. Decreased weight gain of ace2 null mice after weaning. Wild-type
(WT) and knockout mouse body weight was averaged separately for each litter
(4 litters) and compared by a paired t-test. Means ⫾ SE; ace2⫹/y n ⫽ 8, ace2⫺/y
n ⫽ 6. *P ⬍ 0.05.
lowed the growth of ace2 null mice maintained under normal
laboratory diet conditions by weighing male pups from day 1
to day 47. At birth and before weaning, ace2 null mice showed
no weight difference compared with WT littermates. However,
following weaning, ace2 null mice displayed a temporary
slowdown of weight gain similar to that observed in B0AT1
null mice (7) with a maximal difference of ⬃5 g at 30 days
(Fig. 1). The ace2 null mice still displayed a lower weight at 8
wk when the mice were placed in metabolic cages for the
measurement of food and water intake as well as urine and
feces output (Table 1). Under these normal laboratory diet
conditions, ace2 null mice maintained body weight homeostasis despite the intestinal amino acid transport defect that
appeared to be compensated by a small, but statistically insignificant increase in food intake (relative to body weight). An
increase in water intake and a small disturbance in urine pH
were also observed that might be related to the previously
described late-onset glomerulosclerosis (25). To evaluate how
body amino acid homeostasis was affected by the lack of
intestinal B0AT1 in these ace2 null mice, we measured their
plasma amino acid levels at 8 wk. Interestingly, only glycine
(Gly) and Trp were significantly decreased, whereas there was
only a trend towards lower levels of most other neutral plasma
amino acids (Fig. 2).
Defect in luminal neutral amino acid uptake in small intestine of ace2 null mice. We have previously shown in the
Xenopus laevis oocyte expression system that all neutral proteinogenic amino acids are transported by B0AT1-Ace2 (11).
That this transport indeed depends on the presence of Ace2 in
mouse intestine was demonstrated for L-isoleucine (Ile) using
everted rings (11). We now further characterized the defect
in intestinal amino acid transport in ace2 null mice and
show here that the Na⫹-dependent uptake of Trp and of Gly
is either reduced or absent in proximal intestine everted
rings (Fig. 3). Although not statistically significant, part of
the Na⫹-dependant uptake of Gly remained in ace2 null
mice and is probably mediated by either the proton amino
acid transporter PAT1 (SLC36A1) (1), cooperating with
Na⫹/H⫹ exchanger (SLC9A3), or the Na⫹-dependent trans-
Table 1. Summary of metabolic cages and urine data from
8-wk-old male mice
ace2⫹/y
Genotype
Body wt, g
Food, % body wt
Water, % body wt
Urine, % body wt
Feces, % body wt
25.8 ⫾ 0.2
14.7 ⫾ 0.7
19.4 ⫾ 1.3
4.6 ⫾ 0.4
5.3 ⫾ 0.4
ace2⫺/y
24.8 ⫾ 0.3*
15.4 ⫾ 0.5
23.1 ⫾ 1.2*
5.0 ⫾ 0.5
5.4 ⫾ 0.3
Urinary Parameters
Osmolality, mosmol/kg
pH
Creatinine (␮mol/24 h)
3,600 ⫾ 422
6.26 ⫾ 0.05
6.31 ⫾ 0.57
3,716 ⫾ 390
6.01 ⫾ 0.03***
4.69 ⫾ 0.53
Group sizes: ace2⫹/y n ⫽ 10, ace2⫺/y n ⫽ 11. Means ⫾ SE *P ⬍ 0.05,
***P ⬍ 0.001.
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20
30
Age (days)
porter GLYT1, a fraction of which was suggested to be
expressed at the luminal membrane of enterocytes (12). To
measure L-proline (Pro) uptake via the high-affinity, Na⫹dependent, SLC6-family imino acid transporter SIT1
[Slc6a20a, Pro K0.5 ⫽ 0.13 mM (19)], while preventing substantial transport by low affinity transporter PAT1 [Pro K0.5 ⫽
2.8 mM (6)], a relatively low Pro concentration (0.2 mM) was
used (Fig. 3). Our results suggest that like B0AT1, functional
SIT1 expression is also lacking in intestine of ace2 null mice.
The protein expression of SIT1 could, however, not be tested
by immunohistochemistry because of the lack of specific antibodies recognizing mouse SIT1.
Given the downregulation of SGLT1-mediated glucose uptake by angiotensin II in rat everted rings (36), we tested the
possibility that an increased angiotensin II level in ace2 null
mice (14) would lead to a decrease in intestinal glucose
transport. The Na⫹-dependant D-glucose uptake was inhibited
by phlorizin as expected for SGLT1 (data not shown), but no
difference between the WT and the ace2 null mice could be
observed (Fig. 3). Our results show no impact of the lack of
Ace2 on intestinal SGLT1-mediated glucose uptake function
when tested ex vivo.
Impact of Ace2 defect on in vivo amino acid absorption
along the intestine. To characterize the amino acid absorption
defect along the intestine, we designed a new approach consisting of measuring the luminal amino acid content in the
different intestinal segments at a given time after gavage of a
mixture containing all proteinogenic amino acids (10 ␮l/g body
wt), supplemented with radioactive tracers. The time point of
death and content measurement was chosen based on preliminary experiments that indicated that the nonabsorbable solute
mannitol had mostly reached the ileum within ⬃1 h after
gavage, but had not yet accumulated in the caecum. An initial
important observation was made in WT animals (Fig. 4).
Indeed, Ile and Trp showed a differential pattern of absorption
along the small intestine (Fig. 4, A and B). Whereas Ile was
almost no longer detectable in the ileum lumen, suggesting its
efficient absorption along the duodenum and the jejunum,
substantial amounts of Trp were detected in the lumen of
terminal jejunum and ileum. This difference might be due to a
lower affinity of B0AT1 for Trp compared with other neutral
amino acids that compete for this transporter (10). Therefore
Trp would be mostly absorbed after the other neutral amino
acids and thus reach later segments of the small intestine.
These experiments also show that the luminal Ile, Trp, and Gly
G689
INTESTINAL AMINO ACID ABSORPTION
2.0
ace2 +/y
Plasma amino acid ratio
rel. to ace2 +/y
ace2 -/y
1.5
***
**
1.0
0.5
Gly Ala Val Leu Ile
Met Ser Thr Pro Asn Gln Phe Tyr Trp Lys Arg His Asp Glu
Fig. 2. Decrease in L-tryptophan (Trp) and glycine (Gly) plasma levels of ace2 null mice. Plasma was deproteinized and analyzed by UPLC. Groups were
compared by Student’s unpaired t-test. Ala, L-alanine; Val, L-valine; Leu, L-leucine; Ile, L-isoleucine; Met, L-methionine; Ser, L-serine; Thr, L-threonine; Pro,
L-proline; Asn, L-asparagine; Gln, L-glutamine; Phe, L-phenylalanine; Tyr, L-tyrosine; Lys, L-lysine, Arg, L-arginine; His, L-histidine; Asp, L-aspartate; Glu,
L-glutamate. Means ⫾ SE; ace2⫹/y n ⫽ 6, ace2⫺/y n ⫽ 9. **P ⬍ 0.01; ***P ⬍ 0.001.
(Fig. 4, A–C) content of ileum was strongly increased in ace2
null compared with WT mice, which indicated that the absence
of functional B0AT1 along the small intestine prevented their
efficient absorption. These results obtained using tracer amino
acids were confirmed by UPLC measurements of all proteinogenic amino acids reaching the ileum lumen following gavage
(Fig. 4D). Interestingly, the amount of all neutral amino acids
was increased in the lumen of ace2 null mice ileum, two-thirds
of them to a statistically significant extent according to a
stringent multiple comparison posttest. Only the charged
amino acids L-lysine, (Lys), L-arginine (Arg), L-glutamate
(Glu), L-aspartate (Asp), and the imino acid Pro were clearly
not increased in the ileum lumen of ace2 null mice. Taken
together, these results confirm the hypothesis that all neutral
amino acids require functional B0AT1 to be efficiently absorbed along the small intestine.
+
ace2 +/y +Na
+
ace2 +/y -Na
ace2 -/y +Na+
ace2 -/y -Na+
Uptake ratio
+
(rel. to -Na )
5
4
3
2
***
*
***
ns
**
***
*
*
ns
ns
ns
1
0
Trp
Gly
Pro
D-glucose
Fig. 3. Decrease of Na⫹-dependant neutral amino acid transport in small
intestine rings from ace2 null mice. The transport of Gly, Pro, Trp, and
D-glucose into everted proximal small intestine rings was measured in the
presence (white bar) and in the absence (black bar) of sodium. Data points
represent mean values of 3–7 intestinal rings taken from 3–5 mice ⫾ SE.
Groups were compared by one-way ANOVA, followed by Bonferroni’s
posttest on selected pairs of columns. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001;
ns, not significant.
Impact of LP diet on growth and amino acid homeostasis. In
view of the decreased plasma Trp levels observed in ace2 null
mice we additionally fed these mice a very LP diet (0.5%) to
mimick malnutrition conditions under which Hartnup subjects
are prone to develop niacin deficiency and pellagra-like symptoms. This diet had, however, to be interrupted after a few days
because of a too important weight loss. We then opted for a
less severe LP diet (7% casein) lacking niacin (nicotinamide)
addition (niacin content of diet: 1.2 mg/kg) and compared this
diet with normal chow (NP: 20% casein, 30 mg/kg niacin).
Starting at the age of 8 wk, the LP diet was maintained for ⬃10
wk to reveal chronic metabolic disturbances induced by the
lack of Ace2-dependent small intestine amino acid transport
(27). The weight of these mice was followed until the end of
the diet treatment (Fig. 5), when they were placed in metabolic
cages (Table 2). Under the NP diet, ace2 null mice displayed
a relative weight gain that was similar to that of their WT
littermates. In contrast, under the LP diet, WT mice tended to
gain more weight than under the NP diet, whereas ace2 null
mice failed to gain weight, particularly at the beginning of the
LP diet treatment, and this despite increased food consumption
(Fig. 5A, Table 2). The difference in growth rate between the
groups was quantified by comparing the area under the relative
growth curves (Fig. 5B). Unexpectedly, the wet weight of the
gastrocnemius muscle was not affected, either by the diet or by
the genotype, whereas liver weight was decreased by 25%
under LP diet and increased by ⬃10% in ace2 null mice (Table
2). The small increase in water intake and excretion of dilute
urine and creatinine observed in ace2 null mice under the NP
diet (Tables 1 and 2) was enhanced under the LP diet with
urine production reaching as much as ⬃4.5 ml/day. However,
these mice were not dehydrated compared with their WT
littermates, as shown by their normal plasma osmolality values
(Table 2).
To obtain more information on amino acid homeostasis
under LP diet in general and on the impact of intestinal B0AT1
defect in ace2 null mice, we measured amino acid levels in
plasma as well as in liver, kidney, and gastrocnemius muscle
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0.0
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INTESTINAL AMINO ACID ABSORPTION
B
***
150
100
50
0
st.
***
150
100
cae.col.
50
0
st.
s.i.
cae.col.
200
150
100
50
20
15
10
5
0
ace2 +/y
ace2 -/y
***
st.
s.i.
cae.col.
2000
***
1500
***
1000
500
*
***
***
***
***
**
*
0
Gly Ala Val Leu Ile
Met Ser Thr Pro Asn Gln Phe Tyr
Trp Lys Arg His Asp Glu
Fig. 4. Decrease of amino acids’ absorption in ace2 null mice small intestines following gavage. A mixture of proteinogenic amino acids at 10-fold their
approximate mouse plasma concentration was given in a volume of 10 ␮l/g of body weight by gavage. After 1 h, the intestinal content of different segments
was flushed and radiolabeled amino acids (A–C) or the amino acid (AA) content (D) was measured. The content of Ile (A), Trp (B), and Gly (C) was determined
in the stomach and small and large intestine by using radiolabeled tracer. In the ileum segment, the content of all proteinogenic amino acids was measured by
UPLC. The groups (4 – 8 animals) were compared by using one-way ANOVA (A–C) or two-way ANOVA (D), followed by Tukey’s (A–C) or Bonferroni’s
posttest (D). *P ⬍ 0.5, **P ⬍ 0.01, ***P ⬍ 0.001. st, stomach; si, small intestine; cae, caecum; col, colon.
(Fig. 6, A–D). Importantly, the LP dietary challenge also had a
major impact on the amino acid homeostasis in WT mice.
Indeed, the plasma level of several amino acids was decreased
under the LP diet in WT mice, in particular, that of the
beta-branched amino acids L-valine (Val), L-leucine (Leu), and
Ile and also that of the charged amino acids Lys, Asp, and Glu.
In contrast, the level of some other amino acids was increased,
in particular, that of the aromatic amino acids Trp, L-phenylalanine (Phe), and L-tyrosine (Tyr) as well as of L-glutamine
(Gln) and Arg (Fig. 6A). In the liver and kidney of WT mice
under the LP diet, the level of many amino acids [L-alanine (Ala),
Val, Leu, Ile, L-methionine (Met), Pro, L-asparagine (Asn) and
Lys] was decreased, whereas L-threonine (Thr) was increased
(Fig. 6, B–C). Additionally Phe and Glu were also decreased in
the kidney only (Fig. 6C). In gastrocnemius muscle of WT
mice under the LP diet, the results suggested a decrease of the
essential amino acids Leu, Ile, Met, and Trp (Fig. 6D).
Amino acid levels in ace2 null mice under the NP diet were
similar in these 18-wk-old mice to the levels measured previously at 8 wk. In plasma, Gly was significantly decreased, and
Trp and branched-chain neutral amino acid levels tended to be
reduced as well (Fig. 6A). In the liver and kidney of ace2 null
mice, amino acid levels were not different compared with WT
mice (Fig. 6, B–C), whereas in muscle Gly, Met, and Trp levels
were significantly decreased and branched-chain amino acids
showed a similar trend (Fig. 6D). This suggests that the
decreased intestinal amino acid absorption of ace2 null mice
impacts more on the free amino acid content of muscles than
A
120
Area under the curve (a. u.)
ace2 +/y NP
ace2 -/y NP
ace2 +/y LP
ace2 -/y LP
130
Body weight
rel. to day 0 (%)
Fig. 5. Growth defect of ace2 null mice under
low-protein (LP) diet. A: starting at 8 wk,
mouse weight was followed for 64 days while
fed normal protein (NP) or LP diet. B: the area
under the curve represented in A was calculated. Values with different letters are statistically different. Groups were compared by
1-way ANOVA, followed by Bonferroni’s
posttest (P ⬍ 0.05). Means ⫾ SE; n ⫽ 4; nd,
not detected.
B
110
100
90
0
20
40
Time on diet (days)
60
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00140.2012 • www.ajpgi.org
ace2 +/y
ace2 -/y
a
1000
a
a
500
0
b
NP
LP
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AA concentration (µM)
D
s.i.
C
200
Gly content
(pmol/mg tissue)
200
Trp content
(pmol/mg tissue)
Ile content
(pmol/mg tissue)
A
G691
INTESTINAL AMINO ACID ABSORPTION
Table 2. Summary of metabolic cages and urine data from 20-wk-old male mice after an 85-day diet treatment
Dietary Proteins
Genotype
Body wt, g
Food, % body wt
Water, % body wt
Urine, % body wt
NP
LP
ace2⫹/y
ace2⫺/y
ace2⫹/y
ace2⫺/y
28.0 ⫾ 0.8a
10.7 ⫾ 2.1ab
15.2 ⫾ 2.0a
3.0 ⫾ 0.6a
24.0 ⫾ 1.3a
11.9 ⫾ 0.3ab
22.5 ⫾ 2.8ab
6.9 ⫾ 0.8a
35.0 ⫾ 1.3b
7.3 ⫾ 0.8a
12.9 ⫾ 0.7a
4.1 ⫾ 0.3a
27.5 ⫾ 1.8a
13.9 ⫾ 0.5b
32.4 ⫾ 4.2b
18.7 ⫾ 3.5b
1,038 ⫾ 116b
6.16 ⫾ 0.12a
5.87 ⫾ 0.28b
407.3 ⫾ 111c
6.56 ⫾ 0.04a
4.94 ⫾ 0.49b
Urinary Parameters
Osmolality, mosmol/kg
pH
Creatinine, ␮mol/24 h
2,561 ⫾ 143a
6.41 ⫾ 0.11a
2.85 ⫾ 0.49a
1,449 ⫾ 121b
5.73 ⫾ 0.06b
4.77 ⫾ 0.30b
Plasma Parameters
277 ⫾ 6
312 ⫾ 3
Liver, % body wt
Gastrocnemius, % body wt
4.29 ⫾ 0.18a
0.53 ⫾ 0.05
273 ⫾ 4
315 ⫾ 1
274 ⫾ 6
305 ⫾ 3
282 ⫾ 4
306 ⫾ 2
3.25 ⫾ 0.17c
0.50 ⫾ 0.05
3.46 ⫾ 0.14a
0.64 ⫾ 0.08
Organ Weights
4.82 ⫾ 0.09b
0.58 ⫾ 0.03
Group sizes: ace2⫹/y n ⫽ 4, ace2⫺/y n ⫽ 4. NP, normal protein diet of 20% casein and 30 mg/kg niacin; LP, low protein diet of 7% casein and 1.2 mg/kg
niacin. Means ⫾ SE. Values with different letters are statistically different (P ⬍ 0.05).
on that of central organs, such as the liver and kidney. Importantly, under LP diet the lack of Ace2 did not significantly
further decrease amino acid levels in any of the measured
compartments but apparently at the expense of growth.
Absence of clear pellagra-like symptoms in ace2 null mice.
The intestinal neutral amino acid transport defective ace2 null
mice were submitted to LP/LN diet and then tested for possible
manifestations of pellagra-like symptoms, such as cerebellar
ataxia/coordination defects and fatigue. All mice exhibited
normal escape responses with spread limbs when suspended by
their tails (data not shown). In addition, spinning rod experiments, either with increasing speed or with fixed speed, did
not evidence any differences in the latency to fall over five
trials between the WT and the ace2 null mice under either
diet (Fig. 7A).
The severe skin abnormalities associated with pellagra were
also not observed in the ace2 null mice, either in untreated skin
or upon irradiation with a physiological dose of UVB. Gross
observation as well as microscopic analysis of skin morphology did not reveal an increased inflammatory response in the
mutant mice. Moreover, the epidermal alterations seen in
pellagra, including strong hyperkeratosis, parakeratosis, and
acanthosis combined with vacuolation of keratinocytes (35)
were not observed 48 h after UVB exposure (Fig. 7B). A minor
hyperkeratosis was, however, occasionally seen in ace2 null
mice upon treatment with the LP diet (Fig. 7B, lower right).
Finally, sebaceous gland hyperplasia, another common feature
of pellagra (35), did not occur. Other pellagra-like symptoms,
such as gastrointestinal inflammation and diarrhea were also
not observed (data not shown).
Interestingly, and not surprisingly in view of the negative
pellagra symptomatology, no difference in the level of plasma
total niacin (nicotinic acid and nicotinamid acid) was observed
between either group, despite the 75– 85 additional days of
LP and niacin deficient diet. The total niacin levels measured in mice were four times as high as normal levels in
human (17– 85 ␮g/l) (23) (Table 2).
DISCUSSION
Trp is absorbed mostly in later segments of the small
intestine. To follow the absorption of amino acids along the
small intestine and to test the impact of B0AT1 defect, we
designed a new approach consisting of measuring the luminal
amino acid content along the intestine following gavage of an
amino acid mixture. To measure the rate of axial bolus progression, the luminal content of the nonabsorbable substrate
mannitol was first measured at different time points after
gavage along the intestine. These preliminary results indicated
that the bolus mostly reached the ileum within 1 h without yet
being accumulated in the caecum or later large bowel segments
(data not shown). Based on this observation, amino acid
measurements were performed 1 h after gavage. Interestingly,
at this time point most neutral amino acids are found only at
low concentrations along the lumen of the intestine of WT
mice, indicating that they have been absorbed to a large extent
during their progression along the small intestine before reaching the ileum. In contrast, Trp is to some extent accumulated in
the later small intestine segments, suggesting that it is absorbed
to a lesser extent. This observation is in line with the results of
measurements previously made in the Xenopus oocyte expression system that indicated that the apparent affinity of B0AT1
is lower for Trp than for other neutral amino acids (10).
However, unlike expected based on the hypothesis that the
delayed absorption of Trp along the small intestine was due to
competing amino acids given by gavage, the amount of Trp
tracer reaching the ileum was only slightly higher when Trp
was given alone (difference not significant, data not shown).
Importantly, however, total luminal amino acids determined by
UPLCs was to a large extent independent of the amount of
amino acids given by gavage, suggesting that a large part of
luminal amino acids originates from the intestine, presumably
from pancreatic secretion and shedding enterocytes. Thus, the
relative delay in Trp absorption along the small intestine might
be due to the competition by other B0AT1 substrate amino
acids that are to a large extent of endogenous origin. Based on
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Niacin, ␮g/l
Osmolality, mosmol/kg
G692
INTESTINAL AMINO ACID ABSORPTION
Plasma amino acid ratio
rel. to ace2 +/y NP
A
3
ace2 +/y NP
ace2 -/y NP
ace2 +/y LP
ace2 -/y LP
b
1
a
a bbb
a
a bbb
ab
abc c
a ab b
c c
Gly Ala Val Leu Ile
a a bb
a
aa b
a
b
b
b
2
0
a
aa b
a c
ca
a abb
b
a
aa a
a abb
a a
a bb b
Met Ser Thr Pro Asn Gln Phe Tyr Trp Lys Arg His Asp Glu
40
bb
Liver amino acid ratio
rel. to ace2 +/y NP
30
20
10
1.5
aa
b bab
a a
b a bb
2.0
a
babb
aabb
a a
b a bb
aa bb
aabb
aa
a abb
aabb
a abb
1.0
0.5
0.0
Kidney amino acid ratio
rel. to ace2 +/y NP
C
n.d.
Gly Ala Val Leu Ile Met Ser Thr Cys Pro Asn Gln Phe Tyr Trp Lys Arg His Asp Glu
15
bb
10
5
2.0
1.5
1.0
a b
a a a
a a ba c c a
a a
a bbb abbb abbb aabb a bbb
abbb
a
abcb
aa
aabb aabb
aabb
aabb
0.5
0.0
n.d.
Gly Ala Val Leu Ile Met Ser Thr Cys Pro Asn Gln Phe Tyr
Lys Arg His Asp Glu
Gastrocnemius amino acid ratio
rel. to ace2 +/y NP
D
2.5
a
aabb
2.0
1.5
1.0
‡
aa
a a b bab
abb b
†
†
†
a a
babb
aa
abbb
‡
†
†
†
0.5
0.0
a
abbb
‡
n.d.
n.d.
Gly Ala Val Leu Ile Met Ser Thr Cys Pro Asn Gln Phe Tyr Trp Lys Arg His Asp Glu
Fig. 6. Free amino acids of weight and ace2 null mice under NP or LP diet. Plasma (A), liver (B), kidney (C), and gastrocnemius (D) of mice fed the NP or LP
diet were homogenized/deproteinized, and free amino acid concentrations were measured by UPLC. Amino acid levels are represented relative to the
concentrations measured in WT NP mice. Groups were compared by 1-way ANOVA, followed by Bonferroni’s posttest. Means ⫾ SE; n ⫽ 4 (A and D) and
n ⫽ 3 (B and C); †n ⫽ 1, ‡n ⫽ 2. Within a group, columns with one same letter are statistically not different (P ⬎ 0.05), whereas different letters indicate
statistical difference (P ⬍ 0.05).
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B
b
b
INTESTINAL AMINO ACID ABSORPTION
A
Latency to fall (sec)
300
ace2 +/y NP
ace2 -/y NP
ace2 +/y LP
ace2 -/y LP
200
100
0
speed increasing
ace2 +/y
B
ace2 -/y
SG
NP HF
D
LP
Fig. 7. Absence of pellagra-like symptoms in ace2 null mice. A: RotaRod
coordination test. Latency to fall off the rod set with either increasing or fixed
speed during 5 trials was recorded and averaged. Groups were compared by
1-way ANOVA, followed by Bonferroni’s posttest. B: representative PFAfixed hematoxylin and eosin stained sections of UVB-irradiated skin are
shown. D, dermis; E, epidermis; HF, hair follicle; SG, sebaceous gland.
Arrowhead points to mild hyperkeratosis in ace2 null mice fed with a LP diet.
Means ⫾ SE; n ⫽ 4 (A), n ⫽ 3 (B); bar ⫽ 100 ␮m.
these observations, we postulate that the less efficient absorption of Trp along the small intestine, compared with other
neutral amino acids, may increase the risk of its defective
absorption under pathological conditions, such as terminal
ileitis that impair its absorption in the terminal small intestine.
Deficiency of B0AT1 prevents efficient neutral amino acids
absorption along the small intestine. Similarly to the impact of
B0AT1 deficiency in the kidney that leads to aminoaciduria
(15), the deficiency of B0AT1 in small intestine increases the
amount of neutral amino acids that is not absorbed along the
small intestine and thus reaches the ileum and the caecum.
Specifically, we show that following gavage of an amino acid
mixture, the luminal content of the later small intestine segments, in particular, of the ileum, is strongly enriched in
neutral amino acids, whereas charged amino acids and the
imino acid Pro remain unaffected. This experiment confirms
the role of B0AT1 for the absorption of all neutral amino acids
in small intestine.
Long-term LP diet decreases liver amino acid metabolism
and modifies amino acid homeostasis. To reveal the impact of
the lack of B0AT1-mediated intestinal amino acid uptake, mice
were submitted to a long duration LP diet (up to 85 days).
Interestingly, this dietary challenge had a profound effect on
amino acid homeostasis in WT mice and little more effect on
ace2 null mice. Specifically, LP diet produced a strong differential effect on the plasma concentration of amino acids, some
of which were increased and others decreased. It appears that
the common trait of amino acids that were strongly increased
in plasma under long-term LP diet is the important role of the
liver in their metabolism. Indeed, for aromatic amino acids
(Trp, Phe, Tyr) the liver is considered as the major place of
degradation (16) and Arg and Gln are major players for
liver-mediated ammonium removal. Specifically, Arg is the
substrate for urea production within the urea cycle that localizes to the periportal hepatocytes. Ammonium that is not
cleared by this cycle can be used by the glutamine synthetase.
This localizes in perivenous hepatocytes to produce Gln, which
may function as substrate in kidney for ammonium removal
(9). In contrast, amino acids metabolized in other organs, such
as the beta branched amino acids, Lys, and the anionic amino
acids Asp and Glu, were strongly decreased. This observation
suggests that the liver adapts to an LP diet by decreasing its
amino acid metabolism in general, and specifically the activity
of the urea cycle. To what extent this metabolic adaptation is
related to the decrease in liver mass observed under LP diet
(⫺25%, Table 2) needs to be investigated.
Lack of decrease in niacin under LP/LN diet in ace2 null
mice. Our study also shows that in mice, the lack of intestinal
B0AT1 together with long-term LP (7%) diet and the absence
of niacin supplementation do not suffice to lead to nicotinamide deficiency. Accordingly overt pellagra-like symptoms
seen in some Hartnup patients were not recapitulated in these
mice. As discussed above, we observed that the LP diet induced
an unexpected increase in circulating tryptophan in mice, an effect
that was also observed in ace2 null mice, despite the fact that
they displayed a decreased Trp level under NP diet. An
alternative diet that has been shown by Bender (4) 30 years ago
to be pellagragenic in rats contains high leucine and minimally
adequate tryptophan. He showed that an excess of leucine
reduces the rate of nicotinamide nucleotide production from
Trp by reducing the activity of kynureninase and activating
picolinate carboxylase. We did, however, not use this approach
to decrease niacin production, since it had been shown to be
pellagragenic in WT rats and that our aim was to mimick in
ace2 null mice, specifically the pellagragenic effect of amino
acid malabsorption characteristic of Hartnup disorder. The lack
of plasma Trp and niacin depletion that we observed in WT and
ace2 null mice maintained ⬃10 wk under LP diet suggests that
mice are particularly resistant to decreased nutritional Trp.
This is in line with the recently published observation that,
unlike rats, mice submitted to acute Trp depletion do not
develop central serotonin reduction or affective behavioral
changes (34).
Taken together, our study demonstrates that B0AT1 is necessary for the efficient absorption of all neutral amino acids
along the small intestine. We also show that Trp is absorbed
further along the small intestine than most other competing
amino acids and thus potentially reaches its end, thereby
risking incomplete absorption. Additionally, long-term LP diet
is shown to strongly impact on amino acid homeostasis, leading to a decreased plasma level of many amino acids but also
to an increased level of aromatic amino acids as well as of
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E
G693
G694
INTESTINAL AMINO ACID ABSORPTION
L-glutamine and L-arginine, an effect that might result from
altered liver amino acid metabolism. The unexpected lack of
pellagragenic effect exerted by LP diet in mice lacking intestinal B0AT1 suggests compensatory metabolic mechanisms,
some of which might be species specific. In contrast, it is
suggested in view of the similar amino acid affinity profiles of
mouse and human B0AT1 that also in humans, Trp is absorbed
to some extent after the other neutral amino acids along the
small intestine.
14.
15.
16.
GRANTS
This work was supported by Swiss National Science Foundation Grant
31-130471 (to F. Verrey).
17.
DISCLOSURES
18.
AUTHOR CONTRIBUTIONS
Author contributions: D.S., S.M.C., D.W., S.W., J.M.P., and F.V. conception and design of research; D.S., S.M.C., T.R., M.S., L.M., B.H., and K.H.
performed experiments; D.S., S.M.C., and F.V. analyzed data; D.S., S.M.C.,
L.M., and F.V. interpreted results of experiments; D.S. and S.M.C. prepared
figures; D.S., S.M.C., and F.V. drafted manuscript; D.S., S.M.C., T.R., M.S.,
L.M., B.H., K.H., D.W., S.W., J.M.P., and F.V. approved final version of
manuscript.
19.
20.
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