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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 http://www.ajpgi.org Downloaded from http://ajpgi.physiology.org/ by 10.220.33.4 on May 6, 2017 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- AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00140.2012 • www.ajpgi.org Downloaded from http://ajpgi.physiology.org/ by 10.220.33.4 on May 6, 2017 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. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00140.2012 • www.ajpgi.org Downloaded from http://ajpgi.physiology.org/ by 10.220.33.4 on May 6, 2017 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 AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00140.2012 • www.ajpgi.org Downloaded from http://ajpgi.physiology.org/ by 10.220.33.4 on May 6, 2017 0.0 G690 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 Downloaded from http://ajpgi.physiology.org/ by 10.220.33.4 on May 6, 2017 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 AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00140.2012 • www.ajpgi.org Downloaded from http://ajpgi.physiology.org/ by 10.220.33.4 on May 6, 2017 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). AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00140.2012 • www.ajpgi.org Downloaded from http://ajpgi.physiology.org/ by 10.220.33.4 on May 6, 2017 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 AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00140.2012 • www.ajpgi.org Downloaded from http://ajpgi.physiology.org/ by 10.220.33.4 on May 6, 2017 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. 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Downloaded from http://ajpgi.physiology.org/ by 10.220.33.4 on May 6, 2017 AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00140.2012 • www.ajpgi.org