Contents lists available at ScienceDirect

Physiology & Behavior

journal homepage: www.elsevier.com/locate/physbeh

Effects of chronic stress on intestinal amino acid pathways

Xun Yanga,b,c,1, Guowei Wange,1, Xue Gonga,b,c,1, Cheng Huanga,b,c,1, Qiang Maob,c,g, Li Zengb,c,f,Peng Zhenga,b,c, Yinhua Qinb,c, Fei Yea,b,c, Bin Lianb,c, Chanjuan Zhoub,c,d, Haiyang Wangb,c,Wei Zhoub,c, Peng Xiea,b,c,⁎

a Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, Chinab Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing 400016, Chinac Chongqing Key Laboratory of Neurobiology, Chongqing 400016, ChinadDepartment of Neurology, Yongchuan Hospital, Chongqing Medical University, Chongqing 402460, ChinaeNing Xia Medical University, Yin Chuan, Ning Xia 750004, China.fDepartment of Nephrology, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, Chinag The Second Affiliated Hospital of Chongqing Medical University, Department of Pharmacy, Chongqing, China

A R T I C L E I N F O

Keywords:Major depressive disorderChronic unpredictable mild stressGut-brain axisSmall intestineAmino acid absorptive disorderHigh-performance liquid chromatography

A B S T R A C T

Major depressive disorder (MDD) is a debilitating mental disorder with a high prevalence and severe impacts onquality of life. However, the pathophysiological mechanisms underlying MDD remain poorly understood. Here,we used high-performance liquid chromatography with ultraviolet detection-based targeted metabolomics toidentify amino acid changes in the small intestine, in a rat model of chronic unpredictable mild stress (CUMS).Pearson's correlation analysis was conducted to investigate the correlations between amino acid changes andbehavioral outcomes. Western blot analysis was employed to verify intestinal amino acid transport function.Moreover, we performed an integrated analysis of related differential amino acids in the hippocampus, per-ipheral blood mononuclear cells (PBMCs), urine and cerebellum identified in our previous studies using theCUMS rat model to further our understanding of amino acid metabolism in depression. Decreased concentrationsof glutamine and glycine and upregulation of aspartic acid were found in CUMS model rats. These changes weresignificantly correlated with depressive-like behaviors. Western blot analysis revealed that CUMS rats exhibiteda reduction in the expression levels of amino acid transporters ASCT2 and B0AT1, as well as an increase in LAT1expression. Impaired transport of glycine and glutamine into the small intestine may contribute to a centraldeficiency. The current findings suggest that the glycine and glutamine uptake systems may be potential ther-apeutic targets for depression. The integrated analysis strategy used in the current study may provide newinsight into the cellular and molecular mechanisms underlying the gut-brain axis, and help to elucidate thepathophysiological changes in central and peripheral systems in depression.

1. Introduction

Major depressive disorder (MDD) is a chronic, recurrent, debili-tating psychiatric disease with a high prevalence [1]. It is estimatedthat 14.2% individuals suffer from MDD during their lifetime [2]. TheWorld Health Organization predicts that MDD will be the leadingcontributor to disability globally by 2030 [3]. Epidemiological studies

have indicated that MDD significantly increases the risk of several otherdiseases, including diabetes mellitus, heart disease, obesity, cancer andirritable bowel syndrome (IBS) [4,5]. However, knowledge about thepathophysiology of MDD is currently rudimentary.

Changes in physiological metabolic processes involving amino acidshave been linked to the pathophysiology of depression since the 1900s[6]. Amino acids have been found to impact the levels of monoamine

https://doi.org/10.1016/j.physbeh.2019.03.001Received 30 October 2018; Received in revised form 22 February 2019; Accepted 1 March 2019

Abbreviations: CUMS, chronic unpredictable mild stress; MDD, major depressive disorder; HPLC, high-performance liquid chromatography; UV, ultra violet; CON,control; GBA, The gut-brain axis; SPT, sucrose preference test; OFT, open field test; FST, forced swim test; BW, body weight; SEM, standard error; ANOVA, analysis ofvariance; GLY, glycine; GLN, glutamine; ASP, aspartic acid; NacASP, N-acetyl-L-aspartic acid; PBMC, peripheral blood mononuclear cells; NMDAR, N-methyl-D-aspartic acid receptor; DA, dopamine; 5-HT, 5-hydroxytryptamine; NE, norepinephrine; IS, internal standard; IBS, irritable bowel syndrome.

⁎ Corresponding author at: Department of Neurology and Psychiatry, The First Affiliated Hospital of Chongqing Medical University, No. 1 Yixueyuan Road,Chongqing 400016, China.

E-mail address: xiepeng@cqmu.edu.cn (P. Xie).1 These authors contributed equally to this study.

Physiology & Behavior 204 (2019) 199–209

Available online 02 March 20190031-9384/ © 2019 Elsevier Inc. All rights reserved.

T

Downloaded for Christopher Sendi (csendi@sendi.org) at Inova Fairfax Hospital - JCon from ClinicalKey.com by Elsevier on October 20, 2019.For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.

neurotransmitters (NE, 5-HT, DA) [7], as well as regulating mood anddepressive-like behavioral performance [8,9], and are closely bound upwith systemic energy levels [10]. Using clinical and animal models,previous studies in our laboratory revealed several amino acids as po-tential biomarkers of depression [11–13]. However, current under-standing of the roles played by these biomarkers in MDD is limited.

Recent studies suggest that MDD constitutes a systemic disease, withmore widespread effects than impaired brain function alone [14], andthe peripheral inflammatory response and metabolic disorders hy-pothesis of MDD is becoming more widely accepted [15,16]. Moreover,patients with clinical depression are reported to be more likely to de-velop intense somatic symptoms, including fatigue or loss of energy,considerable weight change, and digestive problems [17]. Thus, ex-ploring peripheral systems might provide innovative insight into thepathogenesis of MDD and help to elucidate the intimate relationshipbetween the central and peripheral systems. The small intestine is theprimary site of amino acid absorption [18], and may play a key role inthe amino acid imbalance in depression. Importantly, diseases of thesmall intestine such as IBS and functional gastrointestinal disorders,have been found to exhibit high comorbidity with depression [19,20].The small intestine is a major component of the gastrointestinal tract,which communicates bidirectionally with the brain through the gut-brain axis (GBA), linking the emotional and cognitive centers of thebrain with intestinal functions [21]. Recent studies have emphasizedthe critical role of the GBA in depression, via its ability to modulate theimmune response, gastrointestinal hormone levels and microbial ac-tivity [22,23]. However, the impact of the small intestine on metabo-lism remains unclear.

In the present study, we aimed to gain a better understanding ofsystemic amino acid metabolism in depression by investigating aminoacid metabolism and transport dysfunction in the small intestine. First,depressive-like behaviors in rats were induced via exposure to chronicunpredictable mild stress (CUMS), which is considered to provide aneffective model for mimicking the behavioral and physiological symp-toms of depression [24]. To detect changes in amino acids, targetedmetabolomics approach -high-performance liquid chromatography(HPLC) combined with ultraviolet (UV) detection was applied [25]. The

correlation between these changes and behavioral phenotypes relatedto CUMS exposure was analyzed using Pearson's correlation analysis.Meanwhile, to characterize the changes in intestinal function associatedwith amino acids, three neutral amino acid transporters (ASCT2, LAT1,B0AT1) involved in the uptake of glutamine and glycine in the smallintestine [26] were examined using western blot assays. To extendoverall understanding of systemic amino acid metabolism in depres-sion, we integrated related differential amino acids identified from therat hippocampus, peripheral blood mononuclear cells (PBMCs), urineand cerebellum under CUMS conditions in our previous research[15,27–29]. The current findings provide new insight into the patho-physiology of depression by linking amino acid metabolic changes inthe central and peripheral systems, and identifying new potential drugtargets for the treatment of depression.

2. Methods and materials

2.1. Animals

This study was given consent by the Ethics Committee of ChongqingMedical University (reference number 2017013), and all procedureswere in accordance with the National Institutes of Health Guidelines forAnimal Research (Guide for the Care and Use of Laboratory Animals).Healthy adult male Sprague-Dawley rats (initial weight: 230–280 g;age: 2–3months) were obtained from the animal facility at ChongqingMedical University (Chongqing, China). Before random grouping, allanimals were maintained under standard laboratory conditions (12-hlight/dark cycle, lights on at 8:00 AM; 22 ± 1 °C ambient temperature;52 ± 2% relative humidity; with unrestricted access to food andwater) for a week to acclimatize to the environment. The CUMS pro-tocol was conducted as previously reported (Fig. 1A) [30]. Special carewas taken to minimize the number and suffering of animals.

2.2. CUMS paradigm

To reduce the heterogeneity in the experimental rats, rats with su-crose preferences of lower than 80% in the sucrose preference test (SPT)

Fig. 1. (A) Flow chart of the chronic unpredicted mild stress (CUMS) procedure. (B) Weekly schedule of the CUMS regimen.

X. Yang, et al. Physiology & Behavior 204 (2019) 199–209

200

Downloaded for Christopher Sendi (csendi@sendi.org) at Inova Fairfax Hospital - JCon from ClinicalKey.com by Elsevier on October 20, 2019.For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.

were eliminated from subsequent experiment [31].The remaining 40 rats were then randomly divided into a CUMS

group (n=20) and a control group (CON; n=20). The CUMS proce-dure was conducted as described in our previous study [11]. The ani-mals were exposed to mild stressors for a period of 4 weeks, as shown inFig. 1B. In the CUMS group, the same stressor was not given for twoconsecutive days, and all stressors were applied in a separate procedureroom. CON rats were left undisturbed in their home cages, except forcage cleaning, which was carried out periodically [32].

2.3. Behavioral assessment

2.3.1. SPT and body weightThe SPT was conducted before and after the CUMS procedure. Prior

to the SPT, rats were habituated to 1% sucrose solution for 48 h. On thetesting day, water and food were removed from the cage for 6 h. Therats were then provided with pre-weighed identical bottles of 1% su-crose and water. All fluid consumption was recorded by weighing thetwo bottles before and 1 h after the start of the test. Sucrose preferencewas defined as (sucrose consumption/total consumption)× 100%.Body weight was measured before the CUMS exposure and weeklythroughout the CUMS period.

2.3.2. Open field test (OFT)For the OFT, the rat was placed in the center zone of an open field

apparatus (100×100×40 cm) in a soundproof room, and was al-lowed to move freely for exploration. All behaviors were recorded for aperiod of 5min and 30 s, with the first 30 s used for adaptation.Between each test, the box was cleaned with alcohol and dried toeliminate olfactory cues from the previous rat. Total and central dis-tances traveled, central activity (i.e., [central distance/total dis-tance]× 100%), and number of rearings (defined as an upright posturemaintained on the hind paws) were calculated.

2.3.3. Forced swim test (FST)In the FST, rats were individually placed into cylinders (50 cm high,

20 cm in diameter) filled with water (23 ± 2 °C) to a depth of 30 cm fora 15-min training session, then gently dried and returned to their homecages. They were placed in the cylinders again 24 h later, and the 5-minFST was conducted. The total immobility time in the FST was recordedas an index of depressive-like behaviors. All sessions in the FST and OFTwere recorded with an automated video tracking system (EthoVision XT13, Noldus, Virginia, United States,).

2.4. Sample collection

Animals were euthanized with 10% chloral hydrate (100 g/0.4 ml)by intraperitoneal injection, and the distal small intestine was im-mediately dissected out because of its high expression of amino acidtransporters compared with other parts of the small intestine [33].Feces were gently separated away using tweezers. The tissues were thenrinsed in cold saline solution, and stored at −80 °C until HPLC analysisor protein extraction.

2.5. Preparation of standard solutions

Stock solutions of 20 amino acids and Norleucine (IS) were acquiredby dissolving each standard mixture (18 amino acids AAS18, L-gluta-mine 76,523, L-asparagine 51,363, N-Lorleucine N8513, Sigma-Aldrich,Germany) in 0.1M HCl solution, then labeled and stored at−20 °C. Theworking standard solutions were prepared by diluting the stock solu-tions with water to the desired concentrations of 250 nmol/ml beforeuse, TDPA (0.2%, w/v) was generally added as an antioxidant.

2.6. Dabsylation of standard amino acids and intestinal samples

Dabsylation was performed as previously reported [34,35], withminor modification. In brief, frozen intestine samples (n=7 for eachgroup) were slowly defrosted and transferred to Eppendorf tubes,weighed, homogenized with a Tissue Lyser (Jingxin Co., Ltd., Shanghai,China) at 70 Hz for 2min, and resuspended in 500 μl 0.01M HCl con-taining 0.2% TDPA and internal standard (250 nM). After deproteini-zation by mixing with 40% (w/v) trichloroacetic acid, the mixture waskept in an ice bath for 10min and centrifuged at 10,000g for 15min.The supernatants, containing amino acids, were transferred and filteredthrough a 0.45 μm pore size Millipore filter, then dehydrated in vacuo.The dehydrated samples and amino acid standards were redissolved in50 μl reaction buffer (0.15 mM NaHCO3, pH 8.6). A 5-μl aliquot ofstandard amino acids was blended with 45 μl reaction buffer, and allsamples and standard amino acids were mixed with 50 μl 12.4 mMdabsyl chloride reagent (40mg dabsyl chloride in 10ml of acetone),heated at 70 °C, and vortexed at 1400 rpm for 15min with a thermalshaker (Thermo Fisher Scientific). The reaction was stopped by in-cubating in an ice bath for 5min. Then, 100 μl dilution buffer (50mlacetonitrile, 25ml ethanol and 25ml mobile phase A) was added to thesolution and centrifuged at 15,000g for 5min. The supernatant wastransferred to a 250 μl insert (polypropylene; Agilent Technologies,USA) and subjected to HPLC analysis.

2.7. HPLC gradient system for determination of amino acids

In addition to reacting with amino acids, dabsyl chloride also reactswith water to produce sodium dimethylaminoazobenzene 4-sulfonate,which is detected as a large early peak in the HPLC trace. The de-termination of dabsylated amino acids from intestinal samples wascarried out on an Agilent 1260 HPLC system with a UV detector at436 nm. A reverse-phase column (Hypersil GOLD Thermo,250mm×4mm i.d., 5 μm particle size) coupled with a C18 cartridge(12mm×8mm i.d., 5 μm) was used. The column temperature was setat 50 °C. The flow rate and injection volume were 1.0ml/min and 20 μl,respectively. Mobile phase A (pH=6.55) consisted of 9mM KH2PO4,4% DMF, 0.1% TEA and 0.055% phosphoric acid. Mobile phase Bconsisted of 80/20 acetonitrile (Merck KGaA, Darmstadt, Germany)/ultrapure water. Both mobile phases were passed through a 0.22-μmmembrane filter prior to use. The gradient elution profile is shown inTable 1.

2.8. Western blotting

Frozen small intestinal tissue samples were obtained from both theCUMS group (n=6) and the CON group (n=6). Each sample washomogenized in RIPA Lysis Buffer (Beyotime, Beijing, China), con-taining 0.1% proteases inhibitors, 1% phosphatase inhibitors and 1%PMSF, on ice. The homogenates were then sonicated (70 Hz, pulse for18 s, rest for 20 s; 3×) and centrifuged at 14,000g at 4 °C for 5min. Theprotein concentrations were quantified using a BCA protein assay kit(Beyotime). Solubilized proteins were resolved by electrophoresis on10% SDS-PAGE gels, then transferred onto polyvinylidene difluoridemembranes (Merck, Germany) and subjected to immunoblot analysiswith a rabbit anti-ASCT2 polyclonal antibody (ab84903, Abcam;1:1000), rabbit anti-B0AT1 (ab180516, Abcam; 1:500), mouse

Table 1Gradient elution profile of the HPLC.

Time (min) 0 3 10.5 52.5 67.5 99 100 110 120Solvent B(%) 8 8 20 35 50 100 100 8 8Solvent A(%) 92 92 80 65 50 0 0 92 92

Solvent B: 80/20 acetonitrile; solvent A: 9 mM KH2PO4, 4% DMF, 0.1% TEAand 0.055% phosphoric acid (pH 6.55).

X. Yang, et al. Physiology & Behavior 204 (2019) 199–209

201

Downloaded for Christopher Sendi (csendi@sendi.org) at Inova Fairfax Hospital - JCon from ClinicalKey.com by Elsevier on October 20, 2019.For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.

monoclonal anti-LAT1 (sc-374,232, Santa Cruz, China; 1:1000) or arabbit monoclonal β-actin antibody (generous gift from Bioss, China)overnight at 4 °C. All membranes were washed and incubated with theirrespective horseradish peroxidase-coupled secondary antibody (Bio-Rad). The signal intensities of the immunoreactive bands, detected withan ECL kit (Millipore), were quantified using Quantity One software(Bio-Rad). The proteins were normalized to β-actin, and the controlvalue was set to 1.

2.9. Statistical analysis

Data are presented as mean ± standard error. Repeated measuresanalysis of variance (ANOVA) was used to analyze body weight data.Unless otherwise stated, Student's t-tests were used to analyze sig-nificant differences between the two groups for the OFT, SPT, FST,amino acid concentrations and western blot analysis. All tests were two-tailed. Pearson's correlation analysis was used to evaluate the associa-tion between the behavioral phenotype and the altered amino acidconcentration. The significance threshold for all analyses was set atp < .05. All analyses were performed using SPSS Statistics 21.0 (IBM,Armonk, NY, USA). The plots were produced with GraphPad Prism 6.0(La Jolla, China), Cluster 3.0 (Tokyo) and Venny 2.1.0 (bioinfogp.cnb.csic.es/tools/venny/).

3. Results

3.1. A 4-week CUMS protocol successfully induced depressive-like behavior

Before the CUMS protocol, no significant differences were foundbetween the two groups in body weight or sucrose preference (Figs. 2Aand B). After 4 weeks of exposure to the stressors, rats in the CUMSgroup gained significantly less body weight than control rats(p < .001, repeated measures ANOVA). Body weight was significantlylower in the CUMS group than in the CON group over the last 3 weeksof the CUMS exposure protocol (week 2, p < .05; week 3, p < .001;week 4, p < .001; Fig. 2A). The SPT is considered to be a reliable testfor the evaluation of chronic stress-related anhedonia, a core symptomof depression [36]. At the end of the 4-week CUMS protocol, rats in theCUMS group displayed impaired sucrose preference compared withbaseline period (p < .001; Fig. 2B) and sucrose preference was mark-edly reduced in the CUMS group compared with the CON group(p < .05; Fig. 2B). The FST serves for assessing depression-relateddespair-like behaviors [37,38]. Compared with the CON group, im-mobility time in the FST was significantly elevated in the CUMS group(p < .01; Fig. 2C). Additionally, in the OFT, rats in the CUMS groupshowed decreased exploratory behavior, with significantly reducedcentral distance (p < .05; Fig. 2E) and central activity (p < .01;Fig. 2F). However, there was no significant difference in the total dis-tance (Fig. 2D) or the number of rearings (Fig. 2G) between the twogroups.

3.2. Altered intestinal Asp, Gln, Gly concentration among 20 amino acids

Previous validation studies have confirmed that the HPLC methodexhibits good performance in terms of linearity, accuracy, repeatabilityand stability [35,39]. The chromatograms for the CON and CUMSgroups and the standard curve are presented in Fig. 3. The chromato-grams displayed strong signals as well as a large peak capacity and goodreproducibility in the retention times. Identification of the 20 aminoacids and the dabsyl chloride reagent were performed as previouslyreported [35]. For the determination of the concentration of the re-spective amino acids, the ratio of the peak areas of the amino acid andthe corresponding internal standard was used. Table 2 lists the con-centrations of the 20 amino acids in the small intestinal tissue. Fig. 4Ashows a heat map of the fold-changes in the amino acids. The analysisrevealed a number of differential amino acids between the control and

CUMS groups. Glutamine (p < .05; Fig. 4B) and glycine (p < .05;Fig. 4C) concentrations were reduced, while aspartic acid concentrationwas increased (p < .05; Fig. 4D) in the CUMS group compared with thecontrol group.

3.3. Analysis of the correlation between differential amino acids andbehavioral phenotypes

In the present study, the correlations of glutamine, glycine and as-partic acid levels with behavioral outcomes (body weight, total dis-tance, central distance, central activity, rearing times, sucrose pre-ference, immobility time) were analyzed. Of all the results, a significantnegative correlation was observed between aspartic acid concentrationand body weight in the fourth week (r=−0.65, p= .011; Fig. 5A), andglycine levels were positively associated with sucrose preferenceparameters (r=0.53, p= .047; Fig. 5B). In addition, glutamine levelsexhibited a moderate correlation with immobility time (r=−0.53,p= .049; Fig. 5C) and sucrose preference (r=0.54, p= .045; Fig. 5D).No other significant correlations emerged.

3.4. Dysfunction of three amino acid transporters was uncovered by westernblot analysis

Three candidate proteins participating in the uptake of glutamineand glycine in the small intestine (ASCT2, B0AT1 and LAT1) weresubjected to western blot analysis for validation (Fig. 6A). The ex-pression levels of ASCT2 (p < .01; Fig. 6B) were significantly de-creased in the CUMS group compared with the CON group. In contrast,LAT1 expression was increased (p < .001; Fig. 6C), while B0AT1 ex-pression was reduced (p < .05; Fig. 6D) in the CUMS group. Details ofthe protein bands are included in Supplemental Fig. 1.

3.5. Integrated analysis for amino acid changes induced by CUMS

Previous studies in our laboratory revealed numerous metabolitechanges in CUMS model rats using non-targeted profiling, including aset of differential metabolites in the hippocampus, cerebellum, urineand PBMCs involved in amino acid metabolism. A Venn diagram of allthe significant metabolites in the various tissues in the CUMS rat isshown in Fig. 7A. An overview of changes in glycine (GLY), glutamine(`GLN) and aspartate (ASP) in the hippocampus, cerebellum, PBMCs,urine and small intestine in the CUMS rat model is shown in Fig. 7B.

4. Discussion

MDD is increasingly recognized as a systemic disease that frequentlyoccurs with various metabolic disorders [40]. Although substantial re-search effort has focused on the investigation of amino acid metabolismunder depressed conditions, previous studies have typically focused onthe central nervous system and circulatory system rather than the smallintestine. In the enterocytic apical membrane of the small intestine, aninitial step of amino acid intake takes place, likewise, it acts as the mainsource of amino acids in the body [41]. Herein, we successfully induceda depressive phenotype in rats by exposure to a 4-week CUMS protocol.Afterward, an targeted metabolomics approach was used for the firsttime to identify CUMS-induced amino acid changes in the small intes-tine. Rats in the CUMS group exhibited significantly decreased levels ofneutral amino acids glutamine and glycine, and increased levels of as-partic acid. Subsequently, western blot analysis was carried out todisclose the impaired biological processes of glutamine and glycinetransport in the small intestine by examining the expression levels ofcorresponding transporters.

4.1. Reduced transport of glutamine and glycine in the small intestine

Two Na+-dependent neutral amino acid transporters, B0AT1

X. Yang, et al. Physiology & Behavior 204 (2019) 199–209

202

Downloaded for Christopher Sendi (csendi@sendi.org) at Inova Fairfax Hospital - JCon from ClinicalKey.com by Elsevier on October 20, 2019.For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.

(SLC6A19) and ASCT2 (SLC1A5), undertake glutamine absorption fromthe diet across the apical membrane of intestinal epithelial cells [26].B0AT1 belongs to the B0 system, which accepts a broad variety ofneutral amino acids, with the highest affinity for glutamine [42]. B0AT1plays a critical role in glutamine uptake, as shown by B0AT1-deficientmice, in which Na+-dependent uptake of glutamine in the small in-testine is completely abolished. These mice also display a decrease inglucose uptake of approximately 50%, and a reduction in body weight[43]. Thus, the decrease in B0AT1 expression might underlie the re-duction in body weight during the 4-week CUMS procedure. ASCT2, animportant NA+-dependent transporter of the ASC system that is mainlylocalized in the intestine [44], mediates the obligatory exchange ofamino acids such as glutamine [45]. ASCT2 is a member of the solutecarrier 1 (SLC1) family. Interestingly, the other members of this familyare intimately related to depression. The SLC1 proteins, including ex-citatory amino acid transporters EAAT4 (SLC1A6), EAAT3 (SLC1A1),EAAT2 (SLC1A2) EAAT1 (SLC1A3) [46,47]. LAT1 (SLC7A5) is a NA+

and pH-independent transmembrane transporter in the SLC7 familythat belongs to the L system [48]. LAT1 imports large and neutral es-sential amino acids in exchange for intracellular amino acids (e.g.,glutamine) on the cell membrane surface [49]. Here, we found in-creased expression of LAT1 in the CUMS group compared with controls.This increase might diminish net glutamine uptake via the small in-testine. Interestingly, LAT1 expression is highly upregulated in manytumor cell lines and cancers [50]. Therefore, the elevated expression ofLAT1 in depressed animals might increase their risk of cancer. The

transport pathway for glutamine is shown in Fig. 8A.The majority of glycine appears to be transported by the B0 system

and hPAT1 in brush border epithelial cells in the intestinal mucosa[51]. Moreover, ASCT2 mediates glycine influx across apical mem-branes in the rat jejunum [52]. ASCT2 has substrate specificity, trans-porting small neutral amino acids with Km values of 20 μM. Glycine istransported with the highest Km value (300–500 μM) [53]. This processis depicted in Fig. 8B.

Taken together, our findings suggest that alterations in the expres-sion of transporters might cause a reduction in the absorption of glu-tamine and glycine in the CUMS rat model.

4.2. Systemic amino acid changes induced by CUMS

Glutamine, the most abundant free amino acid in the body, is aconditionally essential nutrient under stressful conditions [54]. Patientswith stress-related major depression typically exhibit reduced brainglutamine levels [40]. In the CUMS rat model, a lack of glutamine wassimultaneously detected in the hippocampus and cerebellum, in linewith the results of another study reporting that glutamine levels aredownregulated in both the rat hippocampus and cerebral cortex afterCUMS exposure [34]. This deficiency in the brain has been reported toincrease despair-like behaviors and anhedonia [9], and these behaviorsare also associated with reduced intestinal glutamine levels as wellthrough correlation analysis. Furthermore, as it easily crosses the blood-brain barrier, it is not surprising that glutamine is frequently used as a

Fig. 2. CUMS-induced depressive-like behaviors. (A) Body weight at baseline and during the consecutive 4-week course of the CUMS protocol. (B) Sucrose preferenceat baseline and after the CUMS procedure. (C) Immobility time in the forced swim test. (D–G) Total distance, central distance, central activity, and number of rearingsin the open field test. *p < .05, **p < .01, ***p < .001. NS, not significant. CON, non-stress control (n=20); CUMS, chronic unpredictable mild stress (n=20).

X. Yang, et al. Physiology & Behavior 204 (2019) 199–209

203

Downloaded for Christopher Sendi (csendi@sendi.org) at Inova Fairfax Hospital - JCon from ClinicalKey.com by Elsevier on October 20, 2019.For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.

biomarker of depression [55], as supported by urine metabolism data inthe CUMS model. Here, we found decreased glutamine caused by ab-sorption disorder in the small intestine. The small intestine is the majorabsorption site for glutamine, providing the vast majority of this aminoacid to the body [56]. Therefore, the markedly reduced transport ofglutamine in the small intestine might contribute to the circulatingglutamine deficiency to regulate depressive-like behavior in the CUMSmodel. Importantly, however, a glutamine-enriched diet is reported todirectly ameliorate the uptake of glutamine in the rat small intestine[57]. Meanwhile, this oral glutamine intervention pattern has beenreported to lead to mood improvement and [58] and alleviation ofanxiety-related behavior [59]. The current finding of decreased ab-sorption of glutamine in the small intestine appears to support thepossibility that a glutamine-enriched diet could be an effective treat-ment for depression.

Glycine is an important inhibitory neurotransmitter in the brain andfunctions as an NMDAR co-agonist [60]. In addition, decreased glycine

levels were found in the hippocampus under CUMS by another group[61]. Here, we observed reduced glycine content in both the PBMCs andcerebellum, and an increased loss of the amino acid via the urine in theCUMS model, likely caused by intestinal dysfunction. The currentlyprevailing view is that mammals may fail to adequately synthesizeglycine under certain conditions [62]. Thus, exogenous supplementa-tion could potentially satisfy this need, and oral glycine has been re-ported to increase serum glycine, as well as ameliorating cognitive anddepressive symptoms [8,63]. However, despite its substantial protectiveeffect [64], there is evidence that excessive glycine intake can lead totoxicity [65]. Therefore, identifying a glycine transport pathway dis-order in the intestine and describing the relationships between in-testinal glycine and depressive behavior could reveal diversified andsafe approaches for the treatment of this symptom.

Increased levels of aspartic acid have also been found in the smallintestine using HPLC [66]. In the current study, we did not find achange in aspartate transport in the small intestine. In the central

Fig. 3. HPLC chromatogram of (A) control rats and (B) CUMS rats. (C) Standard curve (250 nmol/l) peaks: (1) aspartic acid, (2) glutamic acid, (3) DABS-CL, (4)asparagine, (5) glutamine, (6) serine, (7) threonine, (8) glycine, (9) arginine, (10) alanine, (11) proline, (12) valine, (13) methionine, (14) isoleucine, (15) leucine,(16) NorLeu, (17) phenylalanine, (18) ammonia, (19) lysine, (20) histidine, (21) tyrosine.

X. Yang, et al. Physiology & Behavior 204 (2019) 199–209

204

Downloaded for Christopher Sendi (csendi@sendi.org) at Inova Fairfax Hospital - JCon from ClinicalKey.com by Elsevier on October 20, 2019.For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.

Table 2The concentrations of the 20 intestinal amino acids in the CON and CUMS groups.

Amino acids Peaks Retention time (min) CON(ng/mg) CUMS(ng/mg)

Aspartic acida 1 27.92 76.1784 ± 44.1347 131.5736 ± 45.1296Glutamic acid 2 28.821 149.6524 ± 117.5978 115.5661 ± 48.8441DABS-Cl 3 37.117Asparagine 4 39.725 419.9245 ± 197.4087 407.0917 ± 84.5289Glutaminea 5 40.338 190.0276 ± 49.2903 133.1381 ± 25.2770Serine 6 41.796 712.6484 ± 246.2514 525.4560 ± 111.4971Threonine 7 43.004 547.0401 ± 189.0790 456.1394 ± 112.1896Glycinea 8 44.151 388.0036 ± 97.7157 274.2105 ± 57.7137Arginine 9 45.754 2376.6764 ± 1071.9974 2322.9480 ± 534.9061Alanine 10 47.982 905.6265 ± 339.2173 856.8156 ± 232.2708Proline 11 53.105 4608.5908 ± 1513.8826 5014.9110 ± 1226.0399Valine 12 55.253 761.2319 ± 202.2400 611.7856 ± 98.1757Methioine 13 59.668 268.4017 ± 108.7400 211.7385 ± 46.7570IsoLeucine 14 61.97 511.5241 ± 138.0210 417.1333 ± 67.8860Leucine 15 63.306 741.3980 ± 120.5776 956.6248 ± 282.2273Norleucine (IS) 16 64.263 1358.7845 ± 416.9936 1183.4732 ± 146.7748Phenylalanine 17 64.905 499.0420 ± 215.6406 444.2102 ± 136.8661Ammonia 18 66.036 797.2727 ± 212.1280 624.3178 ± 103.4970Lysine 19 79.979 163.0843 ± 91.5155 166.5961 ± 85.7381Histidine 20 80.711 40.7241 ± 15.7827 32.2616 ± 10.2027Tyrosine 21 84.053 62.5686 ± 37.0554 57.8573 ± 24.0056

CON: control; CUMS: chronic unpredictable mild stress; DABS-Cl: dabsyl chloride; IS: internal standard. The concentrations of the amino acids are expressed asmean ± SEM (n=7)

a p < .05.

Fig. 4. Concentrations of amino acids in the small intestine measured by HPLC. (A) Hierarchical clustering was performed using the amino acids in both groups basedon their fold changes. (B, C, D) The concentrations of glutamine and glycine in the CUMS group (n=7) were downregulated, while the concentration of aspartic acidwas upregulated compared with the CON (n=7) group.

X. Yang, et al. Physiology & Behavior 204 (2019) 199–209

205

Downloaded for Christopher Sendi (csendi@sendi.org) at Inova Fairfax Hospital - JCon from ClinicalKey.com by Elsevier on October 20, 2019.For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.

nervous system, aspartate usually functions as a regulator of neuralplasticity by specifically activating the NMDA receptor in its acetylatedform, N-acetyl-L-aspartic acid, which is the second most abundantamino acid in the brain [67]. This process is generally considered toexert antidepressant effects [68]. In the enteric nervous system, whichhas been described as “the brain within the gut” [69], Nagahama et al.reported that aspartate could function as a neuromodulator by detec-tion of Asp-immunoreactive neurons in the small intestine [70]. As-partate also regulates intestinal function via the NMDAR [71]. InCUMS-exposed rats, reduced concentrations of N-acetyl-L-aspartic acidwere found in the hippocampus, and increased levels were found in thesmall intestine. We speculate that the resultant changes in aspartatesignaling might contribute to the intestinal and central symptoms in

depression.

4.3. Amino acids involved in the GBA

The GBA has been increasingly linked to mood and cognition withgut activity in MDD. The existence of reciprocal interactions is unclear,but represents an intriguing possibility [22]. Changes in the brain (oftentriggered by stress) can influence immune activation, entero-endocrinesignaling, epithelial cell functions, as well as intestinal permeability andbarrier function. Conversely, intestinal components, such as cytokines,short-chain fatty acids, nutrients and microflora, may affect centralprocesses, such as neurotransmission and behavior [72]. Moreover,glutamine, glycine and aspartate play a critical role in maintaining gut

Fig. 5. Correlation between differential amino acids (CON, n=7; CUMS, n=7) and corresponding behavioral results (CON, n=7; CUMS, n=7) (A) aspartic acidand body weight (B) glycine and sucrose preference (C) glutamine and immobility time (D) glutamine and sucrose preference.

Fig. 6. Western blot validation of selected proteins involved in the absorption of amino acids. (A) protein bands (30 μg in each line) for ASCT, B0AT1 and LAT1 in theCUMS (n=6) and CON (n=6) groups were analyzed using Quantity One software. (B, D) The expression of ASCT2 and B0AT1 were significantly decreased in theCUMS rats compared with controls. (C) Expression levels of LAT1 were significantly increased in CUMS rats compared with controls. The expression levels arenormalized to β-actin. *p < .05, **p < .01. ***p < .001.

X. Yang, et al. Physiology & Behavior 204 (2019) 199–209

206

Downloaded for Christopher Sendi (csendi@sendi.org) at Inova Fairfax Hospital - JCon from ClinicalKey.com by Elsevier on October 20, 2019.For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.

health and preventing intestinal diseases [73]. These amino acids reg-ulate inflammation by controlling NF-κB signaling and proin-flammatory cytokine secretion, the growth and viability of cells main-tained in culture, and apoptosis [74]. These metabolites mightmodulate other GBA signaling pathways, and exacerbate mental andphysical symptoms in depression.

5. Conclusion

Taken together with our previous findings, the current results sug-gest that the decreased absorption of glycine and glutamine in the smallintestine may contribute to their systemic deficiency in depressive rats.Furthermore, this findings provide insight into the cellular and mole-cular mechanisms underlying the GBA. Therefore, in addition to mi-crobiota, cytokines and gastrointestinal hormones, which have alreadybeen reported to link the GBA with MDD, amino acid disorder mightalso play a pivotal role in the pathophysiology of the disorder.Importantly, the current results indicate novel therapeutic targets forthe treatment of depression, and lays the foundation for future studiesof this debilitating mental disorder.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.physbeh.2019.03.001.

Conflicts of interest

The authors declare no conflict of interest in the submission of thismanuscript.

Acknowledgments

This work was financially supported by the National Basic ResearchProgram of China (973 Program, Grant No. 2017YFA0505700), theNational Natural Science Foundation of China (Grant No. 81701360),the China Postdoctoral Science Foundation funded project (Grant No.2018T110950) and the Chongqing Science & Technology Commission(Grant No. cstc2017jcyjA0207).

References

[1] V. Krishnan, E.J. Nestler, The molecular neurobiology of depression, Nature 455(7215) (2008) 894–902.

[2] R.C. Kessler, M. Angermeyer, J.C. Anthony, R. DEG, K. Demyttenaere, I. Gasquet,G. DEG, S. Gluzman, O. Gureje, J.M. Haro, et al., Lifetime prevalence and age-of-onset distributions of mental disorders in the World Health Organization's worldmental health survey initiative, World Psychiatry 6 (3) (2007) 168–176.

[3] C.D. Mathers, D. Loncar, Projections of global mortality and burden of disease from2002 to 2030, PLoS Med. 3 (11) (2006) e442.

[4] T. Benton, J. Staab, D.L. Evans, Medical co-morbidity in depressive disorders, Ann.Clin. Psychiatry Off. J. Am. Acad. Clin. Psychiatr. 19 (4) (2007) 289.

[5] P.B. Wjh, M. Yuri, L. Femke, V. Nicole, Understanding the somatic consequences ofdepression: biological mechanisms and the role of depression symptom profile, BMCMed. 11 (1) (2013) 129.

[6] G.S. Bennett, G.M. Edelman, Amino acid incorporation into rat brain proteinsduring spreading cortical depression, Science 163 (3865) (1969) 393–395.

[7] C.R. Markus, Dietary amino acids and brain serotonin function; implications forstress-related affective changes, NeuroMolecular Med. 10 (4) (2008) 247–258.

[8] D. Strzelecki, P. Kropiwnicki, J. Rabe-Jablonska, Augmentation of antipsychoticswith glycine may ameliorate depressive and extrapyramidal symptoms in schizo-phrenic patients–a preliminary 10-week open-label study, Psychiatr. Pol. 47 (4)(2013) 609–620.

[9] Y. Lee, H. Son, G. Kim, S. Kim, D.H. Lee, G.S. Roh, S.S. Kang, G.J. Cho, W.S. Choi,H.J. Kim, Glutamine deficiency in the prefrontal cortex increases depressive-like

Fig. 7. (A) Venn diagram of all the significant metabolites in the hippocampus, cerebellum, PBMCs, and urine in the CUMS rat (B) Overview of the changes in glycine(GLY), glutamine (GLN) and aspartate (ASP) in the hippocampus, cerebellum, PBMCs, urine and small intestine in the CUMS rat model.

Fig. 8. (A) Glutamine transport mediated by ASCT2, B0AT1 and LAT1. (B) Reduced expression of ASCT2 and B0AT1 leads to a reduction in glycine.

X. Yang, et al. Physiology & Behavior 204 (2019) 199–209

207

Downloaded for Christopher Sendi (csendi@sendi.org) at Inova Fairfax Hospital - JCon from ClinicalKey.com by Elsevier on October 20, 2019.For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.

behaviours in male mice, J. Psychiatry Neurosci. 38 (3) (2013) 183–191.[10] A. Kugaya, G. Sanacora, Beyond monoamines: glutamatergic function in mood

disorders, CNS spectrums 10 (10) (2005) 808–819.[11] G. Chen, D. Yang, Y. Yang, J. Li, C. Ke, T. Ge, R. Zhang, J. Zhou, W. Li, L. Zhao,

Amino acid metabolic dysfunction revealed in the prefrontal cortex of a rat model ofdepression, Behav. Brain Res. 278 (278C) (2015) 286–292.

[12] C. Rao, H. Shi, C. Zhou, D. Zhu, M. Zhao, Z. Wang, Y. Yang, J. Chen, L. Liao, J. Tang,Hypothalamic proteomic analysis reveals Dysregulation of glutamate balance andenergy metabolism in a mouse model of chronic mild stress-induced depression,Neurochem. Res. 41 (9) (2016) 2443–2456.

[13] P. Zheng, J.J. Chen, C.J. Zhou, L. Zeng, K.W. Li, L. Sun, M.L. Liu, D. Zhu, Z.H. Liang,P. Xie, Identification of sex-specific urinary biomarkers for major depressive dis-order by combined application of NMR- and GC–MS-based metabonomics, Transl.Psychiatry 6 (11) (2016) e955.

[14] P.J. Gianaros, T.D. Wager, Brain-body pathways linking psychological stress andphysical health, Curr. Dir. Psychol. Sci. 24 (4) (2015) 313.

[15] J. Li, G. Tang, K. Cheng, D. Yang, G. Chen, Z. Liu, R. Zhang, J. Zhou, L. Fang,Z. Fang, et al., Peripheral blood mononuclear cell-based metabolomic profiling of achronic unpredictable mild stress rat model of depression, Mol. BioSyst. 10 (11)(2014) 2994–3001.

[16] R.K. Farooq, K. Asghar, S. Kanwal, A. Zulqernain, Role of inflammatory cytokines indepression: focus on interleukin-1beta, Biomed. Reports 6 (1) (2017) 15–20.

[17] G.E. Simon, M. VonKorff, M. Piccinelli, C. Fullerton, J. Ormel, An internationalstudy of the relation between somatic symptoms and depression, N. Engl. J. Med.341 (18) (1999) 1329–1335.

[18] F. Moog, The lining of the small intestine, Sci. Am. 245 (5) (1981) 154–158.[19] E. Urdang, Psychiatric disorder and bowel disease, Am. J. Psychiatr. 148 (7) (1991)

958–959.[20] P.C. Keightley, N.A. Koloski, N.J. Talley, Pathways in gut-brain communication:

evidence for distinct gut-to-brain and brain-to-gut syndromes, Aust. N Z JPsychiatry 49 (3) (2015) 207–214.

[21] C. Marilia, S. Annunziata, M.M. Antonietta, S. Carola, The gut-brain axis: interac-tions between enteric microbiota, central and enteric nervous systems, Ann.Gastroenterol. Q. Publ. Hell. Soc. Gastroenterology 28 (2) (2015) 203–209.

[22] A. Evrensel, M.E. Ceylan, The gut-brain Axis: the missing link in depression, Clin.Psychopharmacol. Neurosci. 13 (3) (2015) 239–244.

[23] T.C. Fung, C.A. Olson, E.Y. Hsiao, Interactions between the microbiota, immune andnervous systems in health and disease, Nat. Neurosci. 20 (2) (2017) 145.

[24] P. Willner, The chronic mild stress (CMS) model of depression: history, evaluationand usage, Neurobiol. Stress 6 (C) (2016) 78.

[25] E. Dudley, M. Yousef, Y. Wang, W.J. Griffiths, Targeted metabolomics and massspectrometry, Adv Protein Chem Struct Biol 80 (2010) 45–83.

[26] S. Bröer, Amino acid transport across mammalian intestinal and renal epithelia,Physiol. Rev. 88 (1) (2008) 249–286.

[27] W.H. Shao, J.J. Chen, S.H. Fan, Y. Lei, H.B. Xu, J. Zhou, P.F. Cheng, Y.T. Yang,C.L. Rao, B. Wu, et al., Combined metabolomics and proteomics analysis of majordepression in an animal model: perturbed energy metabolism in the chronic mildstressed rat cerebellum, Omics. J. Integr. Biol. 19 (7) (2015) 383–392.

[28] Y. Zhang, S. Yuan, J. Pu, L. Yang, X. Zhou, L. Liu, X. Jiang, H. Zhang, T. Teng,L. Tian, Integrated metabolomics and proteomics analysis of Hippocampus in a ratmodel of depression, Neuroscience 371 (2017) 207–220.

[29] X.J. Liu, Z.Y. Li, Z.F. Li, X.X. Gao, Y.Z. Zhou, H.F. Sun, L.Z. Zhang, X.Q. Guo,G.H. Du, X.M. Qin, Urinary metabonomic study using a CUMS rat model of de-pression, Magn. Reson. Chem. 50 (3) (2012) 187–192.

[30] D. Yang, X. Liu, R. Zhang, K. Cheng, J. Mu, L. Fang, P. Xie, Increased apoptosis anddifferent regulation of pro-apoptosis protein bax and anti-apoptosis protein bcl-2 inthe olfactory bulb of a rat model of depression, Neurosci. Lett. 504 (1) (2011)18–22.

[31] H. Akimoto, S. Oshima, T. Sugiyama, A. Negishi, T. Nemoto, D. Kobayashi, Changesin brain metabolites related to stress resilience: Metabolomic analysis of the hip-pocampus in a rat model of depression, Behav. Brain Res. 359 (2019) 342–352.

[32] J. Mu, P. Xie, Z.S. Yang, D.L. Yang, F.J. Lv, T.Y. Luo, Y. Li, Neurogenesis and majordepression: implications from proteomic analyses of hippocampal proteins in a ratdepression model, Neurosci. Lett. 416 (3) (2007) 252.

[33] B.E. Stefan, Epithelial neutral amino acid transporters: lessons from mouse models,Curr. Opin. Nephrol. Hypertens. 22 (5) (2013) 539–544.

[34] P. Yang, X. Li, J. Ni, J. Tian, F. Jing, C. Qu, L. Lin, H. Zhang, Alterations of aminoacid level in depressed rat brain, Korean J. Physiol. Pharmacol. 18 (5) (2014)371–376.

[35] I. Krause, A. Bockhardt, H. Neckermann, T. Henle, H. Klostermeyer, Simultaneousdetermination of amino acids and biogenic amines by reversed-phase high-perfor-mance liquid chromatography of the dabsyl derivatives, J. Chromatogr. A 715 (1)(1995) 67–79.

[36] M.Y. Liu, C.Y. Yin, L.J. Zhu, X.H. Zhu, C. Xu, C.X. Luo, H. Chen, D.Y. Zhu,Q.G. Zhou, Sucrose preference test for measurement of stress-induced anhedonia inmice, Nat. Protoc. 13 (7) (2018) 1686–1698.

[37] F. Bakhtiarzadeh, A. Nahavandi, M. Goudarzi, S. Shirvalilou, K. Rakhshan,S. Niknazar, Axonal transport proteins and depressive like behavior, followingchronic unpredictable mild stress in male rat, Physiol. Behav. 194 (2018).

[38] Yankelevitch-Yahav R, Franko M, Huly A, Doron R: The forced swim test as a modelof depressive-like behavior. J. Vis. Exp. 2015(97).

[39] D. Drnevich, T.C. Vary, Analysis of physiological amino acids using dabsyl deri-vatization and reversed-phase liquid chromatography, J. Chromatogr. 613 (1)(1993) 137.

[40] Y. Shirayama, M. Takahashi, F. Osone, A. Hara, T. Okubo, Myo-inositol, glutamate,and glutamine in the prefrontal cortex, Hippocampus, and amygdala in major

depression, Biol. Psychiatry Cogn. Neurosc. Neuroimaging 2 (2) (2017) 196–204.[41] C.M. Anderson, D.S. Grenade, M. Boll, M. Foltz, K.A. Wake, D.J. Kennedy,

L.K. Munck, S. Miyauchi, P.M. Taylor, F.C. Campbell, H+/amino acid transporter 1(PAT1) is the imino acid carrier: an intestinal nutrient/drug transporter in humanand rat, Gastroenterology 127 (5) (2004) 1410–1422.

[42] A. Bröer, K. Klingel, S. Kowalczuk, J.E.J. Rasko, J. Cavanaugh, S. Bröer, Molecularcloning of mouse amino acid transport system B0, a neutral amino acid transporterrelated to Hartnup disorder, J. Biol. Chem. 279 (23) (2004) 24467.

[43] A. Broer, T. Juelich, J.M. Vanslambrouck, N. Tietze, P.S. Solomon, J. Holst,C.G. Bailey, J.E. Rasko, S. Broer, Impaired nutrient signaling and body weightcontrol in a Na+ neutral amino acid cotransporter (Slc6a19)-deficient mouse, J.Biol. Chem. 286 (30) (2011) 26638–26651.

[44] B.P. Bode, Recent molecular advances in mammalian glutamine transport, J. Nutr.131 (9 Suppl) (2001) 2475S.

[45] Q. Wang, R.A. Hardie, A.J. Hoy, M.V. Geldermalsen, D. Gao, L. Fazli,M.C. Sadowski, S. Balaban, M. Schreuder, Inhibition of ASCT2-mediated glutamineuptake blocks prostate cancer growth, Prostate Cancer World Congress: 2015, 2015,p. 32.

[46] K. Takahashi, J.B. Foster, C.L. Lin, Glutamate transporter EAAT2: regulation,function, and potential as a therapeutic target for neurological and psychiatricdisease, Cell. Mol. Life Sci. 72 (18) (2015) 3489–3506.

[47] Y.B. Wei, P.A. Melas, J.C. Villaescusa, J.J. Liu, N. Xu, S.H. Christiansen,H. Elbrøndbek, D.P.D. Woldbye, G. Wegener, A.A. Mathé, MicroRNA 101b isDownregulated in the prefrontal cortex of a genetic model of depression and targetsthe glutamate transporter SLC1A1 (EAAT3) in vitro, Int. J. Neuropsychopharmacol.19 (12) (2016) pyw069.

[48] D. Fotiadis, Y. Kanai, N.M. Palacã, The SLC3 and SLC7 families of amino acidtransporters, Mol. Asp. Med. 34 (2–3) (2013) 139–158.

[49] N. Singh, G.F. Ecker, Insights into the structure, function, and ligand discovery ofthe large neutral amino acid transporter 1, LAT1, Int. J. Mol. Sci. 19 (5) (2018)1278.

[50] B.C. Fuchs, B.P. Bode, Amino acid transporters ASCT2 and LAT1 in cancer: partnersin crime? Semin. Cancer Biol. 15 (4) (2005) 254–266.

[51] G. Tunnicliff, Membrane glycine transport proteins, J. Biomed. Sci. 10 (1) (2003)30–36.

[52] Y. Kanai, M.A. Hediger, The glutamate and neutral amino acid transporter family:physiological and pharmacological implications, Eur. J. Pharmacol 479 (1) (2003)237–247.

[53] N. Utsunomiyatate, H. Endou, Y. Kanai, Cloning and functional characterization ofa system ASC-like Na+−dependent neutral amino acid transporter, J. Biol. Chem.271 (25) (1996) 14883–14890.

[54] Peng Li, YuLong Yin, Defa Li, W. Kim, Wu Sung, Guoyao: amino acids and immunefunction, Br. J. Nutr. 98 (2) (2007) 237–252.

[55] A. Mayoral-Mariles, C. Cruz-Revilla, X. Vega-Manriquez, R. Aguirre-Hernandez,P. Severiano-Perez, E. Aburto-Arciniega, A. Jimenez-Mendoza, R. Guevara-Guzman,Plasma amino acid levels discriminate between control subjects and mildly de-pressed elderly women, Arch. Med. Res. 43 (5) (2012) 375–382.

[56] D. P, D. D, R. M, H. B, R. O, JF D: absorption and metabolic effects of enterallyadministered glutamine in humans, Am. J. Phys. 260 (1) (1991) 677–682.

[57] R.M. Salloum, W.W. Souba, A. Fernandez, B.R. Stevens, Dietary modulation of smallintestinal glutamine transport in intestinal brush border membrane vesicles of rats,J. Surg. Res. 48 (6) (1990) 635–638.

[58] L.S. Young, R. Bye, M. Scheltinga, T.R. Ziegler, D.O. Jacobs, D.W. Wilmore, Patientsreceiving glutamine-supplemented intravenous feedings report an improvement inmood, JPEN J. Parenter. Enteral Nutr. 17 (5) (1993) 422–427.

[59] D.S. de Lima, E.D. Francisco, C.B. Lima, R.C. Guedes, Neonatal L-glutamine mod-ulates anxiety-like behavior, cortical spreading depression, and microglial im-munoreactivity: analysis in developing rats suckled on normal size- and large sizelitters, Amino Acids 49 (2) (2017) 337–346.

[60] J.W. Johnson, P. Ascher, Glycine potentiates the NMDA response in cultured mousebrain neurons, Nature 325 (6104) (1987) 529–531.

[61] T. Cui, H.M. Qiu, D. Huang, Q.X. Zhou, X.Y. Fu, H.Y. Li, X.H. Jiang, Abnormal levelsof seven amino neurotransmitters in depression rat brain and determination byHPLC-FLD, Biomed. Chromatogr. Bmc 31 (2017).

[62] W. Wang, Z. Wu, Z. Dai, Y. Yang, J. Wang, G. Wu, Glycine metabolism in animalsand humans: implications for nutrition and health, Amino Acids 45 (3) (2013)463–477.

[63] U. Heresco-Levy, D.C. Javitt, M. Ermilov, C. Mordel, G. Silipo, M. Lichtenstein,Efficacy of high-dose glycine in the treatment of enduring negative symptoms ofschizophrenia, Arch. Gen. Psychiatry 56 (1) (1999) 29–36.

[64] Z. Zhong, M.D. Wheeler, X. Li, M. Froh, P. Schemmer, M. Yin, H. Bunzendaul,B. Bradford, J.J. Lemasters, L-Glycine: a novel antiinflammatory, im-munomodulatory, and cytoprotective agent, Curr. Opin. Clin. Nutr. Metab Care 6(2) (2003) 229–240.

[65] S. Shoham, D.C. Javitt, U. Heresco-Levy, High dose glycine nutrition affects glialcell morphology in rat hippocampus and cerebellum, Int. J.Neuropsychopharmacol. 2 (01) (1999) 35–40.

[66] M.W. Fleck, D.A. Henze, G. Barrionuevo, A.M. Palmer, Aspartate and glutamatemediate excitatory synaptic transmission in area CA1 of the hippocampus, J.Neurosci. Off. J. Soc. Neurosci. 13 (9) (1993) 3944.

[67] T.B. Patel, J.B. Clark, Synthesis of N-acetyl-L-aspartate by rat brain mitochondriaand its involvement in mitochondrial/cytosolic carbon transport, Biochem. J. 184(3) (1979) 539–546.

[68] A.E. Autry, M. Adachi, E. Nosyreva, E.S. Na, M.F. Los, P.F. Cheng, E.T. Kavalali,L.M. Monteggia, NMDA receptor blockade at rest triggers rapid behavioural anti-depressant responses, Nature 475 (7354) (2011) 91–95.

X. Yang, et al. Physiology & Behavior 204 (2019) 199–209

208

Downloaded for Christopher Sendi (csendi@sendi.org) at Inova Fairfax Hospital - JCon from ClinicalKey.com by Elsevier on October 20, 2019.For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.

[69] A.M. Goldstein, R. Hofstra, A.J. Burns, Building a brain in the gut: developmentofthe enteric nervous system, Clin. Genet. 83 (4) (2013) 307–316.

[70] M. Nagahama, N. Ma, R. Semba, L -aspartate-immunoreactive neurons in the ratenteric nervous system, Cell Tissue Res. 318 (3) (2004) 483–492.

[71] G.A. Burns, K.E. Stephens, J.A. Benson, Expression of mRNA for the N-methyl-D-aspartate (NMDAR1) receptor by the enteric neurons of the rat, Neurosci. Lett. 170(1) (1994) 87.

[72] G.B. Rogers, D.J. Keating, R.L. Young, M.L. Wong, J. Licinio, S. Wesselingh, From

gut dysbiosis to altered brain function and mental illness: mechanisms and path-ways, Mol. Psychiatry 21 (2016) 738.

[73] W.W. Wang, S.Y. Qiao, D.F. Li, Amino acids and gut function, Amino Acids 37 (1)(2009) 105–110.

[74] G. Chen, J. Shi, M. Qi, H. Yin, C. Hang, Glutamine decreases intestinal nuclearfactor kappa B activity and pro-inflammatory cytokine expression after traumaticbrain injury in rats, Inflamm. Res. 57 (2) (2008) 57–64.

X. Yang, et al. Physiology & Behavior 204 (2019) 199–209

209

Downloaded for Christopher Sendi (csendi@sendi.org) at Inova Fairfax Hospital - JCon from ClinicalKey.com by Elsevier on October 20, 2019.For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.