Differential Effect of Non-Purified and Semi-Purified Standard Diets on Kynurenine and Peripheral Metabolites in Male C57BL/6J Mice

The kynurenine (Kyn) pathway plays crucial roles in several inflammation-induced disorders such as depression. In this study, we measured Kyn and other related molecules in the blood plasma, brain, and urine of male C57BL/6J mice (B6) fed non-purified (MF) and semi-purified (AIN-93G and AIN-93M) standard rodent diets. Mice fed MF had increased plasma Kyn levels compared with those on AIN93-based diets, as well as decreased hippocampal Kyn levels compared with those fed AIN-93G. Previous studies showed that branched chain amino acids (BCAAs) suppress peripheral blood Kyn transportation to the brain, but plasma BCAA levels were not significantly different between the diet groups in our study. Urine metabolome analysis revealed that feed ingredients affected the excretion of many metabolites, and MF-fed mice had elevated excretion of kynurenic and quinolinic acids, pivotal metabolites in the Kyn pathway. Collectively, the level of critical metabolites in the Kyn pathway in the central and peripheral tissues was strongly affected by feed ingredients. Therefore, feed selection is a critical factor to ensure the reproducibility of experimental data in studies involving rodent models.

Indoleamine 2,3-Dioxygenase-1 Expression is Changed During Bladder Cancer Cell Invasion

The severity of the bladder carcinoma (BC) is directly linked to cell invasion and metastasis. Indoleamine 2,3-dioxygenase-1 (IDO-1) is an INF-γ-induced immunomodulating enzyme that has been linked to the cancer cell invasiveness. Because IDO1 is variable among the tumors, we analyzed its expression in the BC invasion using BC mice models and cell culture. MB49 cells were orthotopically or ectopically inoculated in C57Bl6 mice to evaluate IDO1 by immunohistochemistry. For in vitro experiments, expression of IDO1 and INF-γ was evaluated in grade-1 (RT4) and in grade-3 (T24) BC cell lines. Invading and non-invading T24 cells were separated using the Matrigel/Transwell system, of which total RNA was extracted immediately or after 2 weeks of subculture. Finally, IDO1 was silenced in T24 cells to verify its role on cell invasiveness. In both animal models, IDO1 was differentially expressed between non-invading and invading cells. In cell culture, T24 cells expressed more IDO1 than RT4 cells, independently of the INF-γ expression. IDO1 was differentially expressed between non-invading and invading T24 cells, a difference that was lost by long-time subculture. IDO1 silencing resulted in diminished cell invasiveness. In conclusion, IDO1 expression is changed during bladder carcinoma invasion, playing an important role in this process.

The Effects of Maternal Interleukin-17A on Social Behavior, Cognitive Function, and Depression-Like Behavior in Mice with Altered Kynurenine Metabolites

Viral infection and chronic maternal inflammation during pregnancy are correlated with a higher prevalence of autism spectrum disorder (ASD). However, the pathoetiology of ASD is not fully understood; moreover, the key molecules that can cross the placenta following maternal inflammation and contribute to the development of ASD have not been identified. Recently, the pro-inflammatory cytokine, interleukin-17A (IL-17A) was identified as a potential mediator of these effects. To investigate the impact of maternal IL-17A on offspring, C57BL/6J dams were injected with IL-17A-expressing plasmids via the tail vein on embryonic day 12.5 (E12.5), and maternal IL-17A was expressed continuously throughout pregnancy. By adulthood, IL-17A-injected offspring exhibited behavioral abnormalities, including social and cognitive defects. Additionally, maternal IL-17A promoted metabolism of the essential amino acid tryptophan, which produces several neuroactive compounds and may affect fetal neurodevelopment. We observed significantly increased levels of kynurenine in maternal serum and fetal plasma. Thus, we investigated the effects of high maternal concentration of kynurenine on offspring by continuously administering mouse dams with kynurenine from E12.5 during gestation. Obviously, maternal kynurenine administration rapidly increased kynurenine levels in the fetal plasma and brain, pointing to the ability of kynurenine to cross the placenta and change the KP metabolites which are affected as neuroactive compounds in the fetal brain. Notably, the offspring of kynurenine-injected mice exhibited behavioral abnormalities similar to those observed in offspring of IL-17A-conditioned mice. Several tryptophan metabolites were significantly altered in the prefrontal cortex of the IL-17A-conditioned and kynurenine-injected adult mice, but not in the hippocampus. Even though we cannot exclude the possibility or other molecules being related to ASD pathogenesis and the presence of a much lower degree of pathway activation, our results suggest that increased kynurenine following maternal inflammation may be a key factor in changing the balance of KP metabolites in fetal brain during neuronal development and represents a therapeutic target for inflammation-induced ASD-like phenotypes.

Lack of Skeletal Muscle Serotonin Impairs Physical Performance

Low levels of the neurotransmitter serotonin have been associated with the onset of depression. While traditional treatments include antidepressants, physical exercise has emerged as an alternative for patients with depressive disorders. Yet there remains the fundamental question of how exercise is sensed by the brain. The existence of a muscle–brain endocrine loop has been proposed: according to this scenario, exercise modulates metabolization of tryptophan into kynurenine within skeletal muscle, which in turn affects the brain, enhancing resistance to depression. But the breakdown of tryptophan into kynurenine during exercise may also alter serotonin synthesis and help limit depression. In this study, we investigated whether peripheral serotonin might play a role in muscle–brain communication permitting adaptation for endurance training. We first quantified tryptophan metabolites in the blood of 4 trained athletes before and after a long-distance trail race and correlated changes in tryptophan metabolism with physical performance. In parallel, to assess exercise capacity and endurance in trained control and peripheral serotonin–deficient mice, we used a treadmill incremental test. Peripheral serotonin–deficient mice exhibited a significant drop in physical performance despite endurance training. Brain levels of tryptophan metabolites were similar in wild-type and peripheral serotonin–deficient animals, and no products of muscle-induced tryptophan metabolism were found in the plasma or brains of peripheral serotonin–deficient mice. But mass spectrometric analyses revealed a significant decrease in levels of 5-hydroxyindoleacetic acid (5-HIAA), the main serotonin metabolite, in both the soleus and plantaris muscles, demonstrating that metabolization of tryptophan into serotonin in muscles is essential for adaptation to endurance training. In light of these findings, the breakdown of tryptophan into peripheral but not brain serotonin appears to be the rate-limiting step for muscle adaptation to endurance training. The data suggest that there is a peripheral mechanism responsible for the positive effects of exercise, and that muscles are secretory organs with autocrine-paracrine roles in which serotonin has a local effect.

An Investigation into the Temporal Reproducibility of Tryptophan Metabolite Networks Among Healthy Adolescents

Tryptophan and its bioactive metabolites are associated with health conditions such as systemic inflammation, cardiometabolic diseases, and neurodegenerative disorders. There are dynamic interactions among metabolites of tryptophan. The interactions between metabolites, particularly those that are strong and temporally reproducible could be of pathophysiological relevance. Using a targeted metabolomics approach, the concentration levels of tryptophan and 18 of its metabolites across multiple pathways was quantified in 24-hours urine samples at 2 time-points, age 17 years (baseline) and 18 years (follow-up) from 132 (52% female) apparently healthy adolescent participants of the DOrtmund Nutritional and Anthropometric Longitudinally Designed (DONALD) Study. In sex-specific analyses, we applied 2 network approaches, the Gaussian graphical model and Bayesian network to (1) explore the network structure for both time-points, (2) retrieve strongly related metabolites, and (3) determine whether the strongly related metabolites were temporally reproducible. Independent of selected covariates, the 2 network approaches revealed 5 associations that were strong and temporally reproducible. These were novel relationships, between kynurenic acid and indole-3-acetic acid in females and between kynurenic acid and xanthurenic acid in males, as well as known relationships between kynurenine and 3-hydroxykynurenine, and between 3-hydroxykynurenine and 3-hydroxyanthranilic acid in females and between tryptophan and kynurenine in males. Overall, this epidemiological study using network-based approaches shed new light into tryptophan metabolism, particularly the interaction of host and microbial metabolites. The 5 observed relationships suggested the existence of a temporally stable pattern of tryptophan and 6 metabolites in healthy adolescent, which could be further investigated in search of fingerprints of specific physiological states. The metabolites in these relationships may represent a multi-biomarker panel that could be informative for health outcomes.

Temporary Intermediates of L-Trp Along the Reaction Pathway of Human Indoleamine 2,3-Dioxygenase 1 and Identification of an Exo Site

Protein dynamics governs most of the fundamental processes in the human body. Particularly, the dynamics of loops located near an active site can be involved in the positioning of the substrate and the reaction mechanism. The understanding of the functioning of dynamic loops is therefore a challenge, and often requires the use of a multi-disciplinary approach mixing, for example, crystallographic experiments and molecular dynamics simulations. In the present work, the dynamic behavior of the JK-loop of the human indoleamine 2,3-dioxygenase 1 hemoprotein, a target for immunotherapy, is investigated. To overcome the lack of knowledge on this dynamism, the study reported here is based on 3 crystal structures presenting different conformations of the loop, completed with molecular dynamics trajectories and MM-GBSA analyses, in order to trace the reaction pathway of the enzyme. In addition, the crystal structures identify an exo site in the small unit of the enzyme, that is populated redundantly by the substrate or the product of the reaction. The role of this newer reported exo site still needs to be investigated.

Effects of Psychological and Physical Stress on Oxidative Stress, Serotonin, and Fatigue in Young Females Induced by Objective Structured Clinical Examination: Pilot Study of u-8-OHdG, u-5HT, and s-HHV-6

Background: To establish a method to prevent and manage fatigue caused by psychological and physical stress in young females, early detection factors, such as understanding of fatigue and causes of psychological and physical stress, as well as a review of early management of psychiatric disease, are important. With increasing knowledge regarding the diverse causes of stress, it is important to select biomarkers with consideration of the types of stress burden and mechanisms underlying the development of physical symptoms. The methods used to search for stress characteristics is an issue that needs to be addressed. However, consensus regarding objective assessment methods for impaired mental health is lacking. Methods: We examined the effects of an objective structured clinical examination (OSCE), considered to be a uniform source of psychological and physical stress, on biomarkers of oxidative stress and fatigue in 16 third-year female medical university students (21.3 ± 2.1 years old) in Japan with a normal menstrual cycle. A self-administered questionnaire consisting of Zung’s Self-rating Depression Scale (SDS) and State-Trait Anxiety Inventory (STAI) was used to assess subjective stress. Furthermore, stress-related biomarkers (urinary 8-hydroxy-2ʹ-deoxyguanosine [u-8-OHdG], urinary 5-hydroxytryptamine [u-5-HT], and salivary human herpesvirus-6 [s-HHV-6]) were examined at 1 month, 1 week, and 1 day before, and 1 week after the OSCE. Results: The results indicated that the OSCE did not have effects on u-8-OHdG, a biomarker of oxidative stress. However, u-5-HT and s-HHV-6 were found to be elevated in examinations performed prior to the OSCE. Conclusions: The present findings suggest that u-5-HT and s-HHV-6 levels can be used for objective assessment of mental and physical fatigue in young females, including that produced not only by knowledge regarding an upcoming OSCE, but also by skill and attitude aspects related to that examination.

Plasma Anthranilic Acid and Leptin Levels Predict HAM-D Scores in Depressed Women

Objectives: Major depressive disorder (MDD) is associated with dysregulations of leptin and tryptophan–kynurenine (Trp–Kyn) (TKP) pathways. Leptin, a pro-inflammatory cytokine, activates Trp conversion into Kyn. However, leptin association with down-stream Kyn metabolites in MDD is unknown. Methods: Fasting plasma samples from 29 acutely ill drug-naïve (n = 16) or currently non-medicated (⩾6 weeks; n = 13) MDD patients were analyzed for leptin, Trp, Kyn, its down-stream metabolites (anthranilic [AA], kynurenic [KYNA], xanthurenic [XA] acids and 3-hydroxykynurenine [3HK]), C-reactive protein (CRP), neopterin, body mass index (BMI), and insulin resistance (HOMA-IR). Depression severity was assessed by HAM-D-21. Results: In female (n = 14) (but not in male) patients HAM-D-21 scores correlated with plasma levels of AA (but not other Kyn metabolites) (rho = −0.644, P = .009) and leptin (Spearman’s rho = −0.775, P = .001). Inclusion of AA into regression analysis improved leptin prediction of HAM-D from 48.5% to 65.9%. Actual HAM-D scores highly correlated with that calculated by formula: HAM-D = 34.8518−(0.5660 × leptin [ng/ml] + 0.4159 × AA [nmol/l]) (Rho = 0.84, P = .00015). In male (n = 15) (but not in female) patients leptin correlated with BMI, waist circumference/hip ratio, CRP, and HOMA-IR. Conclusions: Present findings of gender specific AA/Leptin correlations with HAM-D are important considering that AA and leptin are transported from plasma into brain, and that AA formation is catalyzed by kynureninase—the only TKP gene associated with depression according to genome-wide analysis. High correlation between predicted and actual HAM-D warrants further evaluation of plasma AA and leptin as an objective laboratory test for the assessment of depression severity in female MDD patients

Neurotransmitter Precursor Amino Acid Ratios Show Differential, Inverse Correlations with Depression Severity in the Low and High Depression Score Range

The immunomodulatory capacity of mental stress is one of the basic concepts of psychoneuroimmunology. The current prospective longitudinal study was designed to evaluate the effect of acute mental stress on neurotransmitter precursor amino acid levels in individuals with depression at 2 time points. Ten physically healthy patients with a diagnosis of major depressive episode and Montgomery–Åsberg Depression Rating Scale scores (MADRAS) ⩾20 points at inclusion were assessed on 2 study days (once with higher MADRAS scores, once with lower MADRAS scores; median 34.5 days apart) and subjected to a standardized acute mental stress test on each study day. Blood was collected at 4 time points: once prior to and at 3 time points (0, 30 minutes, 60 minutes) following mental stress. Neurotransmitter precursor amino acid levels, that is kynurenine/tryptophan (KYN/TRP) and phenylalanine/tyrosine (PHE/TYR), as well as neopterin and nitrite were analyzed in a total of 80 individual blood samples. Regression and correlation analyses were performed. Regression analyses of PHE/TYR ( R2 = .547) and KYN/TRP ( R2 = .440) in relation to MADRAS depression severity showed a quadratic curve fit. This was reflected by a negative linear correlation between MADRAS scores and PHE/TYR as well as KYN/TRP in the lower score range ( r = −.805, P < .001 and r = −.586, P < .001 respectively) and a positive correlation in the higher MADRAS score range ( r = .713, P < .001 and r = .379, P = .016 respectively). No effect of acute mental stress was found. This analysis exemplifies the implications of sampling as well as data distributions on results. The crosstalk of biological mechanisms that orchestrate metabolic and immunological signaling may vary depending on depression severity resulting in non-linear associations that may explain the heterogeneity of results found in the literature.

Blockade of KAT II Facilitates LTP in Kynurenine 3-Monooxygenase Depleted Mice

Excess of brain kynurenic acid (KYNA), a neuroactive metabolite of the kynurenine pathway, is known to elicit cognitive dysfunction. In the present study, we investigated spatial working memory in mice with elevated levels of KYNA, induced by targeted deletion of kynurenine 3-monooxygenase (KMO), as well as long-term potentiation (LTP) of field excitatory postsynaptic potentials (fEPSPs) in hippocampal brain slices from these mice. The KMO knock-out (KMO−/−) mice performed more poorly in the spatial working memory task as compared to their wild-type (WT) counterparts, as reflected by fewer correct choices in a T-maze. Both fEPSPs, or LTP, did not significantly differ between the 2 mouse strains. However, administration of PF-04859989, a kynurenine aminotransferase (KAT) II inhibitor, limiting the production of KYNA, facilitated fEPSP and enhanced LTP to a greater extent in hippocampal slices from KMO−/− mice compared to WT mice. The results of the present study point to an essential role for KYNA in modulating LTP in the hippocampus of KMO−/− mice which may account for their dysfunctional spatial working memory.