Bioavailability and Metabolism of Bioactive Peptide IRW with Angiotensin-Converting Enzyme 2 (ACE2) Upregulatory Activity in Spontaneously Hypertensive Rats.

Peptide IRW is the first food-derived angiotensin-converting enzyme 2 (ACE2) upregulator. This study aimed to investigate the pharmacokinetic characteristics of IRW and identify the metabolites contributing to its antihypertensive activity in spontaneously hypertensive rats (SHRs). Rats were administered 100 mg of IRW/kg of the body weight via an intragastric or intravenous route. The bioavailability (F %) was determined to be 11.7%, and the half-lives were 7.9 ± 0.5 and 28.5 ± 6.8 min for gavage and injection, respectively. Interestingly, significant blood pressure reduction was not observed until 1.5 h post oral administration, or 2 h post injection, indicating that the peptide's metabolites are likely responsible for the blood pressure-lowering activity. Time-course metabolomics revealed a significant increase in the level of kynurenine, a tryptophan metabolite, in blood after IRW administration. Kynurenine increased the level of ACE2 in cells. Oral administration of tryptophan (W), but not dipeptide IR, lowered the blood pressure and upregulated aortic ACE2 in SHRs. Our study supports the key role of tryptophan and its metabolite, kynurenine, in IRW's blood pressure-lowering effects.

Graphene oxide quantum dots (GOQDs) induce behavioral disorders via the disturbance of kynurenine pathway in zebrafish larvae.

The emergence of graphene quantum dots (GQDs) expands the use of graphene derivatives in nanomedicine for its direct therapeutic applications in treating neurodegeneration, inflammation, metabolic dysfunction, and among others. Nevertheless, the biosafety assessment of GQDs remains deficient mostly because of the diverse surface characteristics of the nanoparticles. Our prior work demonstrated that GQDs can induce strong thigmotactic effects in zebrafish larvae over a wide range of concentrations, yet the underlying metabolic mechanisms remain largely unknown. In this study, we conducted a further exploration about graphene oxide quantum dots (GOQDs) for its potential neurotoxic effect on the behaviors of zebrafish larvae by combining neurotransmitter-targeted metabolomics with locomotion analysis. After continuous exposure to a concentration gradient of GOQDs (12.5 - 25 - 50 - 100 - 200 µg/mL) for 7 days, the thigmotactic activities of zebrafish larvae were observed across all exposure concentrations relative to the control group, while the basal locomotor activities, including distance moved and average velocity, were significantly changed by low concentrations of GOQDs. Targeted metabolomics was performed using zebrafish larvae at 7 days post-fertilization (dpf) that were exposed to 12.5 and 200 µg/mL, both of which were found to perturb the kynurenine pathway by regulating the levels of kynurenine, 3-hydroxyanthranilic acid (3-HAA), and quinolinic acid (QA). Furthermore, the thigmotaxis of larval fish induced by GOQDs during exposure could be counteracted by supplementing Ro-61-8048, an agonist acting on kynurenine 3-monooxygenase (KMO). In conclusion, our study establishes the involvement of the kynurenine pathway in GOQDs-induced thigmotaxis, which is independent of the transcriptional modulation of glutamate receptor families.

TDO2-overexpressed Dendritic Cells Possess Tolerogenicity and Ameliorate Collagen-induced Arthritis by Modulating the Th17/Regulatory T Cell Balance.

Tolerogenic dendritic cells are promising for restoring immune homeostasis and may be an alternative therapy for autoimmune diseases such as rheumatoid arthritis. The kynurenine pathway is a vital mechanism that induces tolerance in dendritic cells (DCs). Tryptophan 2,3-dioxygenase (TDO2) is an important rate-limiting enzyme in the kynurenine pathway and participates in immune regulation. However, the role of TDO2 in shaping the tolerogenic phenotypes of DCs remains unclear. In this study, we investigated the effects and mechanisms of TDO2-overexpressed DCs in regulating the T cell balance both in vivo and in vitro. TDO2-overexpressed DC2.4 and TDO2-/- mouse bone marrow-derived DCs (BMDCs) were generated to verify the role of TDO2 in DC maturation and functionality. TDO2 overexpression in BMDCs via PGE2 treatment exhibited an immature phenotype and tolerogenic state, whereas TDO2-/- BMDCs exhibited a mature phenotype and a proinflammatory state. Furthermore, transplant of TDO2-overexpressed BMDCs alleviated collagen-induced arthritis severity in mice, which was correlated with a reduction in Th17 populations and an increase in regulatory T cells. Collectively, these results indicate that TDO2 plays an important role in the tolerogenic phenotype and may be a promising target for the generation tolerogenic DCs for rheumatoid arthritis treatment.

Evaluation of D-amino acid oxidase activity in rat kidney using a D-kynurenine derivative, 6-methylthio-D-kynurenine: An in vivo microdialysis study.

D-Amino acid oxidase (DAO), a D-amino acid metabolizing enzyme, is reportedly associated with the psychiatric disease schizophrenia, suggesting a role for DAO inhibitors in its treatment. We have previously reported that DAO catalyzes the conversion of nonfluorescent 6-methylthio-D-kynurenine (MeS-D-KYN) to fluorescent 5-methylthiokynurenic acid (MeS-KYNA) in vitro. The present study aimed to determine the potential of MeS-D-KYN in evaluating DAO activity in vivo using renal microdialysis technique in rats. Male Sprague-Dawley rats were subjected to linear microdialysis probe implantation in the left kidney. Continuous perfusion of MeS-D-KYN was maintained, and DAO activity in the kidney cortex was evaluated by measuring the MeS-KYNA content in the microdialysate. The microdialysate was collected every 30 min and analyzed by high-performance liquid chromatography with fluorescence detection, monitored at 450 nm with an excitation wavelength of 364 nm. A significant production of MeS-KYNA was observed during, but not before, infusion of MeS-D-KYN, indicating that this compound is not endogenous. MeS-KYNA production was suppressed by the co-infusion of DAO inhibitor, 5-chlorobenzo[d]isoxazol-3-ol (CBIO), suggesting that MeS-D-KYN was converted to MeS-KYNA by renal DAO. Moreover, oral administration of CBIO effectively suppressed DAO activity in a dose-dependent manner. DAO converted MeS-D-KYN to MeS-KYNA in vivo, suggesting the potential of this compound in evaluating DAO activity. The use of the renal microdialysis technique developed in this study facilitates the monitoring of DAO activity in live experimental animals.

Kynurenine attenuates mitochondrial depolarization and neuronal cell death induced by rotenone exposure independently of AhR-mediated parkin induction in SH-SY5Y differentiated cells.

Rotenone is a pesticide commonly used in agriculture that is associated with the risk of developing Parkinson's disease (PD) by inducing mitochondrial damage. As a protective cell response to different challenges, they activate mitophagy, which involves parkin activity. Parkin is an E3 ubiquitin ligase necessary in the initial steps of mitophagy, and its overexpression protects against parkinsonian effects in different models. Recent studies have reported that the aryl hydrocarbon receptor (AHR), a ligand-dependent transcription factor, induces parkin expression. Kynurenine, an endogenous AHR ligand, promotes neuroprotection in chronic neurodegenerative disorders, such as PD, although its neuroprotective mechanism needs to be fully understood. Therefore, we evaluated whether the overexpression of parkin by AHR activation with kynurenine promotes autophagy and reduces the neurotoxicity induced by rotenone in SH-SY5Y cells differentiated to dopaminergic neurons. SH-SY5Y neurons were treated with rotenone or pretreated with kynurenine or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and parkin levels, apoptosis, mitochondrial potential membrane, and autophagy were determined. The results showed that kynurenine and TCDD treatments induced parkin expression in an AHR-dependent manner. Kynurenine pretreatment inhibited rotenone-induced neuronal apoptosis in 17%, and the loss of mitochondrial membrane potential in 30% when compare to rotenone alone, together with a decrease in autophagy. By contrast, although TCDD treatment increased parkin levels, non-neuroprotective effects were observed. The kynurenine protective activity was AHR independent, suggesting that parkin induction might not be related to this effect. On the other hand, kynurenine treatment inhibited alpha amine-3-hydroxy-5-methyl-4-isoxazol propionic acid and N-methyl-D-aspartate receptors, which are well-known excitotoxicity mediators activated by rotenone exposure.

L-Kynurenine participates in cancer immune evasion by downregulating hypoxic signaling in T lymphocytes.

Malignant tumors often escape anticancer immune surveillance by suppressing the cytotoxic functions of T lymphocytes. While many of these immune evasion networks include checkpoint proteins, small molecular weight compounds, such as the amino acid L-kynurenine (LKU), could also substantially contribute to the suppression of anti-cancer immunity. However, the biochemical mechanisms underlying the suppressive effects of LKU on T-cells remain unclear. Here, we report for the first time that LKU suppresses T cell function as an aryl hydrocarbon receptor (AhR) ligand. The presence of LKU in T cells is associated with AhR activation, which results in competition between AhR and hypoxia-inducible factor 1 alpha (HIF-1α) for the AhR nuclear translocator, ARNT, leading to T cell exhaustion. The expression of indoleamine 2,3-dioxygenase 1 (IDO1, the enzyme that leads to LKU generation) is induced by the TGF-ß-Smad-3 pathway. We also show that IDO-negative cancers utilize an alternative route for LKU production via the endogenous inflammatory mediator, the high mobility group box 1 (HMGB-1)-interferon-gamma (IFN-γ) axis. In addition, other IDO-negative tumors (like T-cell lymphomas) trigger IDO1 activation in eosinophils present in the tumor microenvironment (TME). These mechanisms suppress cytotoxic T cell function, and thus support the tumor immune evasion machinery.

NLG-919 combined with cisplatin to enhance inhibitory effect on cell migration and invasion via IDO1-Kyn-AhR pathway in human nasopharyngeal carcinoma cell.

As a common aggressive head and neck cancer, nasopharyngeal carcinoma (NPC) received cisplatin treatment as a first-line chemotherapy. Platinum-induced resistance is a major limitation of current treatment strategy in the advanced NPC. Increased indoleamine 2,3-dioxygenase (IDO1) activities are found in cisplatin-resistant NPC cells versus cisplatin-sensitive NPC cells. As an IDO1 immunosuppressant, NLG-919 has entered clinical phase I to treat advanced solid tumors. To reverse cisplatin resistance, we investigated the combinatory application of cisplatin and NLG-919 in NPC treatment. In vitro biological studies on cisplatin-resistant and cisplatin-sensitive NPC cells were taken to imply that the combination of NLG-919 and cisplatin got a stronger impact on the induction of cell apoptosis and the inhibition of cell migration, exploring superior effect of antitumor over single drug. We proved that the mechanism of the combined therapy could inhibit the activity of IDO1, blocking amino acid tryptophan conversion to kynurenine through the kynurenine pathway, which further inhibited the aryl hydrocarbon receptor expression. Our study underscored the combination of cisplatin and NLG-919 as a potent therapeutic way for the reversal of cisplatin resistance.

Protocatechuic acid prevents isoproterenol-induced heart failure in mice by downregulating kynurenine-3-monooxygenase.

Protocatechuic acid (3,4-dihydroxybenzoic acid) prevents oxidative stress, inflammation and cardiac hypertrophy. This study aimed to investigate the therapeutic effects of protocatechuic acid in an isoproterenol-induced heart failure mouse model and to identify the underlying mechanisms. To establish the heart failure model, C57BL/6NTac mice were given high-dose isoproterenol (80 mg/kg body weight) for 14 days. Echocardiography revealed that protocatechuic acid reversed the isoproterenol-induced downregulation of fractional shortening and ejection fraction. Protocatechuic acid attenuated cardiac hypertrophy as evidenced by the decreased heart-weight-to-body-weight ratio and the expression of Nppb. RNA sequencing analysis identified kynurenine-3-monooxygenase (Kmo) as a potential target of protocatechuic acid. Protocatechuic acid treatment or transfection with short-interfering RNA against Kmo ameliorated transforming growth factor ß1-induced upregulation of Kmo, Col1a1, Col1a2 and Fn1 in vivo or in neonatal rat cardiac fibroblasts. Kmo knockdown attenuated the isoproterenol-induced increase in cardiomyocyte size, as well as Nppb and Col1a1 expression in H9c2 cells or primary neonatal rat cardiomyocytes. Moreover, protocatechuic acid attenuated Kmo overexpression-induced increases in Nppb mRNA levels. Protocatechuic acid or Kmo knockdown decreased isoproterenol-induced ROS generation in vivo and in vitro. Thus, protocatechuic acid prevents heart failure by downregulating Kmo. Therefore, protocatechuic acid and Kmo constitute a potential novel therapeutic agent and target, respectively, against heart failure.

Orchiectomy sensitizes cortical bone in male mice to the harmful effects of kynurenine.

Kynurenine (Kyn) is a tryptophan metabolite that increases with age and promotes musculoskeletal dysfunction. We previously found a sexually dimorphic pattern in how Kyn affects bone, with harmful effects more prevalent in females than males. This raises the possibility that male sex steroids might exert a protective effect that blunts the effects of Kyn in males. To test this, orchiectomy (ORX) or sham surgeries were performed on 6-month-old C57BL/6 mice, after which mice received Kyn (10 mg/kg) or vehicle via intraperitoneal injection, once daily, 5×/week, for four weeks. Bone histomorphometry, DXA, microCT, and serum marker analyses were performed after sacrifice. In vitro studies were performed to specifically test the effect of testosterone on activation of aryl hydrocarbon receptor (AhR)-mediated signaling by Kyn in mesenchymal-lineage cells. Kyn treatment reduced cortical bone mass in ORX- but not sham-operated mice. Trabecular bone was unaffected. Kyn's effects on cortical bone in ORX mice were attributed primarily to enhanced endosteal bone resorption activity. Bone marrow adipose tissue was increased in Kyn-treated ORX animals but was unchanged by Kyn in sham-operated mice. ORX surgery increased mRNA expression of the aryl hydrocarbon receptor (AhR) and its target gene Cyp1a1 in the bone, suggesting a priming and/or amplification of AhR signaling pathways. Mechanistic in vitro studies revealed that testosterone blunted Kyn-stimulated AhR transcriptional activity and Cyp1a1 expression in mesenchymal-linage cells. These data suggest a protective role for male sex steroids in blunting the harmful effects of Kyn in cortical bone. Therefore, testosterone may play an important role in regulating Kyn/AhR signaling in musculoskeletal tissues, suggesting crosstalk between male sex steroids and Kyn signaling may influence age-associated musculoskeletal frailty.

Interleukin-6 promotes skeletal muscle catabolism by activating tryptophan-indoleamine 2,3-dioxygenase 1-kynurenine pathway during intra-abdominal sepsis.

BACKGROUND: Inflammatory cytokine interleukin-6 (IL-6) plays a pivotal role in skeletal muscle degradation after intra-abdominal sepsis (IAS), with mechanism remained to be elucidated. Indoleamine 2,3-dioxygenase 1 (IDO-1), a key enzyme in converting tryptophan into kynurenine, could be activated by IL-6, and kynurenine has been shown to be involved in muscle degradation. We hypothesized that IL-6 could promote muscle degradation via tryptophan-IDO-1-kynurenine pathway in IAS patients. METHODS: Serum and rectus abdominis (RA) were obtained from IAS or non-IAS patients. Mouse model of IAS-induced muscle wasting was generated by caecal ligation and puncture (CLP) and lipopolysaccharide (LPS) injection. IL-6 signalling was blocked by anti-mouse IL-6 antibody (IL-6-AB), and the IDO-1 pathway was blocked by navoximod. To elucidate the role of kynurenine in muscle mass and physiology, kynurenine was administered to IAS mice treated with IL-6-AB. RESULTS: Compared to non-IAS patients, kynurenine levels in serum (+2.30-fold vs. non-IAS, P < 0.001) and RA (+3.11-fold vs. non-IAS, P < 0.001) were elevated, whereas tryptophan levels in serum (-53.65% vs. non-IAS, P < 0.01) and RA (-61.39% vs. non-IAS, P < 0.01) were decreased. Serum IL-6 level of the IAS group was significantly higher compared to non-IAS patients (+5.82-fold vs. non-IAS, P = 0.01), and muscle cross-sectional area (MCSA) was markedly reduced compared to non-IAS patients (-27.73% vs. non-IAS, P < 0.01). In animal experiments, IDO-1 expression was up-regulated in the small intestine, colon and blood for CLP or LPS-treated mice, and there was correlation (R2  = 0.66, P < 0.01) between serum and muscle kynurenine concentrations. Navoximod significantly mitigated IAS-induced skeletal muscle loss according to MCSA analysis (+22.94% vs. CLP, P < 0.05; +23.71% vs. LPS, P < 0.01) and increased the phosphorylated AKT (+2.15-fold vs. CLP, P < 0.01; +3.44-fold vs. LPS, P < 0.01) and myosin heavy chain (+3.64-fold vs. CLP, P < 0.01; +2.13-fold vs. LPS, P < 0.01) protein expression in myocytes. In the presence of anti-IL-6 antibody, a significantly decreased IDO-1 expression was observed in the small intestine, colon and blood in CLP or LPS mice (all P < 0.01), whereas the decrease of MCSA was alleviated (+37.43% vs. CLP + IgG, P < 0.001; +30.72% vs. LPS + IgG, P < 0.001). In contrast, additional supplementation of kynurenine decreased the MCSA in septic mice treated with IL-6-AB (both P < 0.01). CONCLUSIONS: This study provided novel insights into the tryptophan-IDO-1-kynurenine-dependent mechanisms that underlie inflammatory cytokine-induced skeletal muscle catabolism during intra-abdominal sepsis.

Cognitive and hippocampal effects of adult male mice exposure to environmentally relevant doses of phthalates.

We recently showed that chronic exposure of adult male mice to environmental doses of DEHP alone or in a phthalate mixture altered blood brain barrier integrity and induced an inflammatory profile in the hippocampus. Here, we investigate whether such exposure alters hippocampus-dependent behavior and underlying cellular mechanisms. Adult C57BL/6 J male mice were continuously exposed orally to the vehicle or DEHP alone (5 or 50 µg/kg/d) or to DEHP (5 µg/kg/d) in a phthalate mixture. In the Morris water maze, males showed reduced latencies across days to find the platform in the cue and spatial reference memory tasks, regardless of their treatment group. In the probe test, DEHP-50 exposed males displayed a higher latency to find the platform quadrant. In the temporal order memory test, males exposed to DEHP alone or in a phthalate mixture were unable to discriminate between the most recently and previously seen objects. They also displayed reduced ability to show a preference for the new object in the novel object recognition test. These behavioral alterations were associated with a lowered dendritic spine density and protein levels of glutamate receptors and postsynaptic markers, and increased protein levels of the presynaptic synaptophysin in the hippocampus. Metabolomic analysis of the hippocampus indicated changes in amino acid levels including reduced tryptophan and L-kynurenine and elevated NAD + levels, respectively, a precursor, intermediate and endproduct of the kynurenine pathway of tryptophan metabolism. Interestingly, the protein amounts of the xenobiotic aryl hydrocarbon receptor, a target of this metabolic pathway, were elevated in the CA1 area. These data indicate that chronic exposure of adult male mice to environmental doses of DEHP alone or in a phthalate mixture impacted hippocampal function and structure, associated with modifications in amino acid metabolites with a potential involvement of the kynurenine pathway of tryptophan metabolism.

Mitotherapy restores hippocampal mitochondrial function and cognitive impairment in aged male rats subjected to chronic mild stress.

This study aimed to determine the effects of mitotherapy on learning and memory and hippocampal kynurenine (Kyn) pathway, mitochondria function, and dendritic arborization and spines density in aged rats subjected to chronic mild stress. Twenty-eight male Wistar rats (22 months old( were randomly divided into Aged, Aged + Mit, Aged + Stress, and Aged + Stress + Mit groups. Aged rats in the stress groups were subjected to different stressors for 28 days. The Aged + Mit and Aged + stress + Mit groups were treated with intracerebroventricular injection (10 µl) of fresh mitochondria harvested from the young rats' brains, and other groups received 10 µl mitochondria storage buffer. Spatial and episodic-like memories were assessed via the Barnes maze and novel object recognition tests. Indoleamine 2,3-dioxygenase (IDO) expression and activity, Kyn, Tryptophan (TRY), ATP levels, and mitochondrial membrane potential (MMP) were measured in the hippocampus region. Golgi-Cox staining was also performed to assess the dendritic branching pattern and dendritic spines in the hippocampal CA1 subfield. The results showed that mitotherapy markedly improved both spatial and episodic memories in the Aged + Stress + Mit group compared to the Aged + Stress. Moreover, mitotherapy decreased IDO protein expression and activity and Kyn levels, while it increased ATP levels and improved MMP in the hippocampus of the Aged + Stress + Mit group. Besides, mitotherapy restored dendritic atrophy and loss of spine density in the hippocampal neurons of the stress-exposed aged rats. These findings provide evidence for the therapeutic effect of mitotherapy against stress-induced cognitive deterioration in aged rats by improving hippocampal mitochondrial function and modulation of the Kyn pathway.

Activation of the kynurenine-aryl hydrocarbon receptor axis impairs the chondrogenic and chondroprotective effects of human umbilical cord-derived mesenchymal stromal cells in osteoarthritis rats.

It has been proven that intra-articular injection of mesenchymal stromal cells (MSCs) can alleviate cartilage damage in osteoarthritis (OA) by differentiating into chondrocytes and protecting inherent cartilage. However, the mechanism by which the OA articular microenvironment affects MSCs' therapeutic efficiency is yet to be fully elucidated. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in various cellular processes, such as osteogenesis and immune regulation. Tryptophan (Trp) metabolites, most of which are endogenous ligand for AHR, are abnormally increased in synovial fluid (SF) of OA and rheumatoid arthritis (RA) patients. In this study, the effects of kynurenine (KYN), one of the most important metabolites of Trp, were evaluated on the chondrogenic and chondroprotective effects of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs). hUC-MSCs were cultured in conditioned medium containing different proportions of OA/RA SF, or stimulated with KYN directly, and then, AHR activation, proliferation, and chondrogenesis of hUC-MSCs were measured. Moreover, the chondroprotective efficiency of short hairpin-AHR-UC-MSC (shAHR-UC-MSC) was determined in a rat surgical OA model (right hind joint). OA SF could activate AHR signaling in hUC-MSCs in a concentration-dependent manner and inhibit the chondrogenic differentiation and proliferation ability of hUC-MSCs. Similar results were observed in hUC-MSCs stimulated with KYN in vitro. Notably, shAHR-UC-MSC exhibited superior therapeutic efficiency in OA rat upon intra-articular injection. Taken together, this study indicates that OA articular microenvironment is not conducive to the therapeutic effect of hUC-MSCs, which is related to the activation of the AHR pathway by tryptophan metabolites, and thus impairs the chondrogenic and chondroprotective effects of hUC-MSCs. AHR might be a promising modification target for further improving the therapeutic efficacy of hUC-MSCs on treatment of cartilage-related diseases such as OA.

Development and Optimization of a Target Engagement Model of Brain IDO Inhibition for Alzheimer's Disease.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO1) inhibition is a promising target as an Alzheimer's disease (AD) Disease-modifying therapy capable of downregulating immunopathic neuroinflammatory processes. METHODS: To aid in the development of IDO inhibitors as potential AD therapeutics, we optimized a lipopolysaccharide (LPS) based mouse model of brain IDO1 inhibition by examining the dosedependent and time-course of the brain kynurenine:tryptophan (K:T) ratio to LPS via intraperitoneal dosing. RESULTS: We determined the optimal LPS dose to increase IDO1 activity in the brain, and the ideal time point to quantify the brain K:T ratio after LPS administration. We then used a brain penetrant tool compound, EOS200271, to validate the model, determine the optimal dosing profile and found that a complete rescue of the K:T ratio was possible with the tool compound. CONCLUSION: This LPS-based model of IDO1 target engagement is a useful tool that can be used in the development of brain penetrant IDO1 inhibitors for AD. A limitation of the present study is the lack of quantification of potential clinically relevant biomarkers in this model, which could be addressed in future studies.

2-month ketogenic diet preferentially alters skeletal muscle and augments cognitive function in middle aged female mice.

The effect of a ketogenic diet (KD) on middle aged female mice is poorly understood as most of this work have been conducted in young female mice or diseased models. We have previously shown that an isocaloric KD started at middle age in male mice results in enhanced mitochondrial mass and function after 2 months on diet and improved cognitive behavior after being on diet for 14 months when compared with their control diet (CD) fed counterparts. Here, we aimed to investigate the effect of an isocaloric 2-month KD or CD on healthy 14-month-old female mice. At 16 months of age cognitive behavior tests were performed and then serum, skeletal muscle, cortex, and hippocampal tissues were collected for biochemical analysis. Two months on a KD resulted in enhanced cognitive behavior associated with anxiety, memory, and willingness to explore. The improved neurocognitive function was associated with increased PGC1α protein in the gastrocnemius (GTN) muscle and nuclear fraction. The KD resulted in a tissue specific increase in mitochondrial mass and kynurenine aminotransferase (KAT) levels in the GTN and soleus muscles, with a corresponding decrease in kynurenine and increase in kynurenic acid levels in serum. With KAT proteins being responsible for converting kynurenine into kynurenic acid, which is unable to cross the blood brain barrier and be turned into quinolinic acid-a potent neurotoxin, this study provides a potential mechanism of crosstalk between muscle and brain in mice on a KD that may contribute to improved cognitive function in middle-aged female mice.

Kynurenine metabolism is altered in mdx mice: a potential muscle to brain connection.

NEW FINDINGS: What is the central question in this study? Promoting muscle health with regular aerobic exercise can improve mental health through a kynurenine metabolic pathway: do conditions of muscle disease such as muscular dystrophy negatively influence this pathway? What is the main finding and its importance? The DBA/2J mdx model of Duchenne muscular dystrophy exhibits altered kynurenine metabolism with less kynurenic acid and peroxisome proliferator-activated receptor-γ coactivator 1-α and higher levels of tumour necrosis factor α mRNA - results associated with anxiety-like behaviour. ABSTRACT: Regular exercise can direct muscle kynurenine (KYN) metabolism toward the neuroprotective branch of the kynurenine pathway thereby limiting the accumulation of neurotoxic metabolites in the brain and contributing to mental resilience. However, the effect of muscle disease on KYN metabolism has not yet been investigated. Previous work has highlighted anxiety-like behaviours in approximately 25% of patients with Duchenne muscular dystrophy (DMD), possibly due to altered KYN metabolism. Here, we characterized KYN metabolism in mdx mouse models of DMD. Young (8-10 week old) DBA/2J (D2) mdx mice, but not age-matched C57BL/10 (C57) mdx mice, had lower levels of circulating kynurenic acid (KYNA) and lower KYNA:KYN ratio compared with their respective wild-type (WT) controls. While both C57 and D2 mdx mice displayed signs of anxiety-like behaviour, spending more time in the corners of the arena during a novel object recognition test, this effect was more prominent in D2 mdx mice. Correlational analysis detected a significant negative association between KYNA:KYN levels and time spent in corners in D2 mice, but not C57 mice. In extensor digitorum longus muscles from D2 mdx mice, but not C57 mdx mice, we found lowered protein levels of peroxisome proliferator-activated receptor-γ coactivator 1-α and kynurenine amino transferase-1 enzyme when compared with WT. Furthermore, D2 mdx quadriceps muscles had the highest level of tumour necrosis factor α expression, which is suggestive of enhanced inflammation. Thus, our pilot work shows that KYN metabolism is altered in D2 mdx mice, with a potential contribution from altered muscle health.

Dietary AhR Ligands Have No Anti-Fibrotic Properties in TGF-ß1-Stimulated Human Colonic Fibroblasts.

BACKGROUND: Intestinal fibrosis is a common complication in inflammatory bowel disease (IBD) patients without specific treatment. Aryl hydrocarbon receptor (AhR) activation is associated with better outcomes in intestinal inflammation. Development of novel therapies targeting fibrogenic pathways is required and we aimed to screen dietary AhR ligands for their anti-fibrotic properties in TGF-ß1-stimulated human colonic fibroblast cells. METHODS: The study was conducted using TGF-ß1-stimulated CCD-18Co, a human colonic fibroblast cell line in response to increased concentrations of dietary ligands of AhR such as FICZ, ITE, L-kynurenine and curcumin. Fibrosis markers such as α-SMA, COL1A1, COL3A1 and CTGF were assessed. AhR and ANRT RNA were evaluated. RESULTS: TGF-ß1 at 10 ng/mL significantly induced mRNA levels for ECM-associated proteins such as CTGF, COL1A1 and COL3A1 in CCD-18Co cells. FICZ from 10 to 1000 nM, L-kynurenine from 0.1 to 10 µM, ITE from 1 to 100 µM or curcumin from 5 to 20 µM had no significant effect on fibrosis markers in TGF-ß1-induced CCD-18Co. CONCLUSIONS: Our data highlight that none of the tested dietary AhR ligands had an effect on fibrosis markers in TGF-ß1-stimulated human colonic fibroblast cells in our experimental conditions. Further studies are now required to identify novel potential targets in intestinal fibrosis.

Involvement of kynurenine pathway and N-methyl-d-aspartate receptors in the antidepressant-like effect of vilazodone in the tail suspension test in mice.

The present study evaluated the antidepressant-like effects of vilazodone using the tail suspension test in mice. We also investigated the contribution of kynurenine pathway and N-methyl-d-aspartate receptors to this effect. For this purpose, we pretreated animals with sub-effective doses of L-kynurenine, 3-hydroxykynurenine, or quinolinic acid. We then assessed the immobility time, an indicative measure of depressive-like behavior, in the tail suspension test. We also evaluated the possible effects of sub-effective doses of vilazodone combined with sub-effective doses of ketamine (N-methyl-d-aspartate receptor antagonist) in a separate group. Vilazodone (3mg/kg, intraperitoneal) significantly reduced immobility time in the tail suspension test. L-kynurenine (1.7 mg/kg, intraperitoneal), 3-hydroxykynurenine (10 mg/kg, intraperitoneal), and quinolinic acid (3 nmol/site, intracerebroventricular) significantly increased the immobility time in the tail suspension test. The antidepressant-like effects of vilazodone (3mg/kg, intraperitoneal) were inhibited by pre-treatment with non-effective doses of L-kynurenine (0.83 mg/kg, intraperitoneal), 3-hydroxykynurenine (3.33 mg/kg, intraperitoneal), or quinolinic acid (1 nmol/site, intracerebroventricular). Pretreatment of mice with sub-effective doses of ketamine (1 mg/kg, intraperitoneal) optimized the action of a sub-effective dose of vilazodone (0.3mg/kg, intraperitoneal) and reduced the immobility time in the tail suspension test. None of the drugs used in this study induced any changes in locomotor activity in the open field test. The results showed that vilazodone induced an antidepressant-like effect in the tail suspension test, which may be mediated through an interaction with the kynurenine pathway and N-methyl-d-aspartate receptors.

Eucommiae cortex polysaccharides mitigate obesogenic diet-induced cognitive and social dysfunction via modulation of gut microbiota and tryptophan metabolism.

Rationale: The high fat and sucrose diet, known as the obesogenic diet (OD), has been related to low-grade chronic inflammation and neurodevelopmental disorders. Emerging evidence suggests that OD influences cognitive and social function via the gut-brain axis. However, the effects of OD during adolescence on future health have been unclear. Meanwhile, the underlying mechanisms and effective interventions are not fully understood. Polysaccharides, one of the most abundant substances in the Eucommiae cortex, exhibit potential immunomodulatory and neuroprotective effects. Here, we aimed to investigate the impact of OD on adolescents, explore the modulating roles of Eucommiae cortex polysaccharides (EPs) on OD-induced behavioral dysfunction, and elucidate the underlying molecular mechanisms. Methods: In the present study, four-week-old mice were fed with OD for four weeks to simulate persistent OD in adolescents. The behavioral features were accessed by open field test and Morris water maze. The gut bacterial structure was identified by 16S rRNA gene amplicon sequencing. The gene and protein expression in colonic tissues and hippocampus were detected by qRT-PCR, immunoblotting, enzyme-linked immunosorbent assay, and immunofluorescence staining. Detection of biological metabolites in serum and hippocampal tissues was performed by widely targeted metabolomics and targeted metabolomics. Results: We found that OD-fed mice showed cognitive and social-behavioral deficits accompanied by gut dysbiosis and systematic tryptophan (Trp) metabolism disorders, which increased kynurenine (Kyn) concentration in the hippocampus. Bacteria-derived lipopolysaccharide (LPS, endotoxin) induced microglia-mediated neuroinflammation, directing the metabolism of Kyn in the hippocampus toward quinolinic acid (QA), which led to glutamate-mediated hyperactivation of mossy cells (MCs) in hippocampal hilus. Furthermore, OD impaired parvalbumin (PV) interneurons-related local circuits in the hippocampal granule cell layer. These resulted in hippocampal neurogenesis deficits and related behavioral dysfunction in mice. EPs supplementation ameliorated OD-induced gut dysbiosis, as evidenced by inhibiting the expansion of Escherichia coli (E.coli) and reducing the concentration of LPS in colonic contents and serum, thereby inhibiting the subsequent neuroinflammation. In addition, oral EPs suppressed the peripheral Kyn pathway to reduce the concentration of QA and glutamic acid in the hippocampus of OD-fed mice, thereby rescuing the glutamic acid-triggered neuroexcitotoxicity. These contributed to remodeling the rhythm of hippocampal neurogenesis and mitigated behavioral dysfunction in OD-fed mice. Conclusions: The present study addresses a gap in the understanding of neuronal dysfunction associated with OD during adolescence and provides the first evidence that EPs improved cognitive and social behavior via modulation of gut microbiota and tryptophan metabolism in adolescent mice fed with OD, which may represent novel preemptive therapy for neurodevelopmental disorders via manipulation of the tryptophan metabolite.

Kynurenine produced by tryptophan 2,3-dioxygenase metabolism promotes glioma progression through an aryl hydrocarbon receptor-dependent signaling pathway.

The current studies associated with tumor biology continue to describe a high correlation between tryptophan (Trp) metabolism and tumor progression. These findings reflect the complex underlying mechanism of tumor development and highlight the need to explore additional drug targets for carcinoma-associated diseases. In our study, we reported that elevated Trp metabolism was observed in highly malignant glioma tumor tissues from patients. The elevated Trp metabolism in glioma cells were induced by the overexpression of Trp 2,3-dioxygenase 2 (TDO2), which further contributed to the production of the metabolite kynurenine (Kyn). Subsequently, the Kyn derived from Trp metabolism was able to mediate the activation of the aryl hydrocarbon receptor (AhR) and downstream PI3K/AKT signals, resulting in the strengthening of tumor stemness and growth. Meanwhile, the activation of the AhR could promote the process of epithelial-mesenchymal transition in gliomas through a TGF-ß-dependent mechanism, leading to enhanced tumor invasion in vitro and in vivo. Inhibition of the AhR using StemRegenin 1 was demonstrated to suppress glioma growth and improve the outcome of traditional chemotherapy in subcutaneous tumor-bearing mice, representing a promising therapeutic target for clinical glioma treatment.