Dysregulation at multiple points of the kynurenine pathway is a ubiquitous feature of renal cancer: implications for tumour immune evasion.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO), the first step in the kynurenine pathway (KP), is upregulated in some cancers and represents an attractive therapeutic target given its role in tumour immune evasion. However, the recent failure of an IDO inhibitor in a late phase trial raises questions about this strategy. METHODS: Matched renal cell carcinoma (RCC) and normal kidney tissues were subject to proteomic profiling. Tissue immunohistochemistry and gene expression data were used to validate findings. Phenotypic effects of loss/gain of expression were examined in vitro. RESULTS: Quinolate phosphoribosyltransferase (QPRT), the final and rate-limiting enzyme in the KP, was identified as being downregulated in RCC. Loss of QPRT expression led to increased potential for anchorage-independent growth. Gene expression, mass spectrometry (clear cell and chromophobe RCC) and tissue immunohistochemistry (clear cell, papillary and chromophobe), confirmed loss or decreased expression of QPRT and showed downregulation of other KP enzymes, including kynurenine 3-monoxygenase (KMO) and 3-hydroxyanthranilate-3,4-dioxygenase (HAAO), with a concomitant maintenance or upregulation of nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in the NAD+ salvage pathway. CONCLUSIONS: Widespread dysregulation of the KP is common in RCC and is likely to contribute to tumour immune evasion, carrying implications for effective therapeutic targeting of this critical pathway.

Thirteen week toxicity study of dietary l-tryptophan in rats with a recovery period of 5 weeks.

Although l-tryptophan is nutritionally important and widely used in medical applications, toxicity data for its oral administration are limited. The purpose of this study was to evaluate the potential toxicity of an experimental diet containing added l-tryptophan at doses of 0 (basal diet), 1.25%, 2.5% and 5.0% when administered to Sprague-Dawley rats for 13 weeks. There were no toxicological changes in clinical signs, ophthalmology, urinalysis, hematology, necropsy, organ weight and histopathology between control rats and those fed additional l-tryptophan. Body weight gain and food consumption significantly decreased throughout the administration period in males in the 2.5% group and in both sexes in the 5.0% group. At the end of the dosing period, decreases in water intake in males in the 5.0% group and in serum glucose in females in the 5.0% group were observed. The changes described above were considered toxicologically significant; however, they were not observed after a 5 week recovery period, suggesting reversibility. Consequently, the no-observed-adverse-effect level of l-tryptophan in the present study was 1.25% for males and 2.5% for females (mean intake of l-tryptophan: 779 mg kg-1 body weight day-1 [males] and 1765 mg kg-1 body weight day-1 [females]). As the basal diet used in this study contained 0.27% of proteinaceous l-tryptophan, the no-observed-adverse-effect level of overall l-tryptophan was 1.52% for males and 2.77% for females (mean intake of overall l-tryptophan: 948 mg kg-1 body weight day-1 (males) and 1956 mg kg-1 body weight day-1 (females)). We conclude that l-tryptophan has a low toxicity profile in terms of human use.

Inhibition of Large Neutral Amino Acid Transporters Suppresses Kynurenic Acid Production Via Inhibition of Kynurenine Uptake in Rodent Brain.

The tryptophan metabolite, kynurenic acid (KYNA), is a preferential antagonist of the α7 nicotinic acetylcholine receptor and N-methyl-D-aspartic acid receptor at endogenous brain concentrations. Recent studies have suggested that increases of brain KYNA levels are involved in psychiatric disorders such as schizophrenia and depression, and regulation of KYNA production has become a new target for treatment of these diseases. Kynurenine (KYN), the immediate precursor of KYNA, is transported into astrocytes via large neutral amino acid transporters (LATs). In the present study, the effect of LATs regulation on KYN uptake and KYNA production was investigated in vitro and in vivo using an LATs inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). In the in vitro study, cortical slices of rat brain were incubated with a physiological concentration of KYN and 3 µmol/L-3 mmol/L BCH. BCH inhibited KYNA production and KYN uptake in a dose-dependent manner, and their IC50 values were 90.7 and 97.4 µmol/L, respectively. In the in vivo study, mice were administered KYN (50 mg/kg BW) orally and BCH (200 mg/kg BW) intravenously. Administration of KYN increased brain KYN and KYNA levels compared with the mice treated with vehicle, whereas additional administration of BCH suppressed KYN-induced elevations in KYN and KYNA levels to 50 and 70 % in the brain. These results suggest that inhibition of LATs prevented the increase of KYNA production via blockade of KYN uptake in the brain in vitro and in vivo. LATs can be a target to modulate brain function by regulation of KYNA production in the brain.

Thermal conditions influence changes in body temperature induced by intragastric administration of capsaicin in mice.

Capsaicin has been reported to have unique thermoregulatory actions. However, changes in core temperature after the administration of capsaicin are a controversial point. Therefore, we investigated the effects of environmental thermal conditions on changes in body temperature caused by capsaicin in mice. We showed that intragastric administration of 10 and 15 mg/kg capsaicin increased tail temperature and decreased colonic temperatures in the core temperature (CT)-constant and CT-decreasing conditions. In the CT-increasing condition, 15 mg/kg capsaicin increased tail temperature and decreased colonic temperature. However, 10 mg/kg capsaicin increased colonic temperature. Furthermore, the amount of increase in tail temperature was greater in the CT-decreasing condition and lower in the CT-increasing condition, compared with that of the CT-constant condition. These findings suggest that the changes in core temperature were affected by the environmental thermal conditions and that preliminary thermoregulation state might be more important than the constancy of temperature to evaluate the effects of heat diffusion and thermogensis.

High-performance liquid chromatographic method for profiling 2-oxo acids in urine and its application in evaluating vitamin status in rats.

B-group vitamins are involved in the catabolism of 2-oxo acids. To identify the functional biomarkers of B-group vitamins, we developed a high-performance liquid chromatographic method for profiling 2-oxo acids in urine and applied this method to urine samples from rats deficient in vitamins B1 and B6 and pantothenic acid. 2-Oxo acids were reacted with 1,2-diamino-4,5-methylenebenzene to produce fluorescent derivatives, which were then separated using a TSKgel ODS-80Ts column with 30 mmol/L of KH2PO4 (pH 3.0):acetonitrile (7:3) at a flow rate of 1.0 mL/min. Vitamin B1 deficiency increased urinary levels of all 2-oxo acids, while vitamin B6 deficiency only increased levels of sum of 2-oxaloacetic acid and pyruvic acid, and pantothenic acid deficiency only increased levels of 2-oxoisovaleric acid. Profiles of 2-oxo acids in urine samples might be a non-invasive way of clarifying the functional biomarker of B-group vitamins.

Vitamin B1 deficiency inhibits the increased conversion of tryptophan to nicotinamide in severe food-restricted rats.

The conversion of tryptophan (Trp) → nicotinamide (Nam) is an important pathway for supplying vitamin niacin. We reported the following two phenomena: (1) severe food restriction led to an increase in the Trp → Nam conversion compared with free-access control group; (2) the conversion of Trp → Nam is also increased by vitamin B1 deficiency compared with free-access control group. The present study was done to clarify whether or not a true reason about an increase in the Trp → Nam conversion is a vitamin B1 deficiency or severe food restriction. The present results showed that vitamin B1 deficiency suppressed the increased conversion of Trp → Nam induced by severe food restriction, probably by suppressing 3-hydroxyanthranilic acid 3,4-dioxygenase protein synthesis in liver.

Hepatectomy-related hypophosphatemia: a novel phosphaturic factor in the liver-kidney axis.

Marked hypophosphatemia is common after major hepatic resection, but the pathophysiologic mechanism remains unknown. We used a partial hepatectomy (PH) rat model to investigate the molecular basis of hypophosphatemia. PH rats exhibited hypophosphatemia and hyperphosphaturia. In renal and intestinal brush-border membrane vesicles isolated from PH rats, Na(+)-dependent phosphate (Pi) uptake decreased by 50%-60%. PH rats also exhibited significantly decreased levels of renal and intestinal Na(+)-dependent Pi transporter proteins (NaPi-IIa [NaPi-4], NaPi-IIb, and NaPi-IIc). Parathyroid hormone was elevated at 6 hours after PH. Hyperphosphaturia persisted, however, even after thyroparathyroidectomy in PH rats. Moreover, DNA microarray data revealed elevated levels of nicotinamide phosphoribosyltransferase (Nampt) mRNA in the kidney after PH, and Nampt protein levels and total NAD concentration increased significantly in the proximal tubules. PH rats also exhibited markedly increased levels of the Nampt substrate, urinary nicotinamide (NAM), and NAM catabolites. In vitro analyses using opossum kidney cells revealed that NAM alone did not affect endogenous NaPi-4 levels. However, in cells overexpressing Nampt, the addition of NAM led to a marked decrease in cell surface expression of NaPi-4 that was blocked by treatment with FK866, a specific Nampt inhibitor. Furthermore, FK866-treated mice showed elevated renal Pi reabsorption and hypophosphaturia. These findings indicate that hepatectomy-induced hypophosphatemia is due to abnormal NAM metabolism, including Nampt activation in renal proximal tubular cells.

Tissue vitamin concentrations are maintained constant by changing the urinary excretion rate of vitamins in rats' restricted food intake.

We previously reported that mild food restriction induces a reduction in tryptophan-nicotinamide conversion, which helps to explain why death secondary to pellagra is pandemic during the hungry season. In this study, we investigated the levels of B-group vitamins in the liver, kidney, blood, and urine in rats that underwent gradual restriction of food intake (80, 60, 40, and 20% restriction vs. ad libitum food intake). No significant differences in the B-group vitamin concentrations (mol/g tissue) in the liver and kidney were observed at any level of food restriction. However, the urine excretion rates exhibited some characteristic phenomena that differed by vitamin. These results show that the tissue concentrations of B-group vitamins were kept constant by changing the urinary elimination rates of vitamins under various levels of food restriction. Only vitamin B12 was the only (exception).

Moderate food restriction suppresses the conversion of L-tryptophan to nicotinamide in weaning rats.

Calorie restriction leads to a change in the metabolism of nutrients. Nicotinamide is biosynthesized from L-tryptophan. We attempted to determine the effects of food restriction on the biosynthesis of nicotinamide from L-tryptophan. Weaning male rats were fed a conventional chemically defined diet without preformed niacin for 63 d. However, the food intake was restricted to 80 and 65% of the intake of the ad libitum-fed control group of rats. The 24-h urine samples were periodically collected, and the urinary excretion of nicotinamide and its catabolites was measured. The conversion percentages were lower in both restricted groups than in the ad libitum-fed control group during the experimental period (control group, 1.37 ± 0.24%; 80%-restricted group, 0.20 ± 0.04%; 65%-restricted group, 0.15 ± 0.02%; control vs. restricted groups, p < 0.01). Food restriction, even at mild level, suppressed the conversion of L-tryptophan to nicotinamide when compared to the ad libitum-fed control group.

Simultaneous measurement of nicotinamide and its catabolites, nicotinamide N-oxide, N(1)-methyl-2-pyridone-5-carboxamide, and N(1)-methyl-4-pyridone-3-carboxamide, in mice urine.

Nicotinamide N-oxide is a major nicotinamide catabolite in mice but not in humans and rats. A high-performance liquid chromatographic method for the simultaneous measurement of nicotinamide, nicotinamide N-oxide, N(1)-methyl-2-pyridone-5-carboxamide, and N(1)-methyl-4-pyridone-3-carboxamide in mice urine was developed by modifying the mobile phase of a reported method for measurement of nicotinamide N-oxide.

Contributions of tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase to the conversion of D-tryptophan to nicotinamide analyzed by using tryptophan 2,3-dioxygenase-knockout mice.

We investigated the contribution percentage of tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) to the conversion of D-tryptophan to nicotinamide in TDO-knockout mice. The calculated percentage conversions indicated that TDO and IDO oxidized 70 and 30%, respectively, of the dietary L-tryptophan. These results indicate that both TDO and IDO biosynthesize nicotinamide from D-tryptophan and L-tryptophan in mice.

Adrenalectomy exacerbates stress-induced impairment in fear discrimination: A causal role for kynurenic acid?

Impaired activity of the hypothalamic-pituitary axis and reduced blood levels of glucocorticoids (GCs) are signature features of stress-related maladies. Recent evidence suggests a possible role of the tryptophan metabolite kynurenic acid (KYNA) in this context. Here we investigated possible causal relationships in adult male rats, using stress-induced fear discrimination as a translationally relevant behavioral outcome measure. One week following adrenalectomy (ADX) or sham surgery, animals were for 2 h either physically restrained or exposed to a predator odor, which caused a much milder stress response. Extracellular KYNA levels were determined before, during and after stress by in vivo microdialysis in the prefrontal cortex. Separate cohorts underwent a fear discrimination procedure starting immediately after stress termination. Different auditory conditioned stimuli (CS) were either paired with a foot shock (CS+) or non-reinforced (CS-). One week later, fear was assessed by re-exposing the animals to each CS. Separate groups of rats were treated with the KYNA synthesis inhibitor BFF-816 prior to stress initiation to test a causal role of KYNA in fear discrimination. Restraint stress raised extracellular KYNA levels by ∼85 % in ADX rats for several hours, and these animals were unable to discriminate between CS+ and CS-. Both effects were prevented by BFF-816 and were not observed after exposure to predator odor or in sham-operated rats. These findings suggest that a causal connection exists between adrenal function, stress-induced KYNA increases, and behavioral deficits. Pharmacological inhibition of KYNA synthesis may therefore be an attractive, novel option for the treatment of stress-related disorders.

Chronic Ethanol Intake Impairs Niacin Nutritional Status in Mice.

Niacin is involved in many biological reactions relating energy metabolism, redox reactions, DNA repair and longevity. Since niacin deficiency has been reported in alcoholic patients, and niacin coenzyme NAD is used as substrate to dehydrogenate ethanol in the liver, ethanol consumption can be a factor to impair niacin nutritional status. We have recently established the niacin insufficient model mice using kynurenine 3-monooxygenase knock out (KMO-/-) mice with niacin-limited diet, which lack the de novo NAD synthesis pathway from tryptophan. To evaluate the effects of chronic ethanol intake on niacin nutritional status, 4 wk old KMO-/- mice were fed 4 or 30 mg/kg nicotinic acid containing diets with or without 15% ethanol for 35 d. The mice fed 4 mg/kg nicotinic acid diet with ethanol showed lower body weight gain and niacin nutritional markers such as liver and blood NAD, and urine nicotinamide metabolites than the mice without ethanol. These animals did not show any difference in the NAD synthesis, NAD salvage and nicotinamide catabolic pathways. Chronic ethanol intake failed to affect any indices in the mice fed the 30 mg/kg nicotinic acid diet. When the diet was exchanged the 4 mg/kg for 30 mg/kg nicotinic acid diet to the mice showed chronic ethanol-induced growth retardation, their body weight rapidly increased. These results show that chronic ethanol intake impairs niacin nutritional status in the niacin insufficient mice, and enough niacin intake can prevent this impairment. Our findings also suggest that chronic ethanol intake increases niacin requirement by increase of NAD consumption.

Chronic Endurance Exercise Impairs Niacin Nutritional Status in Mice.

Niacin is a cofactor in many biological reactions related to energy metabolism, redox reactions, DNA repair and longevity. Although it has been considered that increasing energy expenditure increases NAD consumption, little study has directly demonstrated the effect of exercise on niacin nutritional status. We have recently established the niacin insufficient model mice using kynurenine 3-monooxygenase knock out (KMO-/-) mice with niacin-limited diet, which lack the de novo NAD synthesis pathway from tryptophan. To evaluate the effects of chronic endurance exercise on niacin nutritional status, 4 wk old KMO-/- mice were fed 4 or 30 mg/kg nicotinic acid containing diets, and forced to swim in a running water pool every other day for 35 d. The swim-exercised mice fed 4 mg/kg nicotinic acid diet showed lower body weight gain and niacin nutritional markers such as liver and blood NAD, and urine nicotinamide metabolites than the sedentary mice. These animals did not show any difference in the NAD synthesis, NAD salvage and nicotinamide catabolic pathways. Chronic endurance exercise failed to affect any indices in the mice fed the 30 mg/kg nicotinic acid diet. When the diet was exchanged the 4 mg/kg for 30 mg/kg nicotinic acid diet to the mice showed chronic endurance exercise-induced growth retardation, their body weight rapidly increased. These results show that chronic endurance exercise impairs niacin nutritional status in the niacin insufficient mice, and enough niacin intake can prevent this impairment. Our findings also suggest that chronic endurance exercise increases niacin requirement by increase of NAD consumption.

Supplementing healthy women with up to 5.0 g/d of L-tryptophan has no adverse effects.

Because of the frequent use of L-tryptophan (L-Trp) in dietary supplements, determination of the no-observed-adverse-effect-level is desirable for public health purposes. We therefore assessed the no-observed-adverse-effect-level for L-Trp and attempted to identify a surrogate biomarker for excess L-Trp in healthy humans. A randomized, double-blind, placebo-controlled, crossover intervention study was performed in 17 apparently healthy Japanese women aged 18-26 y with a BMI of ≈ 20 kg/m(2). The participants were randomly assigned to receive placebo (0 g/d) or 1.0, 2.0, 3.0, 4.0, or 5.0 g/d of L-Trp for 21 d each with a 5-wk washout period between trials. Food intake, body weight, general biomarkers in blood and urine, and amino acid composition in blood and urine were not affected by any dose of L-Trp. Administration of up to 5.0 g/d L-Trp had no effect on a profile of mood states category measurement. The urinary excretion of nicotinamide and its catabolites increased in proportion to the ingested amounts of L-Trp, indicating that participants could normally metabolize this amino acid. The urinary excretion of L-tryptophan metabolites, including kynurenine (Kyn), anthranilic acid, kynurenic acid, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and quinolinic acid (QA), all of which are intermediates of the L-TRP→Kyn→QA pathway, was in proportion to L-Trp loading. The response of 3-HK was the most characteristic of these L-Trp metabolites. This finding suggests that the urinary excretion of 3-HK is a good surrogate biomarker for excess L-Trp ingestion.

The niacin required for optimum growth can be synthesized from L-tryptophan in growing mice lacking tryptophan-2,3-dioxygenase.

In mammals, nicotinamide (Nam) is biosynthesized from l-tryptophan (l-Trp). The enzymes involved in the initial step of the l-Trp→Nam pathway are l-Trp-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO). We aimed to determine whether tdo-knockout (tdo(-/-)) mice fed a diet without preformed niacin can synthesize enough Nam to sustain optimum growth. Wild-type (WT) and tdo(-/-) mice were fed a chemically defined 20% casein diet with or without preformed niacin (30 mg nicotinic acid/kg) for 28 d. Body weight, food intake, and liver NAD concentrations did not differ among the groups. In the groups of mice fed the niacin-free diet, urinary concentrations of the upstream metabolites kynurenine (320% increase, P < 0.0001), kynurenic acid (270% increase, P < 0.0001), xanthurenic acid (770% increase, P < 0.0001), and 3-hydroxyanthranilic acid (3-HA; 450% increase, P < 0.0001) were higher in the tdo(-/-) mice than in the WT mice, while urinary concentrations of the downstream metabolite quinolinic acid (QA; 50% less, P = 0.0010) and the sum of Nam and its catabolites (10% less, P < 0.0001) were lower in the tdo(-/-) mice than in the WT mice. These findings show that the kynurenine formed in extrahepatic tissues by IDO and subsequent enzymes can be metabolized up to 3-HA, but not into QA. However, the tdo(-/-) mice sustained optimum growth even when fed the niacin-free diet for 1 mo, suggesting they can synthesize the minimum necessary amount of Nam from l-Trp, because the liver can import blood kynurenine formed in extrahepatic tissues and metabolize it into Nam via NAD and the resulting Nam is then distributed back into extrahepatic tissues.

Increased conversion of tryptophan to nicotinamide in rats by dietary valproate.

Valproic acid (VPA) is a short-chained, branched fatty acid that is widely used in humans as an anticonvulsant and mood stabilizer, and has been reported to increase the liver NAD concentration. We investigated the effects of VPA on the conversion of tryptophan to nicotinamide. Rats were fed diets containing various amounts of VPA (0, 0.5, and 1.0% in the diets) for 14 d, 24-h urine samples were collected, and tryptophan and its catabolites were measured. We found that the conversion of tryptophan to nicotinamide was increased by feeding a diet containing VPA (p<0.01; 0% vs. 1.0% VPA). Of the intermediates formed during the conversion of tryptophan to nicotinamide, the tryptophan to 3-hydroxyanthranilic acid step was not affected by the administration of VPA, while such metabolites beyond quinolinic acid as nicotinamide and its catabolites were significantly increased (p<0.01; 0% vs. 1.0% VPA). This increase was dependent on the intake of VPA.

Changes in B-group vitamin status in adenine-induced chronic renal failure rats.

The purpose of this study was to determine the effects of chronic renal failure (CRF) on B-group vitamin status using model rats in which adenine-induced CRF. We measured B-groups vitamins in the urine, blood, liver, and kidney. These results showed that renal failure affected the distribution, metabolism, and renal clearance of water-soluble vitamins, and that the effects were different with each vitamin.

Enzymes that control the conversion of L-tryptophan-nicotinamide and the urinary excretion ratio (N(1)-methyl-2-pyridone-5-carboxamide + N(1)-methyl-4-pyridone-3-carboxamide)/N(1)-methylnicotinamide in mice.

There is little information on L-tryptophan→nicotinamide metabolism in mice. In the present study, we investigated the two important nutritional factors involved in metabolism L-tryptophan→nicotinamide; one is the amount of nicotinamide synthesized from L-tryptophan, and the other is the urine ratio (N(1)-methyl-2-pyridone-5-carboxamide + N(1)-methyl-4-pyridone-3-carboxamide)/N(1)-methylnicotinamide. The order of the percentages of nicotinamide synthesized from L-tryptophan was as follows: CBA strain mice (conversion percentage 0.41%) < BALB strain mice (0.82%) < C57BL/6 strain mice (1.13%) < ICR strain mice (1.70%). Urinary excretion of quinolinic acid was correlated with urinary excretion of the sum of nicotinamide and its catabolites (p<0.0001). The urine sum, which reflects the conversion of L-tryptophan→nicotinamide, correlated well with the activity of 3-hydroxyanthranilic acid dioxygenase (p=0.040). A nutritional indicator, the urine ratio (N(1)-methyl-2-pyridone-5-carboxamide + N(1)-methyl-4-pyridone-3-carboxamide)/N(1)-methylnicotinamide, was controlled by the activity of N(1)-methyl-2-pyridone-5-carboxamide-forming N(1)-methylnicotinamide oxidase.

Establishment of Model Mice to Evaluate Low Niacin Nutritional Status.

Niacin is involved in many biological reactions relating energy metabolism, redox reactions, DNA repair and longevity, and low NAD levels with aging and feeding high fat diets develop and progress age-related diseases. Although recent findings suggest the requirement of niacin insufficient animal model to further study, appropriate animal models have not been established yet because niacin is biosynthesized from tryptophan via tryptophan-nicotinamide pathway. To establish model mice to evaluate niacin nutritional status, we used kynurenine 3-monooxygenase knock out (KMO-/-) mice which lack NAD biosynthesis pathway from tryptophan. To determine the niacin requirement and assess niacin nutritional markers, 4 wk old KMO-/- mice were fed 2-30 mg/kg nicotinic acid containing diets for 28 d. More than 4 mg/kg but not less than 3 mg/kg nicotinic acid containing diets induced maximum growth, and niacin nutritional markers in the blood, liver and urine increased with increase of dietary nicotinic acid. These results showed that several niacin nutritional markers reflect niacin nutritional status, niacin nutritional status can be controlled by dietary nicotinic acid, and niacin requirement for maximum growth is 4 mg/kg nicotinic acid diets in the KMO-/- mice. This animal model useful to investigate pathophysiology and mechanism of niacin deficiency, clarify the relationships between niacin nutritional status and age-related and lifestyle diseases, and evaluate factors affecting niacin nutritional status.