Tryptophan Hydroxylase Isoform Research Articles

Night-Restricted Feeding Improves Gut Health by Synchronizing Microbe-Driven Serotonin Rhythm and Eating Activity-Driven Body Temperature Oscillations in Growing Rabbits.

The circadian misalignment of the gut microbiota caused by unusual eating times in adult animals is related to disease development. However, whether the composition and diurnal rhythm of gut microbiota can be optimized by synchronizing the window period of eating with natural eating habits to reduce the risk of diarrhea remains unclear, especially in growing animals. In this study, 108 5-week-old weaned rabbits (nocturnal animals) were randomly subjected to daytime feeding (DF) and night-restricted feeding (NRF). At age 12 weeks, six rabbits were selected from each group, and caecum and cecal contents, as well as serum samples were collected at 4-h intervals during 24 h. Overall, NRF was found to reduce the risk of diarrhea in growing rabbits, improved the diurnal rhythm and abundance of beneficial microorganisms, along with the production of beneficial metabolites, whereas reduced the abundance of potential pathogens (Synergistes, Desulfovibrio, and Alistipes). Moreover, NRF improved diurnal rhythm of tryptophan hydroxylase isoform 1 and serotonin. Furthermore, NRF strengthened the diurnal amplitude of body core temperature, and promoted the diurnal expression of intestinal clock genes (BMAL1, CLOCK, REV-ERBα, and PER1), and genes related to the regulation of the intestinal barrier (CLAUDIN-1), and intestinal epithelial cell self-proliferation and renewal (BMI1). In vitro simulation experiments further revealed that synchronization of microbial-driven serotonin rhythm and eating activity-driven body temperature oscillations, which are important zeitgebers, could promote the diurnal expression of clock genes and CLAUDIN-1 in rabbit intestinal epithelial cells (RIEC), and enhance RIEC proliferation. This is the first study to reveal that NRF reprograms the diurnal rhythm of the gut microbiome, promotes the diurnal expression of clock genes and tight junction genes via synchronization of microbial-driven serotonin rhythm and eating activity-driven body temperature oscillations, thereby improving intestinal health and reducing the risk of diarrhea in growing rabbits. Collectively, these results provide a new perspective for the healthy feeding and management of growing animals.

Distribution of two isoforms of tryptophan hydroxylase in the brain of rainbow trout (Oncorhynchus mykiss). An in situ hybridization study

Serotonin (5-HT) is one of the principal neurotransmitters in the nervous system of vertebrates. It is initially synthesized by hydroxylation of tryptophan (Trp) by means of tryptophan hydroxylase or TPH which is the rate-limiting enzyme in the production of 5-HT. In most vertebrates, there are two isoforms of TPH present, TPH1 and TPH2, which exhibit different catalytic or substrate specificity as well as different expression domains. Studies carried out in mammals show that only tph2 is expressed in the brain whereas tph1-mRNA is primarily localized in the enterochromaffin cells and pineal gland. A large number of neurons are also considered to be serotonergic or “pseudo-serotonergic” as they accumulate and release 5-HT yet do not produce it as no amine-synthetic enzymes are expressed, yet a combination of 5-HT transporters is observed. Therefore, tph expression is considered to be the only specific marker of 5-HT-producing neurons that can discriminate true 5-HT from pseudo-serotonergic neurons. This work examined in situ hybridization to study the mRNA distribution of one paralogue for tph1 and tph2 in the central nervous system of rainbow trout. Results show a segregated expression for both paralogues that predominantly match previous immunocytochemical studies. This study thus adds valuable information to the scarce analyses focusing on the central distribution of the expression of serotonergic markers, particularly tphs, in the vertebrate brain thus characterizing the true serotonergic brain territories.

Phenylalanine hydroxylase contributes to serotonin synthesis in mice.

Serotonin is an important signaling molecule in the periphery and in the brain. The hydroxylation of tryptophan is the first and rate-limiting step of its synthesis. In most vertebrates, two enzymes have been described to catalyze this step, tryptophan hydroxylase (TPH) 1 and 2, with expression localized to peripheral and neuronal cells, respectively. However, animals lacking both TPH isoforms still exhibit about 10% of normal serotonin levels in the blood demanding an additional source of the monoamine. In this study, we provide evidence by the gain and loss of function approaches in in vitro and in vivo systems, including stable-isotope tracing in mice, that phenylalanine hydroxylase (PAH) is a third TPH in mammals. PAH contributes to serotonin levels in the blood, and may be important as a local source of serotonin in organs in which no other TPHs are expressed, such as liver and kidney.

Cell-Free Synthesis of Dopamine and Serotonin in Two Steps with Purified Enzymes.

The synthesis of serotonin and dopamine with purified enzymes is described. Both pathways start from an amino acid substrate and synthesize the monoamine neurotransmitter in two enzymatic steps. The enzymes human tryptophan hydroxylase isoform 2, Rattus norvegicus tyrosine hydroxylase, Chlamydia pneumoniae Cpn1046, and aromatic amino acid decarboxylase from Drosophila melanogaster are recombinantly expressed, purified, and shown to be functional in vitro. The hydroxylases efficiently convert L-DOPA (L-dihydroxy-phenylalanine) and 5-HTP (5-hydroxytryptophan) from L-tyrosine and L-tryptophan, respectively. A single aromatic amino acid decarboxylase is capable of converting both hydroxylated intermediates into the final neurotransmitter. The platform described here may facilitate future efforts to generate medically useful artificial cells and nanofactories.

A119 AN EX VIVO MODEL TO STUDY THE GUT SEROTONERGIC SYSTEM RESPONSE TO LIVE AND HEAT-KILLED LACTOBACILLUS RHAMNOSUS STRAIN JB-1

Abstract Background Enterochromaffin (EC) cells are one of the most abundant enteroendocrine cells in the intestinal epithelium, responsible for producing and storing the largest pool of serotonin or 5-hydroxytryptamine (5-HT) in the body. 5-HT has been shown to be important for modulating a large number of gastrointestinal reflexes in health and disease. 5-HT can stimulate extrinsic (vagal and spinal afferents) or intrinsic primary afferent neurons (IPANs) which are involved in motility, secretion and vasodilation within the intestines. Where EC cell localized enzyme tryptophan hydroxylase (TpH) isoform 1 is responsible for 5-HT synthesis, serotonin reuptake transporter (Sert) and monoamine oxidase A (Mao A) are responsible for termination by uptake and metabolism of 5-HT respectively. Our previous research has demonstrated the effects Lactobacillus rhamnosus (JB-1) on the firing frequency of spinal nerve fibres and motility. Increasing interest is being focused on potential health benefits of heat-inactivated microbes and purified bacterial components. However, the effect of these heat-killed bacteria on the intestinal epithelium cells, particularly on EC cells, is unknown. Aims Small intestinal organoids are shown to recapitulate in vivo characteristics of the small intestine epithelium. The present study aims to assess the suitability of intestinal organoids to study bacterial effects on the serotonergic system in the gut. Here we determined changes in the gene expression of key mediators in the serotonergic system [serotonin reuptake transporter (Sert), tryptophan hydroxylase-1 (Tph-1) and monoamine oxidase A (Mao A)] in response to live and heat-killed JB-1. Methods Male C57bl/6 mice aged 6–8 weeks were used for both ex vivo and in vivo experiments. Jejunal organoids were grown from whole crypts isolated using DTT-EDTA solution. Live and heat-killed JB-1 bacteria were used as treatments. Gene expression analysis was performed on jejunal organoids and jejunum tissue using qRT-PCR. Results JB-1 induced a significant increase in gene expression of Sert, Mao A and Tph-1. No significant difference was observed between the effects of live and heat-killed bacteria. In contrast the JB-1 increased expression of the peptide hormone CCK. Effects of JB-1 on gene expression in organoid culture were reflective of changes observed in in vivo experiments involving feeding of the bacteria. Conclusions Ex vivo organoid culture could be a useful tool in studying mechanisms underlying bacterial effects on serotonergic signalling. The observation that heat-killed bacteria produced comparable effects to the live organism suggests the possibility of isolating active 5-HT modulating components from these strains. Future research will focus on identifying such bacterial components and how their effects on gene expression influence serotonin availability Funding Agencies None

Inhibition of serotonin synthesis: A novel therapeutic paradigm.

The rate-limiting enzyme in serotonin synthesis is tryptophan hydroxylase (TPH). There are two independent serotonin systems in the body characterized by two isoforms of TPH, TPH1 and TPH2. While TPH2 synthesizes serotonin in the brain, TPH1 is expressed in the gut and in other peripheral tissues and supplies platelets in the circulation with serotonin. This duality of the serotonin system is enforced by the blood-brain barrier which is impermeable for serotonin. In the brain serotonin acts as neurotransmitter and is a main target for the treatment of psychiatric disorders. In the periphery it is released by platelets at the site of activation and elicits numerous physiological effects. TPH1 deficient mice were shown to be protected from diverse diseases including hemostatic, inflammatory, fibrotic, gastrointestinal, and metabolic disorders and therefore serotonin synthesis inhibition emerged as a reasonable therapeutic paradigm. Recently the first TPH inhibitor, telotristat ethyl, came on the market for the treatment of carcinoid syndrome. This review summarizes the state of development and the therapeutic opportunities of such compounds.

Maternal and infant characteristics connected to shared pleasure in dyadic interaction.

The aim of the study was to analyze which maternal factors (depressive symptoms, effect of life events, maternal sensitivity and structuring) and infant characteristics (temperament, social withdrawal symptoms, interactive behavior, genotype, gender) contribute to shared pleasure (SP) in parent-infant interaction. Participants were 113 mother-infant dyads. The mothers filled in the Edinburgh Postnatal Depression Scale, the Infant Behavior Questionnaire, and the Life Events Questionnaire. The dyads were videotaped in a free-play situation, and the videos were analyzed using the Alarm Distress Baby Scale and the Emotional Availability Scales. The infants were genotyped for four genes involved in emotion regulation. The occurrence and duration of SP (SP-MD) in mother-infant interactions were analyzed from the videotapes. Higher maternal sensitivity and depressive symptoms, better infant responsiveness, and the infant having the GG variant of the gene tryptophan hydroxylase isoform 2 (TPH2) -307 were associated with the occurrence of SP. Lower level depressive symptoms, better maternal structuring, and greater infant involvement were associated with the longer duration of SP. Those dyads where the mother and infant were best able to read each other's positive cues and to respond to them were more likely to experience mutual positive affect, as seen in SP.

Targeted Manipulation of Brain Serotonin: RNAi-Mediated Knockdown of Tryptophan Hydroxylase 2 in Rats.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of the biogenic monoamine serotonin (5-hydroxytryptamine, 5-HT). Two existing TPH isoforms are responsible for the generation of two distinct serotonergic systems in vertebrates. TPH1, predominantly expressed in the gastrointestinal tract and pineal gland, mediates 5-HT biosynthesis in non-neuronal tissues, while TPH2, mainly found in the raphe nuclei of the brain stem, is accountable for the production of 5-HT in the brain. Neuronal 5-HT is a key regulator of mood and behavior and its deficiency has been implicated in a variety of neuropsychiatric disorders, e.g., depression and anxiety. To gain further insights into the complexity of central 5-HT modulations of physiological and pathophysiological processes, a new transgenic rat model, allowing an inducible gene knockdown of Tph2, was established based on doxycycline-inducible shRNA-expression. Biochemical phenotyping revealed a functional knockdown of Tph2 mRNA expression following oral doxycycline administration, with subsequent reductions in the corresponding levels of TPH2 enzyme expression and activity. Transgenic rats showed also significantly decreased tissue levels of 5-HT and its degradation product 5-Hydroxyindoleacetic acid (5-HIAA) in the raphe nuclei, hippocampus, hypothalamus, and cortex, while peripheral 5-HT concentrations in the blood remained unchanged. In summary, this novel transgenic rat model allows inducible manipulation of 5-HT biosynthesis specifically in the brain and may help to elucidate the role of 5-HT in the pathophysiology of affective disorders.

The journey from gene knockout to clinical medicine: telotristat and sotagliflozin.

Gene knockout has been a powerful technique to evaluate the physiologic role of selected gene products. Lexicon pioneered high-throughput gene knockout technology and went further in designing agents to inhibit products of gene expression. Two agents have entered late-stage development. Telotristat is an inhibitor of tryptophan hydroxylase (TPH), preventing the production of serotonin. Although this agent blocks the two isoforms of TPH, it does not cross the blood–brain barrier, thus avoiding central neurologic manifestations. It inhibits the peripheral production of serotonin, and in particular prevents serotonin action in the intestines, resulting in decreased peristaltic action. Lexicon successfully developed telotristat to treat carcinoid syndrome not responding adequately to somatostatin inhibitors. Sotagliflozin development proceeded from the observation that dual inhibition of SGLT2 in the kidneys and SGLT1 in the intestines resulted in increased renal glucose excretion, reduced early-phase glucose absorption, as well as increased blood levels of GLP-1 and PYY. Initial development efforts focused on type 1 diabetes and have shown reduced postprandial glucose levels, less tendency to hypoglycemia, and lower HbA1c. Several other SGLT2 inhibitors have been associated with increased frequency of diabetic ketoacidosis (DKA). In the type 1 trials, sotagliflozin-treated individuals experienced DKA at a higher rate than placebo-treated patients. The sotagliflozin development program has now been extended to trials on type 2 diabetes. Long-term clinical trials will determine the benefits and risks of the agent in comparison to other currently marketed SGLT2 inhibitors.

Brain Aromatase Modulates Serotonergic Neuron by Regulating Serotonin Levels in Zebrafish Embryos and Larvae.

Teleost fish are known to express two isoforms of P450 aromatase, a key enzyme for estrogen synthesis. One of the isoforms, brain aromatase (AroB), cyp19a1b, is highly expressed during early development of zebrafish, thereby suggesting its role in brain development. On the other hand, early development of serotonergic neuron, one of the major monoamine neurons, is considered to play an important role in neurogenesis. Therefore, in this study, we investigated the role of AroB in development of serotonergic neuron by testing the effects of (1) estradiol (E2) exposure and (2) morpholino (MO)-mediated AroB knockdown. When embryos were exposed to E2, the effects were biphasic. The low dose of E2 (0.005 µM) significantly increased serotonin (5-HT) positive area at 48 hour post-fertilization (hpf) detected by immunohistochemistry and relative mRNA levels of tryptophan hydroxylase isoforms (tph1a, tph1b, and tph2) at 96 hpf measured by semi-quantitative PCR. To test the effects on serotonin transmission, heart rate and thigmotaxis, an indicator of anxiety, were analyzed. The low dose also significantly increased heart rate at 48 hpf and decreased thigmotaxis. The high dose of E2 (1 µM) exhibited opposite effects in all parameters. The effects of both low and high doses were reversed by addition of estrogen receptor (ER) blocker, ICI 182,780, thereby suggesting that the effects were mediated through ER. When AroB MO was injected to fertilized eggs, 5-HT-positive area was significantly decreased, while the significant decrease in relative tph mRNA levels was found only with tph2 but not with two other isoforms. AroB MO also decreased heart rate and increased thigmotaxis. All the effects were rescued by co-injection with AroB mRNA and by exposure to E2. Taken together, this study demonstrates the role of brain aromatase in development of serotonergic neuron in zebrafish embryos and larvae, implying that brain-formed estrogen is an important factor to sustain early development of serotonergic neuron.

Vitamin D Stimulates Serotonin Production via Induction of the Tryptophan Hydroxylase 2 Isoform in B14 Rat Medullary Neurons

The active form of vitamin D3, 1,25‐dihydroxyvitamin D (1,25D), and serotonin (5‐HT) are postulated to play significant roles in abnormal social behavior associated with psychological conditions including autism spectrum disorders (ASDs) and depression. Moreover, we have observed that 1,25D regulates synthesis of two isoforms of tryptophan hydroxylase (TPH), the rate‐limiting enzyme in the 5‐HT biosynthetic pathway, including induction of the tryptophan hydroxylase 2 (TPH2) gene in the central nervous system (CNS) and suppression of tryptophan hydroxylase 1 (TPH1) expression in some peripheral tissues. Interestingly, major characteristics associated with autism may include low concentrations of serotonin in the CNS and elevated levels of serotonin in the periphery. Thus, in addition to assessing the effect of 1,25D on TPH gene expression, we also examined, for the first time, the ability of 1,25D to stimulate production of 5‐HT in neuronal cells. Rat serotonergic medullary cells (RN46A‐B14; B‐14) were cultured to confluency and plated in six‐well plates. After allowing cells to attach in the plates overnight, the cells were cultured in differentiation medium, followed by treatment of differentiated B‐14 cells with ethanol vehicle or 10−8 M 1,25D. At time zero, a 200 μL sample of media from each well was collected to serve as a baseline for 5‐HT concentration. The cells were then incubated over the next three days, and additional 200 μL samples were collected from each well at the 24‐, 48‐ and 72‐hour time points. A 5‐HT ELISA assay was then conducted using the collected samples. The results of the ELISA revealed a statistically significant increase (57%, p < 0.05) in 5‐HT production following treatment with 1,25D for 24 hours versus vehicle, with subsequently larger percent‐increases in 5‐HT synthesis observed at 48 and 72 hours (64% and 103%, respectively, p < 0.05). These results suggest that 1,25D stimulates the production of serotonin, likely through the induction of the neuronal TPH2 gene, and the time‐course of this effect appears to lag behind the induction of TPH mRNA which peaks at 24 hours. In summary, this study illuminates a novel coordinated biochemical and regulatory interaction between two neuromodulators, vitamin D and serotonin, thereby identifying a potential connection between serum vitamin D levels and the occurrence of psychiatric disorders commonly linked to an imbalance in CNS serotonin production, including autism and depression.Support or Funding InformationThis project was funded by the New College of Interdisciplinary Arts and Sciences at Arizona State University and the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dorsal raphé nucleus glucocorticoid receptors inhibit tph2 gene expression in male C57BL/6J mice.

The serotonergic dorsal raphé nucleus (DRN) expresses glucocorticoid receptors (GR), and systemic glucocorticoids have been shown to regulate expression and activity of tryptophan hydroxylase isoform 2, the rate-limiting enzyme for serotonin synthesis in brain. We have used intra-DRN injection of pseudotyped adeno-associated virus AAV2/9 transducing either green fluorescent protein (GFP control) or Cre recombinase (DRN GR deletion) in floxed GR mice to determine if DRN GR directly regulate DRN mRNA levels of tryptophan hydroxylase 2 (tph2). In a separate set of similarly-treated floxed GR mice, we also measured limbic forebrain region concentrations of serotonin (5-hydroxytryptamine; 5-HT) and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA). DRN GR deletion increased tph2 mRNA levels in the dorsal, lateral wing, and caudal parts of the DRN without altering tissue concentrations of 5-HT, 5-HIAA, or the 5-HIAA/5-HT ratio in limbic forebrain regions. We conclude that DRN GR inhibit DRN tph2 gene expression in mice without marked effects on serotonin metabolism, at least under basal conditions at the circadian nadir. These data provide the first evidence of localized control of DRN tph2 mRNA expression by DRN GR in mice.

Isoform-Specific Substrate Inhibition Mechanism of Human Tryptophan Hydroxylase

Tryptophan hydroxylase (TPH) catalyzes the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression and irritable bowel syndrome. TPH exists in two isoforms: TPH1 and TPH2. TPH1 catalyzes the initial step in the synthesis of serotonin in the peripheral tissues, while TPH2 catalyzes this step in the brain. In this study, the steady-state kinetic mechanism for the catalytic domain of human TPH1 has been determined. Varying substrate tryptophan (Trp) and tetrahydrobiopterin (BH4) results in a hybrid Ping Pong-ordered mechanism in which the reaction can either occur through a Ping Pong or a sequential mechanism depending on the concentration of tryptophan. The catalytic domain of TPH1 shares a sequence identity of 81% with TPH2. Despite the high sequence identity, differences in the kinetic parameters of the isoforms have been identified; i.e., only TPH1 displays substrate tryptophan inhibition. This study demonstrates that the difference can be traced to an active site loop which displays different properties in the TPH isoforms. Steady-state kinetic results of the isoforms, and variants with point mutations in a loop lining the active site, show that the kinetic parameters of only TPH1 are significantly changed upon mutations. Mutations in the active site loop of TPH1 result in an increase in the substrate inhibition constant, Ki, and therefore turnover rate. Molecular dynamics simulations reveal that this substrate inhibition mechanism occurs through a closure of the cosubstrate, BH4, binding pocket, which is induced by Trp binding.

Human placenta expresses both peripheral and neuronal isoform of tryptophan hydroxylase

The role of placental serotonin has been an active topic of research notably because of its crucial role in brain development. However, which cell types synthesize serotonin in human placenta remains unknown. Moreover, it is not known if the two tryptophan hydroxylase isoforms (TPH1 and TPH2), the rate-limiting enzymes in serotonin biosynthesis, are expressed in placenta. Human placentas were obtained in first trimester or at term, and trophoblast cells were isolated and purified using a magnetic cell sorter and placed in primary culture. The tissue sublocalization of each TPH was determined by immunohistochemistry. TPH expression in primary villous trophoblasts was determined by PCR and immunoblotting, and serotonin secretion by LC-MS/MS. Villous cytotrophoblasts, syncytiotrophoblast, fetal capillaries, extravillous cytotrophoblasts, and decidual cells co-expressed TPH1 and TPH2. Moreover, mRNA and protein levels of both TPHs were detected in human primary trophoblast as well as in mouse placental tissues. Finally, human trophoblast cells were shown to produce serotonin de novo. This study demonstrates that both TPH1 and TPH2 are expressed in human and mouse placenta throughout pregnancy and helps to better understand the placental serotonin system, which is crucial for healthy pregnancy and fetal development. It is therefore important to further understand regulation of the placental serotonin system and how its disruption during pregnancy may impact the developing fetus and subsequent child programming.

Stress inhibits tryptophan hydroxylase expression in a rat model of depression.

Serotonin (5-hydroxytryptamine, 5-HT) dysfunction is associated with the pathophysiology of depression. Tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, is believed to have essential role in many mental disorders, including depression. In the present study, we generated a rat model of depression by exposing the animals to stress, and the rats were then treated with paroxetine. The results indicated that the concentration of 5-HT in the brain and liver tissues were significantly lower in the rat model of depression than in healthy or treated rats. Immunohistochemical analyses of TPH1/2 showed less TPH1 and TPH2 expression, specifically TPH2, in the brain, liver and kidney of the depressive rats than in the healthy rats; In addition, the two TPH isoforms, TPH1 and TPH2, had different spatial distributions,the mRNAs of the TPH1/2 genes were significantly decreased and TPH1/2 were highly methylated in the depressive model rat, but treatment with paroxetine ameliorated the expression and methylation of TPH1/2. All together, stress was able to inhibit expression of TPH1/2 in brain tissue and decrease concentration of 5-HT, the mechanism maybe involve in increasing the methylation of TPH2 genes promoter; Paroxetine has a role in confronting the effect of stress in depressive rat model.

Perinatal SSRI exposure permanently alters cerebral serotonin receptor mRNA in mice but does not impact adult behaviors

Purpose: Associations have been made between maternal selective serotonin reuptake inhibitor (SSRI) use during pregnancy and altered behavior in offspring, including an increased risk of autism. Given the important role serotonin plays in behavior, we hypothesized SSRI exposure in the perinatal period would alter central serotonin receptor expression and program adult behaviors in mice.Methods: Female mice were injected with sertraline or saline throughout pregnancy. Offspring continued to receive injections on postnatal days 1–14, a time period in mice similar to the third trimester in human pregnancy. Adult offspring underwent behavioral testing, and serotonin receptor mRNA levels were quantified.Results: Compared to controls, SSRI exposed mice did not have a reduction in social interactions, spatial learning, or exploratory behavior. As adults, sertraline exposed mice had significantly increased mRNA levels of multiple 5-HT receptors, serotonin transporter (5-HTT), and tryptophan hydroxylase isoform 2 in the cerebral cortex.Conclusion: Although no behavioral phenotype was observed, SSRI exposure in the perinatal period permanently alters cerebral receptor mRNA levels. We speculate these shifts in mRNA expression provide important compensation during SSRI exposure. Further pre-clinical and clinical investigation into additional serotonin-regulated phenotypes is necessary to further assess the long-term implications of perinatal SSRI exposure.

Central anti-fatigue activity of verbascoside

In this study, the effects of verbascoside on treadmill exercise endurance, 5-hydroxytryptamine (5-HT) concentrations, the second isoforms of tryptophan hydroxylase (TPH2) and serotonergic type 1B inhibitory autoreceptors (5-HT1B) protein expression in the caudate putamen of exercised rats were investigated. Sixty Sprague-Dawley male rats were randomly divided into six groups: normal group, exercise group, exercise and verbascoside (0.1mg/kg)–treated group, exercise and verbascoside (1mg/kg)–treated group, exercise and verbascoside (10mg/kg)–treated group, exercise and caffeine (10mg/kg)–treated group. In exercise groups, rats were put on treadmill and forced to run for 30min once a day for 6 consecutive days. On the 7th day of the experiment, the time to exhaustion in treadmill exercise was determined for the trained groups. Immediately after the determination of the exhaustion time, all rats were sacrificed. 5-HT concentrations were detected by HPLC analysis. TPH2 and 5-HT1B protein expression were measured by western blot analysis. We found that verbascoside could prolong the time to exhaustion in treadmill exercise and suppress the exercise-induced increase of 5-HT synthesis and TPH2 protein expression, and prevent the exercise-induced decrease of 5-HT1B protein expression in the caudate putamen. Verbascoside was found as effective as caffeine in these effects. Verbascoside at 10mg/kg improved endurance of exercised rats. The mechanism of verbascoside' s anti-fatigue activity might be related to the inhibition of the exercise-induced synthesis of 5-HT and TPH2 expression, and to the increase of the 5-HT1B expression in the caudate putamen of exercised rats.

Fluoxetine impairs insulin secretion without modifying extracellular serotonin levels in MIN6 β-cells.

Pancreatic β-cells synthetize and store Serotonin (5-Hydroxytriptamine, 5HT) which is co-released with insulin. It has been proposed that extracellular 5HT binds to specific cell surface receptors and modulate insulin secretion. On the other hand, Selective Serotonin Reuptake Inhibitor (SSRI) fluoxetine seems to reduce Glucose-Stimulated Insulin Secretion (GSIS). However, it is unknown whether this effect results from changes in extracellular 5HT concentration owed to the blockade of 5HT transporter (SERT) or from non-5HT dependent actions. The aims of this work were: 1) to quantify extracellular 5HT levels and GSIS in β-cell lines, 2) to determine whether extracellular 5HT levels and GSIS are changed by fluoxetine or 5-Hydroxytryptophan (5HTP, the immediate 5HT biosynthetic precursor), and 3) to quantify the expression of Slc6a4 gene (encoding SERT) in β-cell lines in relation to other genes involved in 5HT system. β-cell lines MIN6 and RINm5f were subjected to GSIS protocols, after treatment with fluoxetine, 5HTP or 5HT. Insulin and 5HT were quantified by ELISA and HPLC, respectively. Relative mRNA expression was quantified by RT-qPCR. MIN6 β-cells secretes 5HT in response to glucose, showing a sharp increase in 5HT release when cells were preloaded with 5HTP. Treatment with 5HT or fluoxetine reduces GSIS. Fluoxetine fails to further increases 5HTP-induced elevation of secreted 5HT. MIN6 β-cells express both isoforms of Tryptophan Hydroxylase (Tph1 and Tph2), and have high expression levels of L-Dopa decarboxylase (Ddc), both enzymes involved in 5HT biosynthetic pathway, but do not express the 5HT transporters Slc6a4 or Slc6a3 (the Dopamine-5HT transporter) genes. The inhibitory effect of fluoxetine on β-cell glucose stimulated insulin secretion is not mediated by blockage of 5HT transporter through SERT.

Association analysis of TPH-1 and TPH-2 genes with suicidal behavior in patients with attempted suicide in Mexican population

BackgroundSuicidal behavior is a worldwide health problem. Tryptophan hydroxylase (TPH) is a rate limiting enzyme in the biosynthesis of serotonergic neurotransmission. TPH-1 and TPH-2 genes encode for TPH isoforms and have been implicated as candidate genes for suicidal behavior. The aim of this study was to evaluate the association between the genetic variants of the TPH-1 (rs21102 and 1607395) and TPH-2 (rs4290270, rs7305115 and rs1007023) genes and suicidal behavior in a Mexican population. MethodsWe conducted a case-control study including 200 cases with suicide attempt and 263 controls. Patients were evaluated by a trained psychiatrist or clinical psychologists. Five polymorphisms were genotyped and assessed for allele, genotype and haplotype association with suicide attempt. ResultsThe rs7305115 polymorphism of the TPH-2 gene was associated with suicidal behavior in a Mexican population in genotype (χ2=6.02, df=2, p=0.04) and allele (OR=1.39, 95%IC=1.06–1.81, p=0.01) frequencies. The THP-2 haplotypes GTA (χ2=5.68, p=0.01) and ATT (χ2=5.0, p=0.02) were associated with risk for suicide attempt. ConclusionOur results provide evidence for an association between the rs7305115 polymorphism of the TPH-2 gene and suicidal behavior in a Mexican population. However, more studies are necessary to replicate these results using larger samples.

Association study of TPH2 polymorphisms and bipolar disorder in the Han Chinese population.

Bipolar disorder (BPD) is a serious and common mental disorder with high heritability. The serotonergic system is known to be implicated in the etiology of the disorder. Tryptophan hydroxylase isoform-2 (TPH2), which controls the synthesis of serotonin in the brain, has been suggested as a candidate gene for BDP. The aim of this study was to examine the association between the polymorphisms in TPH2 and BPD. We conducted a case-control study by genotyping six SNPs (rs10784941, rs1386494, rs2171363, rs4760816, rs1386486, and rs1872824) in 506 bipolar patients and 507 controls of Chinese Han origin. rs10784941 was not in the Hardy-Weinberg equilibrium and therefore excluded from further analysis. rs1386486 and rs1872824 showed statistically significant differences between cases and controls in genotype frequencies (rs1386486: p=0.043351; rs1872824: p=0.016563), but no association in allele frequencies. Strong LD was found among rs1386494, rs2171363 and rs4760816, but no positive association with BPD was found for haplotypes. Our results indicate that in the Han Chinese population TPH2 may be a potential susceptibility gene for bipolar disorder. Further studies are needed to validate this association.