Repeated 3,3-Dimethyl-1-butanol exposure alters social dominance in adult mice.

The influence of gut microbiota on brain function and brain disorders has been attracted more and more attention. Trimethylamine N-oxide (TMAO), an indirect metabolite of gut microbiota, has been linked to aging, cognitive impairment, and other brain disorders. However, the relationship between TMAO and social behaviors are still poorly understood. Adult male mice were exposed to drinking water containing 3,3- Dimethyl-1-butanol (DMB), an indirect inhibitors of TMAO, for 21 continuous days followed by a series of behavioral tests to detect the effect of DMB exposure on social behaviors, mainly including social dominance test (SDT), bedding preference test (BP), sexual preference test (SP), social interaction test (SI), open field test (OFT), tail suspension test (TST), forced swim test (FST), novelty suppressed feeding test (NSF), and novel object recognition (NOR) task. In the SDT, compared with the control group, the mice treated with DMB (both 0.2% and 1.0%), both high-ranked and low-ranked mice, showed a reduction in the number of victories. There is no statistical difference on sexual preference, anxiety, depression-like behavior phenotype, and memory formation. In conclusion, the present findings provide direct evidence, for the first time, that repeated DMB exposure produces significant effects on social dominance of adult mice, without any effects on sexual preference, anxiety, depression-like behavior phenotype or memory formation, highlighting the regulatory effects of gut-brain interaction on social behaviors.

Use of dietary phytochemicals for inhibition of trimethylamine N-oxide formation.

Trimethylamine-N-oxide (TMAO) has been reported as a risk factor for atherosclerosis development, as well as for other cardiovascular disease (CVD) pathologies. The objective of this review is to provide a useful summary on the use of phytochemicals as TMAO-reducing agents. This review discusses the main mechanisms by which TMAO promotes CVD, including the modulation of lipid and bile acid metabolism, and the promotion of endothelial dysfunction and oxidative stress. Current knowledge on the available strategies to reduce TMAO formation are discussed, highlighting the effect and potential of phytochemicals. Overall, phytochemicals (i.e., phenolic compounds or glucosinolates) reduce TMAO formation by modulating gut microbiota composition and/or function, inhibiting host's capacity to metabolize TMA to TMAO, or a combination of both. Perspectives for design of future studies involving phytochemicals as TMAO-reducing agents are discussed. Overall, the information provided by this review outlines the current state of the art of the role of phytochemicals as TMAO reducing agents, providing valuable insight to further advance in this field of study.

Discovery of a Histidine-Based Scaffold as an Inhibitor of Gut Microbial Choline Trimethylamine-Lyase.

Anaerobic choline metabolism by human gut microbiota to produce trimethylamine (TMA) has recently evolved as a potential therapeutic target because of its association with chronic kidney disease and increased cardiovascular risks. Limited examples of choline analogues have been reported as inhibitors of bacterial enzyme choline TMA-lyase (CutC), a key enzyme regulating choline anaerobic metabolism. We used a new workflow to discover CutC inhibitors based on focused screening of a diversified library of small molecules for intestinal metabolic stability followed by in vitro CutC inhibitory assay. This workflow identified a histidine-based scaffold as a CutC inhibitor with an IC50 value of 1.9±0.2 µM. Remarkably, the identified CutC inhibitor was able to reduce the production of TMA in whole-cell assays using various bacterial strains as well as in complex gut microbiota environment. The improved efficiency of the new scaffold identified in this study in comparison to previously reported CutC inhibitors would enable optimization of potential leads for in vivo screening and clinical translation. Finally, docking studies and molecular-dynamic simulations were used to predict putative interactions created between inhibitor and CutC.

GW9508 ameliorates cognitive impairment via the cAMP-CREB and JNK pathways in APPswe/PS1dE9 mouse model of Alzheimer's disease.

GPR40 was utilized as the drug target to the treatment of diabetes, but the function and mechanisms ameliorating the Alzheimer's disease (AD) remain unknown. In present study, the typical APP/PS1 mouse model was applied to explore the function and mechanism of GPR40 in AD. GPR40 agonist GW9508 and antagonist GW1100 were respectively given by i.c.v. injection to activate/inhibit the GPR40 in the brain of APP/PS1 mice which illustrated the function and mechanism of GPR40 in ameliorating AD symptoms. Morris water maze test, step-through test, Y-maze spontaneous alternation test, open field test and new object recognition test were used to test the cognitive function and memory ability of mice, while molecular biology experiments such as Western blot, immunofluorescence, JC-1 were used to detect the corresponding changes of signal pathways. The results revealed that treatment with GW9508 could significantly ameliorate cognitive deficits of APP/PS1 mice, upregulate the expression levels of cAMP, p-CREB and neurotrophic factors in vivo, while GW9508 also ameliorate Aß1-42-induced neuron damage and downregulate the expression levels of pathological protein such as p-JNK, JNK and apoptosis-related proteins such as IL-6, IL-1ß, TNF-α and caspase-3 in vitro. Meanwhile, high-content screening also showed that GW9508 promoted the cellular differentiation of SH-SY5Y cells, while GW1100 reversed the effects of GW9508. These results suggested that GPR40 was an underlying therapeutic target for the treatment of AD and GPR40 agonist could be explored as the emerging AD therapeutic drug.

Amelioration of TMAO through probiotics and its potential role in atherosclerosis.

Atherosclerosis is a major cause of mortalities and morbidities worldwide. It is associated with hyperlipidemia and inflammation, and become chronic by triggering metabolites in different metabolic pathways. Disturbance in the human gut microbiota is now considered a critical factor in the atherosclerosis. Trimethylamine-N-oxide (TMAO) attracts attention and is regarded as a vital contributor in the development of atherosclerosis. TMAO is generated from its dietary precursors choline, carnitine, and phosphatidylcholine by gut microbiota into an intermediate compound known as trimethylamine (TMA), which is then oxidized into TMAO by hepatic flavin monooxygenases. The present review focus on advances in TMAO preventing strategies through probiotics, including, modulation of gut microbiome, metabolomics profile, miRNA, or probiotic antagonistic abilities. Furthermore, possible recommendations based on relevant literature have been presented, which could be applied in probiotics and atherosclerosis-preventing strategies.

Reductions in gut microbiota­derived metabolite trimethylamine N­oxide in the circulation may ameliorate myocardial infarction­induced heart failure in rats, possibly by inhibiting interleukin­8 secretion.

Myocardial infarction (MI) is a common cause of chronic heart failure (HF). Increasing evidence has revealed that trimethylamine N­oxide (TMAO), a gut­microbiota­derived metabolite, contributes to the pathogenesis of cardiovascular disease by promoting inflammation. Elevated levels of circulating TMAO have been reported in patients following MI and were associated with unfavorable outcomes. The present study examined whether reductions in circulating TMAO could attenuate the progression of HF in rats following MI. Sprague­Dawley rats underwent coronary ligation to induce MI or a sham operation. Echocardiography confirmed MI and cardiac dysfunction one day following coronary ligation. MI and sham rats were then treated with either vehicle (tap water) or 1.0% 3,3­dimethyl­1­butanol (DMB, a trimethylamine formation inhibitor) in tap water, for 8 weeks. At the end of the experiment, TMAO plasma levels were markedly elevated in vehicle­treated MI rats compared with vehicle­treated sham rats; however, TMAO plasma levels were reduced in DMB­treated MI rats compared with vehicle­treated MI rats. Both MI groups exhibited cardiac hypertrophy, lung congestion, left ventricular remodeling and impaired cardiac function, according to the results of anatomical analysis, echocardiography and left ventricular hemodynamics; however, these manifestations of MI­induced HF were significantly improved in DMB­treated MI rats compared with vehicle­treated MI rats. The plasma levels of the chemokine interleukin (IL)­8, and cardiac expression of IL­8 and its receptors were significantly increased in vehicle­treated MI rats compared with vehicle­treated sham rats; however, these were normalized in DMB­treated MI rats. In addition, elevated TMAO plasma level was positively correlated with increased IL­8 plasma level in MI groups. Notably, DMB treatment of sham rats also reduced plasma TMAO, but did not alter other parameters. These results indicated that reducing circulating TMAO may ameliorate the development of chronic HF following MI in rats, potentially by inhibiting IL­8 secretion. The results from the present study suggested that inhibition of TMAO synthesis may be considered as a novel therapeutic approach for the prevention and treatment of patients with chronic MI­induced HF.

Gut microbe-derived metabolite trimethylamine N-oxide induces cardiac hypertrophy and fibrosis.

Trimethylamine N-oxide (TMAO), a gut microbe-derived metabolite of dietary choline and other trimethylamine-containing nutrients, has been linked to increased cardiovascular disease risk. It is unknown whether TMAO plays a role in the development of cardiac hypertrophy. Transverse aortic constriction (TAC) was performed to induce cardiac hypertrophy in Sprague-Dawley (SD) rats. We observed that TMAO levels were significantly elevated in SD rats after 6 weeks of TAC, suggesting the potential role of TMAO in regulating cardiac hypertrophy. In cultured cardiomyocytes, TMAO treatment stimulated cardiac hypertrophy, as indicated by increased cell area of cardiomyocytes and expression of hypertrophic markers including atrial natriuretic peptide (ANP) and beta-myosin heavy chain (ß-MHC). Additionally, TMAO treatment induced cardiac hypertrophy and cardiac fibrosis in SD rats. Reducing TMAO synthesis by antibiotics (Abs) attenuated TAC-induced cardiac hypertrophy and fibrosis. Furthermore, pharmacological inhibition of Smad3 by SIS3 significantly reduced the expression of ANP and ß-MHC, and cardiomyocyte cell size in TMAO-treated group. These data for the first time demonstrate that gut microbe-derived metabolite TMAO induces cardiac hypertrophy and fibrosis involving Smad3 signaling, suggesting that inhibition of gut microbes or generation of TMAO may become a potential target for the prevention and treatment of cardiac hypertrophy.

Trimethylamine N-oxide (TMAO) as a New Potential Therapeutic Target for Insulin Resistance and Cancer.

BACKGROUND: The intake of animal products in food has been associated with both the development of insulin resistance and gastrointestinal cancers (GIC). Through the digestion of animal protein and other constituents of animal products, the commensal bacteria in the gut (the gut microbiota) forms metabolites that can contribute to the development of both insulin resistance and cancer. Trimethylamine-N-Oxide (TMAO) is such a molecule and has recently drawn a lot of attention as it may be a risk factor for - and a link between - the gut microbiota and cardiovascular and renal disease. Further, TMAO is anticipated to have significance as a biomarker of - or even an independent risk factor for - other undesirable conditions, including insulin resistance and GIC. TMAO originates from a precursor, trimethylamine (TMA) that is a metabolite of various precursors; mainly choline and carnitine from ingested foods. METHODS: We review the literature on TMAO as a shared risk factor and/or pathway between insulin resistance and GIC risk and take the reader through the literature of interventions that could reduce formation of TMAO and thereby the risk of insulin resistance and GIC. The purpose of the work is to generate a hypothesis to be tested in preclinical and clinical studies. RESULTS: TMAO seems to be associated with both insulin resistance and GIC risk and also with atherosclerotic cardiovascular disease. One shared pathway is the formation of N-Nitroso compounds, a group of metabolites that can cause DNA-damage and epigenetic changes. Levels of TMAO can be reduced by limiting the dietary intake of certain foods, most importantly animal products. Further, certain drugs, namely Meldonium and 3,3-dimethyl- 1-butanol, may inhibit the formation of TMAO by inhibiting bacterial enzymes. CONCLUSIONS: The TMAO pathway and its metabolites are possibly involved in the development of two major health problems: insulin resistance and cancer. Within these pathways novel therapeutic targets may be identified. Further research is needed in order to verify existing or develop new pharmacological agents that modify these pathways and reduce the risk of insulin resistance and GIC.

The activation of supraspinal GPR40/FFA1 receptor signalling regulates the descending pain control system.

BACKGROUND AND PURPOSE: The ω-3 polyunsaturated fatty acids exert antinociceptive effects in inflammatory and neuropathic pain; however, the underlying mechanisms remain unclear. Docosahexaenoic acid-induced antinociception may be mediated by the orphan GPR40, now identified as the free fatty acid receptor 1 (FFA1 receptor). Here, we examined the involvement of supraspinal FFA1 receptor signalling in the regulation of inhibitory pain control systems consisting of serotonergic and noradrenergic neurons. EXPERIMENTAL APPROACH: Formalin-induced pain behaviours were measured in mice. Antinociception induced by FFA1 receptor agonists was examined by intrathecal injections of a catecholaminergic toxin, 5-HT lowering drug or these antagonists. The expression of FFA1 receptor protein and c-Fos was estimated by immunohistochemistry, and the levels of noradrenaline and 5-HT in the spinal cord were measured by LC-MS/MS. KEY RESULTS: FFA1 receptors colocalized with NeuN (a neuron marker) in the medulla oblongata and with tryptophan hydroxylase (TPH; a serotonergic neuron marker) and dopamine ß-hydroxylase (DBH; a noradrenergic neuron marker). A single i.c.v. injection of GW9508, a FFA1 receptor agonist, increased the number of c-Fos-positive cells and the number of neurons double-labelled for c-Fos and TPH and/or DBH. It decreased formalin-induced pain behaviour. This effect was inhibited by pretreatment with 6-hydroxydopamine, DL-p-chlorophenylalanine, yohimbine or WAY100635. Furthermore, GW9508 facilitated the release of noradrenaline and 5-HT in the spinal cord. In addition, GW1100, a FFA1 receptor antagonist, significantly increased formalin-induced pain-related behaviour. CONCLUSION AND IMPLICATIONS: Activation of the FFA1 receptor signalling pathway may play an important role in the regulation of the descending pain control system.

QSAR study for a novel series of ortho disubstituted phenoxy analogues of alpha1-adrenoceptor antagonist WB4101.

On the basis of the affinities at the alpha1a-, alpha1b- and alpha1d-adrenoceptors and the 5-HT1A receptor of a previous series of sixteen 2-[(2-phenoxyethyl)aminomethyl]-1,4-benzodioxanes ortho monosubstituted at the phenoxy moiety, a number of ortho disubstituted analogues were designed, synthesized in both the enantiomeric forms and tested in binding assays on the same receptors. The affinity values of the new compounds 1-11 were compared with those of the enantiomers of the 2,6-dimethoxyphenoxy analogue, the well-known alpha1 antagonist WB4101, and of the ortho monosubstituted derivatives, suggesting some distinctive aspects of the interaction of the phenoxy moiety, in particular with the alpha1a-AR and the 5-HT1A receptor, of the monosubstituted and the disubstituted compounds. A classical quantitative structure-activity relationship (Hansch) analysis was applied to the whole set of the S enantiomers of the ortho mono- and disubstituted WB4101 analogues (26 compounds), finding a very good correlation for the alpha1a affinity. For this latter, a significant parabolic relationship was also found with the volume of the two ortho substituents. Diametrically opposite, the same relationships for the 5-HT1A exhibit low or insignificant correlation coefficients.

Ammonium (methylammonium) transport by heterocysts and vegetative cells of Anabaena variabilis.

Transport of the ammonium analogue [(14)C]methylammonium was similar in non-growing, fully differentiated heterocysts as compared to vegetative, multiplying cells of the filamentous cyanobacterium Anabaena variabilis. NH(4)(+) inhibited uptake into the cells and released accumulated methylammonium from the cells. These observations suggest that the main function of ammonium transport in heterocysts may not be NH(4)(+) acquisition but cyclic retention of ammonia produced by nitrogenase.

Teratogenic and macromolecular synthesis inhibitory effects of trimethylamine on mouse embryos in culture.

Trimethylamine (TMA) is an aliphatic amine, and its blood levels can increase after ingestion of certain foods, such as fish, and during disease states, such as chronic renal failure. We recently reported that TMA can inhibit fetal development in vivo and in vitro in mice. The present studies were done to find out if the inhibitory effects of TMA on embryonic development are caused by a decrease in macromolecular synthesis, using mouse embryo cultures as the experimental model. At a submaximally toxic concentration (0.75mM), TMA inhibited the growth of embryos to approximately 70% of control and caused neural-tube defects in 73% of embryos. By 42 h of culture, DNA, RNA, and protein content of TMA-treated embryos were approximately 50% of the control values. Embryotoxic effects of TMA were not caused by changes in pH and osmolarity of the culture media. The inhibitory effects of TMA on embryonic growth were time dependent and apparent at 2-4 h of culture. The inhibition of growth was accompanied by a decrease in the incorporation of tritium-labeled thymidine, uridine, and leucine into DNA, RNA, and proteins, respectively. Thiols (L- and D-cysteine, glutathione) and the antioxidant L-ascorbic acid did not cause significant antagonism of embryotoxic effects of TMA. It is concluded that TMA exerts teratogenic effects on mouse embryos in culture and inhibits their growth by reducing macromolecular synthesis; these effects may not involve glutathione depletion or generation of free radicals.

Nitrogen regulation of synthesis of the high affinity methylammonium transport system of Escherichia coli.

Uptake of 14CH3NH+3 (methylammonium) was measured as a probe of NH+4 transport in intact Escherichia coli cells and derivatives impaired in the Ntr regulatory system. The results suggest that expression of the high affinity 14CH3NH+3 transport system (a) requires de novo polypeptide synthesis, (b) is activated by the glnG and glnF regulatory products under nitrogen limitation, and (c) is repressed under nitrogen excess by the glnL product. Cells deficient in glutamate synthase activity by virtue of their harbouring the gltB31 mutation were unable to activate synthesis of 14CH3NH+3 transport. This could explain the inability of cells carrying gltB mutations to grow on low concentrations of NH+4.

An analysis of the action of cations of the lanthanide series on the mechanical responses of guinea-pig ileal longitudinal muscle.

1. The inhibitory effects of lanthanide cations (Ln3+) on mechanical responses and 45Ca uptake in guinea-pig ileal longitudinal smooth muscle were studied. 2. Ln3+ strongly inhibited the phasic and tonic component of the response to the muscarinic agonist cis-2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide (CD) the two components being affected to the same extent. Inhibition was also obtained for the responses evoked by high K+ but here the effect was mainly on the phasic response, the tonic component was merely delayed. 3. Other members of the Ln3+ series, with the exception of cerium, were found to be more effective than lanthanum in their ability to inhibit the CD response. Thulium, Tm3+, the thirteenth member of the series was the most effective cation. 4. Analysis of 170Tm uptake revealed at least two components. The concentration-dependence of one component, saturating at 2-5 x 10(-6) Tm, corresponded closely to that of the inhibitory effect of Tm3+ on contraction. 5. 170Tm uptake as a function of time showed a secondary rise after 30 min of exposure to the lanthanide. 6. Although 2-5 x 10(-6) M-Tm3+ produced 90% inhibition of the CD and the high K+ induced responses significant reduction of 45Ca uptake by the muscle was only detected when much higher Tm3+ concentrations (greater than or equal 10(-3) M-Tm3+) were used. 7. It is concluded that Ln3+ combine with membrane sites specifically involved in Ca2+ translocation during excitation-contraction coupling.