Pharmacology of a Potent and Novel Inhibitor of the NOD-Like Receptor Pyrin Domain-Containing Protein 3 (NLRP3) Inflammasome that Attenuates Development of Nonalcoholic Steatohepatitis and Liver Fibrosis.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a multiprotein complex and component of the innate immune system that is activated by exogenous and endogenous danger signals to promote activation of caspase-1 and the maturation and release of the proinflammatory cytokines interleukin (IL)-1ß and IL-18. Inappropriate activation of NLRP3 has been implicated in the pathophysiology of multiple inflammatory and autoimmune diseases, including cardiovascular disease, neurodegenerative diseases, and nonalcoholic steatohepatitis (NASH), thus increasing the clinical interest of this target. We describe in this study the preclinical pharmacologic, pharmacokinetic, and pharmacodynamic properties of a novel and highly specific NLRP3 inhibitor, JT001 (6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonylurea). In cell-based assays, JT001 potently and selectively inhibited NLRP3 inflammasome assembly, resulting in the inhibition of cytokine release and the prevention of pyroptosis, a form of inflammatory cell death triggered by active caspase-1. Oral administration of JT001 to mice inhibited IL-1ß production in peritoneal lavage fluid at plasma concentrations that correlated with mouse in vitro whole blood potency. Orally administered JT001 was effective in reducing hepatic inflammation in three different murine models, including the Nlrp3A350V /+CreT model of Muckle-Wells syndrome (MWS), a diet-induced obesity NASH model, and a choline-deficient diet-induced NASH model. Significant reductions in hepatic fibrosis and cell damage were also observed in the MWS and choline-deficient models. Our findings demonstrate that blockade of NLRP3 attenuates hepatic inflammation and fibrosis and support the use of JT001 to investigate the role of NLRP3 in other inflammatory disease models. SIGNIFICANCE STATEMENT: Persistent inflammasome activation is the consequence of inherited mutations of NLRP3 and results in the development of cryopyrin-associated periodic syndromes associated with severe systemic inflammation. NLRP3 is also upregulated in nonalcoholic steatohepatitis, a metabolic chronic liver disease currently missing a cure. Selective and potent inhibitors of NLRP3 hold great promise and have the potential to overcome an urgent unmet need.

Absence of Either Ripk3 or Mlkl Reduces Incidence of Hepatocellular Carcinoma Independent of Liver Fibrosis.

Nonalcoholic fatty liver disease (NAFLD) is one of the etiologies that contribute to hepatocellular carcinoma (HCC), and chronic inflammation is one of the proposed mediators of HCC. Because necroptosis is a cell death pathway that induces inflammation, we tested whether necroptosis-induced inflammation contributes to the progression of NAFLD to HCC in a mouse model of diet-induced HCC. Male and female wild-type (WT) mice and mouse models where necroptosis is blocked (Ripk3-/- or Mlkl-/- mice) were fed either a control diet, choline-deficient low-fat diet or choline-deficient high-fat diet. Blocking necroptosis reduced markers of inflammation [proinflammatory cytokines (TNFα, IL6, and IL1ß), F4/80+ve macrophages, CCR2+ve infiltrating monocytes], inflammation-associated oncogenic pathways (JNK, PD-L1/PD-1, ß-catenin), and HCC in male mice. We demonstrate that hepatic necroptosis promotes recruitment and activation of liver macrophages leading to chronic inflammation, which in turn trigger oncogenic pathways leading to the progression of NAFLD to HCC in male mice. Whereas in female mice, blocking necroptosis reduced HCC independent of inflammation. Our data show a sex-specific difference in the development of inflammation, fibrosis, and HCC in WT mice. However, blocking necroptosis reduced HCC in both males and females without altering liver fibrosis. Thus, our study suggests that necroptosis is a valid therapeutic target for NAFLD-mediated HCC. IMPLICATIONS: Necroptosis is a major contributor to hepatic inflammation that drives the progression of NAFLD to HCC and therefore represents a valid target for NAFLD-mediated HCC.

Diet-Induced High Serum Levels of Trimethylamine-N-oxide Enhance the Cellular Inflammatory Response without Exacerbating Acute Intracerebral Hemorrhage Injury in Mice.

Trimethylamine-N-oxide (TMAO), an intestinal flora metabolite of choline, may aggravate atherosclerosis by inducing a chronic inflammatory response and thereby promoting the occurrence of cerebrovascular diseases. Knowledge about the influence of TMAO-related inflammatory response on the pathological process of acute stroke is limited. This study was designed to explore the effects of TMAO on neuroinflammation, brain injury severity, and long-term neurologic function in mice with acute intracerebral hemorrhage (ICH). We fed mice with either a regular chow diet or a chow diet supplemented with 1.2% choline pre- and post-ICH. In this study, we measured serum levels of TMAO with ultrahigh-performance liquid chromatography-tandem mass spectrometry at 24 h and 72 h post-ICH. The expression level of P38-mitogen-protein kinase (P38-MAPK), myeloid differentiation factor 88 (MyD88), high-mobility group box1 protein (HMGB1), and interleukin-1ß (IL-1ß) around hematoma was examined by western blotting at 24 h. Microglial and astrocyte activation and neutrophil infiltration were examined at 72 h. The lesion was examined on days 3 and 28. Neurologic deficits were examined for 28 days. A long-term choline diet significantly increased serum levels of TMAO compared with a regular diet at 24 h and 72 h after sham operation or ICH. Choline diet-induced high serum levels of TMAO did not enhance the expression of P38-MAPK, MyD88, HMGB1, or IL-1ß at 24 h. However, it did increase the number of activated microglia and astrocytes around the hematoma at 72 h. Contrary to our expectations, it did not aggravate acute or long-term histologic damage or neurologic deficits after ICH. In summary, choline diet-induced high serum levels of TMAO increased the cellular inflammatory response probably by activating microglia and astrocytes. However, it did not aggravate brain injury or worsen long-term neurologic deficits. Although TMAO might be a potential risk factor for cerebrovascular diseases, this exploratory study did not support that TMAO is a promising target for ICH therapy.

Excess Vitamins or Imbalance of Folic Acid and Choline in the Gestational Diet Alter the Gut Microbiota and Obesogenic Effects in Wistar Rat Offspring.

Excess vitamin intake during pregnancy leads to obesogenic phenotypes, and folic acid accounts for many of these effects in male, but not in female, offspring. These outcomes may be modulated by another methyl nutrient choline and attributed to the gut microbiota. Pregnant Wistar rats were fed an AIN-93G diet with recommended vitamin (RV), high 10-fold multivitamin (HV), high 10-fold folic acid with recommended choline (HFol) or high 10-fold folic acid without choline (HFol-C) content. Male and female offspring were weaned to a high-fat RV diet for 12 weeks post-weaning. Removing choline from the HFol gestational diet resulted in obesogenic phenotypes that resembled more closely to HV in male and female offspring with higher body weight, food intake, glucose response to a glucose load and body fat percentage with altered activity, concentrations of short-chain fatty acids and gut microbiota composition. Gestational diet and sex of the offspring predicted the gut microbiota differences. Differentially abundant microbes may be important contributors to obesogenic outcomes across diet and sex. In conclusion, a gestational diet high in vitamins or imbalanced folic acid and choline content contributes to the gut microbiota alterations consistent with the obesogenic phenotypes of in male and female offspring.

Pathobiological Relationship of Excessive Dietary Intake of Choline/L-Carnitine: A TMAO Precursor-Associated Aggravation in Heart Failure in Sarcopenic Patients.

The microecological environment of the gastrointestinal tract is altered if there is an imbalance between the gut microbiota phylases, resulting in a variety of diseases. Moreover, progressive age not only slows down physical activity but also reduces the fat metabolism pathway, which may lead to a reduction in the variety of bacterial strains and bacteroidetes' abundance, promoting firmicutes and proteobacteria growth. As a result, dysbiosis reduces physiological adaptability, boosts inflammatory markers, generates ROS, and induces the destruction of free radical macromolecules, leading to sarcopenia in older patients. Research conducted at various levels indicates that the microbiota of the gut is involved in pathogenesis and can be considered as the causative agent of several cardiovascular diseases. Local and systematic inflammatory reactions are caused in patients with heart failure, as ischemia and edema are caused by splanchnic hypoperfusion and enable both bacterial metabolites and bacteria translocation to enter from an intestinal barrier, which is already weakened, to the blood circulation. Multiple diseases, such as HF, include healthy microbe-derived metabolites. These key findings demonstrate that the gut microbiota modulates the host's metabolism, either specifically or indirectly, by generating multiple metabolites. Currently, the real procedures that are an analogy to the symptoms in cardiac pathologies, such as cardiac mass dysfunctions and modifications, are investigated at a minimum level in older patients. Thus, the purpose of this review is to summarize the existing knowledge about a particular diet, including trimethylamine, which usually seems to be effective for the improvement of cardiac and skeletal muscle, such as choline and L-carnitine, which may aggravate the HF process in sarcopenic patients.

Enterobacter aerogenes ZDY01 inhibits choline-induced atherosclerosis through CDCA-FXR-FGF15 axis.

Atherosclerosis is the leading cause of cardiovascular diseases worldwide. Trimethylamine N-oxide (TMAO), a metabolite of intestinal flora from dietary quaternary amines, has been shown to be closely related to the development of atherosclerosis. Previous studies have shown that Enterobacter aerogenes ZDY01 significantly reduces the serum levels of TMAO and cecal trimethylamine (TMA) in Balb/c mice; however, its role in the inhibition of choline-induced atherosclerosis in ApoE-/- mice remains unclear. Here, we demonstrated that E. aerogenes ZDY01 inhibited choline-induced atherosclerosis in ApoE-/- mice fed with 1.3% choline by reducing cecal TMA and modulating CDCA-FXR/FGF15 axis. We observed that E. aerogenes ZDY01 decreased the cecal TMA and serum TMAO levels by utilizing cecal TMA as a nutrient, not by changing the expression of hepatic FMO3 and the composition of gut microbiota. Furthermore, E. aerogenes ZDY01 enhanced the expression of bile acid transporters and reduced the cecal CDCA levels, thereby attenuating the FXR/FGF15 pathway, upregulating the expression of Cyp7a1, promoting reverse cholesterol transport. Taken together, E. aerogenes ZDY01 attenuated choline-induced atherosclerosis in ApoE-/- mice by decreasing cecal TMA and promoting reverse cholesterol transport, implying that E. aerogenes ZDY01 treatment might have therapeutic potential in atherosclerosis.

Berberine attenuates choline-induced atherosclerosis by inhibiting trimethylamine and trimethylamine-N-oxide production via manipulating the gut microbiome.

Trimethylamine-N-oxide (TMAO), a derivative from the gut microbiota metabolite trimethylamine (TMA), has been identified to be an independent risk factor for promoting atherosclerosis. Evidences suggest that berberine (BBR) could be used to treat obesity, diabetes and atherosclerosis, however, its mechanism is not clear mainly because of its poor oral bioavailability. Here, we show that BBR attenuated TMA/TMAO production in the C57BL/6J and ApoE KO mice fed with choline-supplemented chow diet, and mitigated atherosclerotic lesion areas in ApoE KO mice. Inhibition of TMA/TMAO production by BBR-modulated gut microbiota was proved by a single-dose administration of d9-choline in vivo. Metagenomic analysis of cecal contents demonstrated that BBR altered gut microbiota composition, microbiome functionality, and cutC/cntA gene abundance. Furthermore, BBR was shown to inhibit choline-to-TMA conversion in TMA-producing bacteria in vitro and in gut microbial consortium from fecal samples of choline-fed mice and human volunteers, and the result was confirmed by transplantation of TMA-producing bacteria in mice. These results offer new insights into the mechanisms responsible for the anti-atherosclerosis effects of BBR, which inhibits commensal microbial TMA production via gut microbiota remodeling.

Inhibition of microbiota-dependent TMAO production attenuates chronic kidney disease in mice.

Patients with chronic kidney disease (CKD) have elevated circulating levels of trimethylamine N-oxide (TMAO), a metabolite derived from gut microbes and associated with cardiovascular diseases. High circulating levels of TMAO and its dietary precursor, choline, predict increased risk for development of CKD in apparently healthy subjects, and studies in mice fed TMAO or choline suggest that TMAO can contribute to kidney impairment and renal fibrosis. Here we examined the interactions between TMAO, kidney disease, and cardiovascular disease in mouse models. We observed that while female hyperlipidemic apoE KO mice fed a 0.2% adenine diet for 14 weeks developed CKD with elevated plasma levels of TMAO, provision of a non-lethal inhibitor of gut microbial trimethylamine (TMA) production, iodomethylcholine (IMC), significantly reduced multiple markers of renal injury (plasma creatinine, cystatin C, FGF23, and TMAO), reduced histopathologic evidence of fibrosis, and markedly attenuated development of microalbuminuria. In addition, while the adenine-induced CKD model significantly increased heart weight, a surrogate marker for myocardial hypertrophy, this was largely prevented by IMC supplementation. Surprisingly, adenine feeding did not increase atherosclerosis and significantly decreased the expression of inflammatory genes in the aorta compared to the control groups, effects unrelated to TMAO levels. Our data demonstrate that inhibition of TMAO production attenuated CKD development and cardiac hypertrophy in mice, suggesting that TMAO reduction may be a novel strategy in treating CKD and its cardiovascular disease complications.

Salicylates enhance CRM1 inhibitor antitumor activity by induction of S-phase arrest and impairment of DNA-damage repair.

Chromosome region maintenance protein 1 (CRM1) mediates protein export from the nucleus and is a new target for anticancer therapeutics. Broader application of KPT-330 (selinexor), a first-in-class CRM1 inhibitor recently approved for relapsed multiple myeloma and diffuse large B-cell lymphoma, have been limited by substantial toxicity. We discovered that salicylates markedly enhance the antitumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. Using salicylates in combination enables targeting of a range of blood cancers with a much lower dose of selinexor, thereby potentially mitigating prohibitive clinical adverse effects. Choline salicylate (CS) with low-dose KPT-330 (K+CS) had potent, broad activity across high-risk hematological malignancies and solid-organ cancers ex vivo and in vivo. The K+CS combination was not toxic to nonmalignant cells as compared with malignant cells and was safe without inducing toxicity to normal organs in mice. Mechanistically, compared with KPT-330 alone, K+CS suppresses the expression of CRM1, Rad51, and thymidylate synthase proteins, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, cell-cycle arrest in S-phase, and cell apoptosis. Moreover, the addition of poly (ADP-ribose) polymerase inhibitors further potentiates the K+CS antitumor effect. K+CS represents a new class of therapy for multiple types of blood cancers and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for cancer therapy in general.

Relationship between dietary choline intake and diabetes mellitus in the National Health and Nutrition Examination Survey 2007-2010.

BACKGROUND: Little is known about the relationship between dietary intake of choline, which is a major dietary precursor for gut microbiome-derived trimethylamine N-oxide (TMAO), and diabetes mellitus (DM) in the general population. The present study aims to explore the relationship between dietary choline intake and DM in the US adult population. METHODS: Cross-sectional data were derived from the National Health and Nutrition Examination Survey (NHANES) 2007-2010 of 8621 individuals aged 20 years or older. Multivariable logistic regression models were used to determine odds ratios (ORs) and 95% confidence intervals (CIs) for DM of each quartile category of energy-adjusted choline intakes. The restricted cubic spline model was used for the dose-response analysis. The receiver operating characteristic (ROC) curve was used to determine the optimal cutoff value of choline intake for predicting DM. RESULTS: A linear dose-response relationship between dietary choline intake and the odds of DM was found after adjustment for multiple potential confounding factors. With the lowest quartile category of choline as the reference, the multivariable-adjusted ORs and 95% CIs of the second, third, and highest quartile categories were 1.23 (0.99-1.53), 1.27 (1.02-1.58), and 1.49 (1.20-1.85), respectively, P for trend =0.0004. The ROC analysis identified energy-adjusted choline of 331.7 mg/8.37-MJ per day as the optimal cutoff value for predicting DM, with 52.5% sensitivity and 60.7% specificity. CONCLUSION: This study supports a positive and linear relationship between dietary choline intake and DM in the US adult population.

Effects of choline supplementation on liver biology, gut microbiota, and inflammation in Helicobacter pylori-infected mice.

AIMS: Diet is one of the factors affecting the pathogenicity of Helicobacter pylori (H. pylori) infection. Choline is a dietary component that is crucial for normal cellular function. However, choline intake imbalance can lead to liver injury, inflammation, and changes of the gut microbiota composition. The study aimed to explore the effects of choline supplementation on liver biology, gut microbiota, and inflammation in H. pylori-infected mice. MAIN METHODS: Liver function was detected by biochemical and histopathological analysis. Serum inflammatory markers were measured using ELISA. Fecal microbial profiles were determined via 16S rRNA sequencing. KEY FINDINGS: The results showed that choline supplementation decreased serum LDL level, while increased the activities of serum AST and ALT in normal BALB/c mice. Besides, choline also reduced hepatic SOD and GSH-Px activities, and elevated hepatic MDA level of H. pylori-infected mice. Moreover, choline markedly enhanced the concentrations of inflammatory factors including LPS, CRP, IL-6, TNF-α, and CXCL1 in H. pylori-infected mice. Meanwhile, choline and H. pylori cotreatment altered the richness and diversity of the mice gut microbiota, and increased the relative abundance of Escherichia_Shigella, which had a significant positive correlation with the levels of LPS, CRP, IL-6, TNF-α and CXCL1. SIGNIFICANCE: Our data suggest, for the first time, that choline can aggravate H. pylori-induced inflammation, which may be associated with the alterations of gut microbiota. This study may provide novel insights into the possible effects of food-derived choline on H. pylori infection-related diseases.

Protective Effects of Black Raspberry (Rubus occidentalis) Extract against Hypercholesterolemia and Hepatic Inflammation in Rats Fed High-Fat and High-Choline Diets.

Choline is converted to trimethylamine by gut microbiota and further oxidized to trimethylamine-N-oxide (TMAO) by hepatic flavin monooxygenases. Positive correlation between TMAO and chronic diseases has been reported. Polyphenols in black raspberry (BR), especially anthocyanins, possess various biological activities. The objective of this study was to determine the effects of BR extract on the level of choline-derived metabolites, serum lipid profile, and inflammation markers in rats fed high-fat and high-choline diets. Forty female Sprague-Dawley (SD) rats were randomly divided into four groups and fed for 8 weeks as follows: CON (AIN-93G diet), HF (high-fat diet), HFC (HF + 1.5% choline water), and HFCB (HFC + 0.6% BR extract). Serum levels of TMAO, total cholesterol, and low-density lipoprotein (LDL)-cholesterol and cecal trimethylamine (TMA) level were significantly higher in the HFC than in the HFCB. BR extract decreased mRNA expression of pro-inflammatory genes including nuclear factor-κB (NF-κB), interleukin (IL)-1ß, IL-6, and cyclooxygenase-2 (COX-2), and protein expression of NF-κB and COX-2 in liver tissue. These results suggest that consistent intake of BR extract might alleviate hypercholesterolemia and hepatic inflammation induced by excessive choline with a high-fat diet via lowering elevated levels of cecal TMA and serum TMAO in rats.

The gut microbial metabolite trimethylamine N-oxide aggravates GVHD by inducing M1 macrophage polarization in mice.

The diversity of the human microbiome heralds the difference of the impact that gut microbial metabolites exert on allogenic graft-versus-host (GVH) disease (GVHD), even though short-chain fatty acids and indole were demonstrated to reduce its severity. In this study, we dissected the role of choline-metabolized trimethylamine N-oxide (TMAO) in the GVHD process. Either TMAO or a high-choline diet enhanced the allogenic GVH reaction, whereas the analog of choline, 3,3-dimethyl-1-butanol reversed TMAO-induced GVHD severity. Interestingly, TMAO-induced alloreactive T-cell proliferation and differentiation into T-helper (Th) subtypes was seen in GVHD mice but not in in vitro cultures. We thus investigated the role of macrophage polarization, which was absent from the in vitro culture system. F4/80+CD11b+CD16/32+ M1 macrophage and signature genes, IL-1ß, IL-6, TNF-α, CXCL9, and CXCL10, were increased in TMAO-induced GVHD tissues and in TMAO-cultured bone marrow-derived macrophages (BMDMs). Inhibition of the NLRP3 inflammasome reversed TMAO-stimulated M1 features, indicating that NLRP3 is the key proteolytic activator involved in the macrophage's response to TMAO stimulation. Consistently, mitochondrial reactive oxygen species and enhanced NF-κB nuclear relocalization were investigated in TMAO-stimulated BMDMs. In vivo depletion of NLRP3 in GVHD recipients not only blocked M1 polarization but also reversed GVHD severity in the presence of TMAO treatment. In conclusion, our data revealed that TMAO-induced GVHD progression resulted from Th1 and Th17 differentiation, which is mediated by the polarized M1 macrophage requiring NLRP3 inflammasome activation. It provides the link among the host choline diet, microbial metabolites, and GVH reaction, shedding light on alleviating GVHD by controlling choline intake.

A high methyl donor diet affects physiology and behavior in Peromyscus polionotus.

Folic acid and other dietary methyl donors are widely supplemented due to their ability to prevent neural tube defects. Dietary methyl donors are also added to other consumables such as energy drinks due to energy-promoting attributes and other perceived benefits. However, there is mounting evidence that indicates developmental exposure to high levels of dietary methyl donors may have deleterious effects. We assessed whether behavior was affected in the social North American rodent species Peromyscus polionotus exposed to a diet enriched with folic acid, Vitamin B12, choline, and betaine/trimethylglycine(TMG). P. polionotus (PO) animals are very social and exhibit little repetitive behavior, particularly compared to their sister species, P. maniculatus. We assayed the effects of dietary methyl-donor supplementation on anxiety-like repetitive and social behaviors by testing young adult animals for novel cage behavior and in social interaction tests. Animals of both sexes exposed to the diet had increased repetitive behaviors and reduced social interactions. Males exposed to the diet became more aggressive compared to their control counterparts. Since methyl-diet animals were larger than control animals, DEXA scans and hormone analyses were performed. Animals exposed to the diet had increased body fat percentages and experienced hormonal changes typically associated with excess fat storage and anxiety-like behavior changes. Therefore, these data suggest the wide use of these dietary supplements makes further investigation imperative.

Dietary choline is positively related to overall and cause-specific mortality: results from individuals of the National Health and Nutrition Examination Survey and pooling prospective data.

Little is known about the association between dietary choline intake and mortality. We evaluated the link between choline consumption and overall as well as cause-specific mortality by using both individual data and pooling prospective studies by meta-analysis and systematic review. Furthermore, adjusted means of cardiometabolic risk factors across choline intake quartiles were calculated. Data from the National Health and Nutrition Examination Survey (1999-2010) were collected. Adjusted Cox regression was performed to determine the risk ratio (RR) and 95 % CI, as well as random-effects models and generic inverse variance methods to synthesise quantitative and pooling data, followed by a leave-one-out method for sensitivity analysis. After adjustments, we found that individuals consuming more choline had worse lipid profile and glucose homeostasis, but lower C-reactive protein levels (P < 0·001 for all comparisons) with no significant differences in anthropometric parameters and blood pressure. Multivariable Cox regression models revealed that individuals in the highest quartile (Q4) of choline consumption had a greater risk of total (23 %), CVD (33 %) and stroke (30 %) mortality compared with the first quartile (Q1) (P < 0·001 for all comparison). These results were confirmed in a meta-analysis, showing that choline intake was positively and significantly associated with overall (RR 1·12, 95 % CI 1·08, 1·17, I2 = 2·9) and CVD (RR 1·28, 95 % CI 1·17, 1·39, I2 = 9·6) mortality risk. In contrast, the positive association between choline consumption and stroke mortality became non-significant (RR 1·18, 95 % CI 0·97, 1·43, P = 0·092, I2 = 1·1). Our findings shed light on the potential adverse effects of choline intake on selected cardiometabolic risk factors and mortality risk.

Improvement of Language in Children with Autism with Combined Donepezil and Choline Treatment.

The safety and efficacy of a novel combination treatment of AChE inhibitors and choline supplement was initiated and evaluated in children and adolescents with autism spectrum disorder (ASD). Safety and efficacy were evaluated on 60 children and adolescents with ASD during a 9-month randomized, double-blind, placebo-controlled trial comprising 12 weeks of treatment preceded by baseline evaluation, and followed by 6 months of washout, with subsequent follow-up evaluations. The primary exploratory measure was language, and secondary measures included core autism symptoms, sleep and behavior. Significant improvement was found in receptive language skills 6 months after the end of treatment as compared to placebo. The percentage of gastrointestinal disturbance reported as a side effect during treatment was higher in the treatment group as compared to placebo. The treatment effect was enhanced in the younger subgroup (younger than 10 years), occurred already at the end of the treatment phase, and was sustained at 6 months post treatment. No significant side effects were found in the younger subgroup. In the adolescent subgroup, no significant improvement was found, and irritability was reported statistically more often in the adolescent subgroup as compared to placebo. Combined treatment of donepezil hydrochloride with choline supplement demonstrates a sustainable effect on receptive language skills in children with ASD for 6 months after treatment, with a more significant effect in those under the age of 10 years.

Intestinal Microbiota Protects against MCD Diet-Induced Steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in western countries, with a continuously rising incidence. Gut-liver communication and microbiota composition have been identified as critical drivers of the NAFLD progression. Hence, it has been shown that microbiota depletion can ameliorate high-fat diet or western-diet induced experimental Non-alcoholic steatohepatitis (NASH). However, its functional implications in the methionine-choline dietary model, remain incompletely understood. Here, we investigated the physiological relevance of gut microbiota in methionine-choline deficient (MCD) diet induced NASH. Experimental liver disease was induced by 8 weeks of MCD feeding in wild-type (WT) mice, either with or without commensal microbiota depletion, by continuous broad-spectrum antibiotic (AB) treatment. MCD diet induced steatohepatitis was accompanied by a reduced gut microbiota diversity, indicating intestinal dysbiosis. MCD treatment prompted macroscopic shortening of the intestine, as well as intestinal villi in histology. However, gut microbiota composition of MCD-treated mice, neither resembled human NASH, nor did it augment the intestinal barrier integrity or intestinal inflammation. In the MCD model, AB treatment resulted in increased steatohepatitis activity, compared to microbiota proficient control mice. This phenotype was driven by pronounced neutrophil infiltration, while AB treatment only slightly increased monocyte-derived macrophages (MoMF) abundance. Our data demonstrated the differential role of gut microbiota, during steatohepatitis development. In the context of MCD induced steatohepatitis, commensal microbiota was found to be hepatoprotective.

Methodological considerations for the identification of choline and carnitine-degrading bacteria in the gut.

The bacterial formation of trimethylamine (TMA) has been linked to cardiovascular disease. This review focuses on the methods employed to investigate the identity of the bacteria responsible for the formation of TMA from dietary choline and carnitine in the human gut. Recent studies have revealed the metabolic pathways responsible for bacterial TMA production, primarily the anaerobic glycyl radical-containing, choline-TMA lyase, CutC and the aerobic carnitine monooxygenase, CntA. Identification of these enzymes has enabled bioinformatics approaches to screen both human-associated bacterial isolate genomes and whole gut metagenomes to determine which bacteria are responsible for TMA formation in the human gut. We centre on several key methodological aspects for identifying the TMA-producing bacteria and report how these pathways can be identified in human gut microbiota through bioinformatics analysis of available bacterial genomes and gut metagenomes.

Choline and Choline alphoscerate Do Not Modulate Inflammatory Processes in the Rat Brain.

Choline is involved in relevant neurochemical processes. In particular, it is the precursor and metabolite of acetylcholine (ACh). Choline is an essential component of different membrane phospholipids that are involved in intraneuronal signal transduction. On the other hand, cholinergic precursors are involved in ACh release and carry out a neuroprotective effect based on an anti-inflammatory action. Based on these findings, the present study was designed to evaluate the effects of choline and choline precursor (Choline alphoscerate, GPC) in the modulation of inflammatory processes in the rat brain. Male Wistar rats were intraperitoneally treated with 87 mg of choline chloride/kg/day (65 mg/kg/day of choline), and at choline-equivalent doses of GPC (150 mg/kg/day) and vehicle for two weeks. The brains were dissected and used for immunochemical and immunohistochemical analysis. Inflammatory cytokines (Interleukin-1ß, IL-1ß; Interleukin-6 , IL-6 and Tumor Necrosis Factor-α, TNF-α) and endothelial adhesion molecules (Intercellular Adhesion Molecule, ICAM-1 and Vascular cell Adhesion Molecule, VCAM-1) were studied in the frontal cortex, hippocampus, and cerebellum. The results clearly demonstrated that treatment with choline or GPC did not affect the expression of the inflammatory markers in the different cerebral areas evaluated. Therefore, choline and GPC did not stimulate the inflammatory processes that we assessed in this study.

Dietary Choline and Betaine and Risk of CVD: A Systematic Review and Meta-Analysis of Prospective Studies.

Studies implicate choline and betaine metabolite trimethylamine N-oxide (TMAO) in cardiovascular disease (CVD). We conducted a systematic review and random-effects meta-analysis to quantify a summary estimated effect of dietary choline and betaine on hard CVD outcomes (incidence and mortality). Eligible studies were prospective studies in adults with comprehensive diet assessment and follow-up for hard CVD endpoints. We identified six studies that met our criteria, comprising 18,076 incident CVD events, 5343 CVD deaths, and 184,010 total participants. In random effects meta-analysis, incident CVD was not associated with choline (relative risk (RR): 1.00; 95% CI: 0.98, 1.02) or betaine (RR: 0.99; 95% CI: 0.98, 1.01) intake. Results did not vary by study outcome (incident coronary heart disease, stroke, total CVD) and there was no evidence for heterogeneity among studies. Only two studies provided data on phosphatidylcholine and CVD mortality. Random effects meta-analysis did not support an association between choline and CVD mortality (RR: 1.09, 95% CI: 0.89, 1.35), but one study supported a positive association and there was significant heterogeneity (I² = 84%, p-value < 0.001). Our findings do not support an association between dietary choline/betaine with incident CVD, but call for further research into choline and CVD mortality.