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Ingested choline is processed by gut commensal bacteria into trimethylamine (TMA). TMA is then enzymatically converted into trimethylamine N-oxide (TMAO) by members of Flavin-containing monooxygenases (FMOs) in liver. FMO3, TMA and TMAO are linked with metabolic syndrome, and cardiovascular diseases. We employed in vitro model of murine hepatocytes (AML12) treated with TMA and TMAO and C57BL/6 mice treated with A. mucinphila to investigate the health beneficial effect of this bacterium. Quantitative polymerase chain reaction, western blot, and lipid enzymatic assays were used to evaluate the changes of lipid genes expression, ER stress markers and inflammatory signalling molecules upon TMA, TMAO and A. mucinphila treatment. Our studies showed that TMA promotes lipogenic processes through upregulation of fmo genes expression (p<0.05). TMA treated hepatocytes had increased mRNA expression of cholesterol and triglycerides synthesis regulators: srebp2 and srebp1c (p<0.05) and their downstream target genes (p < 0.05) comparing to untreated controls. This promoted elevated triglyceride and cholesterol contents in cell media and in the treated cells (p < 0.05). These changes were associated with the upregulation of miR-125a and miR-125b (p<0.05). miR-125 has been shown to target mRNA of an anti-inflammatory protein A20. A20 protein level was significantly decreased in the TMA treated AML12 compared to untreated ones. TMAO induces lipogenesis (p<0.05) and increase ER stress (p < 0.05). A. mucinphila (Akk) treatment of mice upregulated A20 protein level in liver while reduced level of miR-125b (p<0.05). This can be associated with AMUC_1100 membrane protein of Akk which showed to prevent TMA mediated reduction in A20 in vitro. Conclusion: TMA has a pro-lipogenic properties associated with FMOs activity which mediate miR-125a/b dependent A20 downregulation. On the other hand, TMAO has pro-lipogenic properties, while inducing ER stress. TMA and TMAO effect on hepatocytes could be prevented by Akk treatment of mice or AMUC_1100 transfection of TMA treated AML12 cells.

Neurological pathologies provide a distinctively challenging region for new therapeutic targets due to extra physical protection, resident brain cell responses, and the fragility of the irreplaceable brain itself. Novel ways to approach neurological diseases include considering smaller molecules such as metabolites and their effects and origins, and exploring broader, more complex connections such as the gut-brain-microbiome axis. In Chapter 1, I discuss gut-brain-microbial pathways and how these unique connections may provide us with myriad therapeutic targets in disease, including glioblastoma and Alzheimer's disease (AD). Chapter 2 explores the enrichment of trimethylamine-N oxide (TMAO), a metaorganismal metabolite, in the olfactory bulb, indicating an impactful role in olfactory perception. The evidence supporting this observation comes from orthogonal studies using both pharmacological inhibition and genetic loss of function (LOF) models. These findings carry implications for potential dietary and sensory interventions associated with neural changes in AD. Chapter 3 includes investigation into a new mechanism of immune suppression in the tumor microenvironment (TME) via the small molecule metabolite spermidine (SPD). Metabolites such as SPD that are produced and secreted by tumor cells have an indirect effect on tumor growth by targeting immune cells in the TME. This novel connection provides a window into yet another manner glioblastoma cells are able to manipulate their surroundings, building hope for new therapeutic targets. Although not fully comprehensive, these studies highlight some unique mechanisms regarding how some metabolites function in brain cancer and how other metabolites are working through the microbe-host connection to influence neurological symptoms.

Type II Diabetes Mellitus (T2D) and cardiovascular diseases (CVD) are non-communicable chronic diseases that involves impairments in glucose metabolism and vascular function. Multiple factors may increase the risk for T2D, including but not limited to genetics, obesity and lifestyle, such as physical inactivity and diet. The gut microbiota, the human's largest population of microorganisms, plays an essential role in health and disease. The physiology and function of the gastrointestinal tract can be influenced by the diet. Phosphatidylcholine (PC), a source of choline in the diet, is rich in Western-type diets. Gut microbiota metabolize choline to trimethylamine (TMA) which circulates and is oxidized in the liver to form trimethylamine N-oxide (TMAO). As a result, ingestion of PC or choline could increase levels of TMAO. Preclinical studies indicate a role of TMAO in the development of atherosclerosis. Likewise, multiple observations support a potential role of TMAO in the development of insulin resistance and T2D. Much of the research has been conducted on rodent models, while others are observational human studies. Whether acute and short-term increases in TMAO contribute to impairments in insulin sensitivity in humans remains unknown. To address this, we performed two studies utilizing a double-blind, placebo controlled, crossover design. Eligible participants consumed a 1000mg/day dose of choline bitartrate and placebo (maltodextrin) the night before each testing session (for the acute choline study) or for 4 weeks (for the short-term choline ingestion study). Oral glucose tolerance test, continuous glucose monitoring, flow-mediated dilation, and applanation tonometry was performed the day after the acute choline load and before and after the short-term choline ingestion period. We hypothesized that gut microbiota-generated increase in TMAO will impair insulin sensitivity, glucose tolerance, endothelial function and arterial stiffness in healthy sedentary humans. Following acute choline ingestion, significant increases in plasma TMAO (p = 0.013) and choline (p = 0.003) were evident. There was no statistically significant difference in insulin sensitivity, glucose tolerance or in any of the endothelial function and arterial stiffness measurements. Four weeks of 1000mg choline ingestion per day, significantly increased plasma (p = 0.042) and urine (p = 0.008) TMAO concentrations compared to the placebo. However, no significant differences were observed for any other measurements of insulin sensitivity, glucose tolerance, glycemic variability, endothelial function, and arterial stiffness. More research is needed to elucidate the mechanisms behind the mechanistic observations between elevated TMAO concentrations and T2D and CVD.

Cardiovascular disease (CVD) is a major cause of mortality and morbidity worldwide, often with heart failure as the terminal stage. Clinical studies have associated elevated levels of trimethylamine N-oxide (TMAO), a gut-derived metabolite, with adverse outcomes of CVD. As of today, TMAO's effects on cardiac structure and function are not well understood. In this study, both male and female TMAO-treated hearts showed functional deficits based on electrocardiography and echocardiography results. Immunohistochemistry results showed signs of hypertrophic cardiomyopathy in TMAO-treated male hearts while female TMAO-treated hearts showed signs of dilated cardiomyopathy. Neither TMAO group showed signs of fibrosis. Overproduction of reactive oxygen species was only observed in male TMAO-treated hearts. At the level of individual cardiomyocytes, significant delays in time to reach maximum contraction and dilation were only seen in TMAO-treated male hearts along with higher contractile force. Overall, TMAO-treated hearts show significant functional deficits with altered structure in a sex-specific way. Our study utilizes a variety of methods to comprehensively characterize features of TMAO-induced heart failure in both males and females which extends our current knowledge from human clinical associations.

Cardiovascular diseases (CVDs) are the leading cause of death and disability worldwide. The aging population and the rapidly increasing prevalence of obesity and type 2 diabetes will contribute to a growing epidemic of CVDs globally. Despite the extensive investigations in etiology, the pathogenesis of CVDs still not fully understand, and the treatment and prevention for CVDs are still limited. Significant interest has been raised in gut microbiota-host interaction since increasing evidence revealed that gut microbiomes play an important role in human health and diseases, including CVDs. Among more than two thousand gut microbiota metabolites, a compound named trimethylamine N-oxide (TMAO) was revealed to be closely related to CVDs. However, the impact of TMAO on cardiovascular health is still full of controversy and the direct impact of TMAO on heart tissue and cardiomyocytes has not been fully understood yet. In the first chapter, we reviewed the literature on TMAO-related atherosclerosis and cardiomyopathy to give us a general aspect of current research progress in the role of TMAO on CVDs. In this context, we provide an overview of the potential mechanisms underlying TMAO-induced cardiovascular diseases at the cellular and molecular levels, with a focus on atherosclerosis and cardiomyopathy. We also address the direct effects of TMAO on cardiomyocytes (a new and under-researched area) and finally propose TMAO as a potential biomarker and/or therapeutic target for the diagnosis and treatment of patients with CVDs. In the second chapter, the direct impact of TMAO on cardiac function was tested in vivo using wild-type C57B6L mice model. Four experiment groups were enrolled in the feeding protocol, which included 3w (different time points), 6w, and 13w feeding time to reveal the impact of short and longer periods of TMAO consumption on cardiac function. The plasma TMAO was measured by liquid chromatography-tandem mass spectrometry (LC/MS/MS) method at the end of the feeding protocol. Echocardiography and electrocardiography (ECG) were performed to assess the overall heart function. The histopathology staining was used to evaluate the cardiac microstructure change. By the end of the feeding protocol, the plasma TMAO all increased significantly in the TMAO group compared to the control no matter the TMAO feeding period. Echocardiography showed that 6w and 13w TMAO intake could significantly decrease cardiac contractility evidenced by decreased eject fraction (EF) and fraction shortening (FS). The electrocardiography (ECG) showed decreased R wave aptitude in 6w and 13w TMAO feed group with sinus rhythm. However, 3w TMAO intake had no impact on both cardiac contractability and ECG. Moreover, chronic TMAO supplement (13w) showed increased left ventricle (LV) mass on echocardiography and increased LV thickness on the tissue section. Further histology analysis revealed cardiomyocyte hypertrophy in the 13w TMAO-treated male group. Notably, the female mice showed significantly higher TMAO levels both in the control and treated group compared to the male, however, no gender difference was observed as to the ECG and echocardiography. In addition, the plasma inflammation cytokines were also analyzed and the tumor necrosis factor-α (TNF- α), interleukin 10 (IL-10), Fibroblast growth factor 2 (FGF β) and leptin were all increased in the 13w TMAO treated group compared to the control. These results suggest that chronic TMAO exposure led to increased plasma TMAO levels, which contribute to system inflammation and cardiac dysfunction due to cardiac hypertrophy in mice models. Research in chapter 3 demonstrates the potential underlying mechanisms of TMAO-induced cardiac dysfunction using adult mouse cardiomyocytes. In this study, we examined the direct effect of TMAO on reactive oxidative species (ROS) generation and factors related to cardiomyocyte contractibility, including, microtubule, Connexin43 (Cx43) expression, and gap junction intracellular communication (GJIC), intracellular calcium dynamics and transversal-tubule (T-tubule) both in acute and chronic TMAO challenge. Moreover, we also tested whether TMAO can enter cardiomyocytes directly. The results suggested that TMAO could enter cardiomyocytes through organic cation transporters (OCTs) and promote increased ROS generation via augmentation of NADPH oxidase 4 (Nox4). Moreover, both acute and chronic TMAO exposure could induce microtubule densification, which plays a critical role in intracellular protein transportation and cardiomyocyte morphology maintenance. We also demonstrated chronic TMAO exposure could inhibit the Cx43 expression at both cellular and tissue level, and therefore impact the GJIC for the first time. Besides, we also revealed that TMAO could interrupt intracellular calcium handling both acutely and chronically, especially documented by decreased efficiency in intracellular calcium removal, related to decreased sarcoplasmic reticulum Ca2+-ATPase (Serca2) expression. However, TMAO showed no impact on cardiomyocyte T-tubule network organization. Taken together, we demonstrated a direct destructive role of TMAO on cardiomyocytes' functional properties and provided a novel potential mechanism for TMAO-induced cardiac dysfunction. Overall, the research in this dissertation demonstrated the direct impact of TMAO on cardiomyocytes and cardiac function both in vivo and in vitro and evaluated the effect of TMAO both acutely and chronically. The TMAO can enter cardiomyocytes and induce Nox4-mediated oxidative stress, which could connect to multiple intracellular pathways, including microtubule densification, decreased Cx43 expression, and GJIC, as well as calcium handling dysfunction. Meanwhile, all these changes were closely related to the cardiomyocyte swelling observed in mice cardiac tissue after chronic TMAO consumption, which could ultimately contribute to cardiac contractile dysfunction and electrophysiology change in mice models.

Trimethylamine (TMA)’s downstream oxidized form, trimethylamine n-oxide (TMAO), has been associated with diseases ranging from autism to stroke. However, not much research has been conducted on trimethylamine, a metabolite of choline. The Western diet contains a high amount of choline, especially in highly consumed meat and dairy products. Once consumed, this choline is readily transformed by a liver enzyme, flavin monooxygenase 3 (FMO-3) to TMAO. It is reported that TMA can exist as a uremic toxin and cardiotoxic form. In this study, we show preliminary data that confirms TMA’s function in the caco-2 cells. Caco-2 cells are derived from human colon carcinoma and are commonly used for in vitro models of the intestinal epithelium. Looking at its mechanism prior to its metabolism into TMAO by flavin-containing monooxygenases 3 (FMO3) in the liver gives a better understanding of the mechanistic regulation of the epithelial barrier. As the epithelial barrier has physiological and pharmacological significance due to its ability to regulate the absorption of drugs, nutrients, electrolytes, small solutes, and bacterial metabolites.

Abstract: Choline, as an essential nutrient, is needed for a variety of biological processes such as phospholipid synthesis, cell-membrane signaling, lipoprotein secretion, acetylcholine biosynthesis, and one-carbon metabolism. In the North American population diet the two most common forms of choline in foods are phosphatidylcholine (PC) and free-choline (FC). The recommended adequate intake (AI) is 550 and 425 mg/d for men and women, respectively. However, it has been reported that choline intake is below the AI in various populations and it has been recommended to increase the consumption of foods that are rich in choline. During the last decade epidemiological studies have suggested that consumption of choline rich foods (specifically PC from animal source) might increase cardiovascular disease (CVD) risk. Excess dietary choline is metabolized by gut microorganisms to trimethylamine (TMA) and then is further oxidized in the liver to form trimethylamine N-oxide (TMAO). It has been proposed that TMAO might enhance atherosclerosis development and be a biomarker for CVD risk. Therefore, the objective of this thesis was to understand the role of dietary choline supplementation on atherosclerosis development. We conducted a series of feeding trials to investigate whether the form of dietary choline, free choline or PC supplementation influences atherosclerosis development in atherogenic mouse models. It was observed that in Ldlr-/- mice, dietary supplementation with choline or TMAO increased plasma TMAO levels by 1.6- and 4-fold, respectively after 8 wk. Meanwhile, after 16 wk there was an increase of 2-fold at TMAO supplementation. In Apoe-/- mice, plasma TMAO levels were significantly (p

Background: Acute Coronary Syndrome (ACS) is a leading cause of morbidity and mortality. Metabolic syndrome and obesity are major risk factors for atherosclerosis and ACS. Dysbiosis plays an important role in metabolic syndrome and obesity. Studies show a markedly increased risk of heart attacks in patients with high levels of the pro-atherogenic metabolite trimethylamine-N-Oxide (TMAO). TMAO is produced by the intestinal microbial flora through metabolism of dietary phospholipids; Gram-negative bacteria (Phylum Proteobacteria) is the major source of TMAO metabolism. Patients with obesity and metabolic syndrome have a defective intestinal tight-junctional (TJ) barrier, which allows paracellular permeation of luminal antigens such as lipopolysaccharides (LPS; endotoxins); leading to endotoxemia. Endotoxins are also associated with increased risk of atherosclerosis and cardiovascular disease. We aim to study the hypothesis that patients with ACS have dysbiosis, including higher proportion of Gram-negative bacterial species capable of vi producing the pro-atherogenic metabolite TMAO, and test if dysbiosis in ACS patients is associated with increased intestinal permeability and endotoxemia. Methods: This is a prospective case-control study conducted at a single university hospital. We enrolled ACS patients (within 72 hours of acute cardiac events) and healthy controls. Relevant clinical and demographic data were collected. Stool microbiome composition was examined using the 16S ribosomal RNA (Illumina) method to identify microbiota taxonomic genera. LPS serum level was measured by an ELISA-based method. The intestinal TJ barrier function was evaluated using lactulose-to-mannitol urinary excretion ratio (L/M ratio). Serum TMAO was measured using a mass spectrometry. Results: We enrolled 19 patients and 19 controls. ACS patients had increased relative abundance of Gammaproteobacteria and Proteobacteria compared to healthy controls, with mean proportions of 1.8 ± 3.0 vs 0.2 ± 0.4 % (P = .04) and 4.1 ± 3.8 vs 2.1 ± 1.7% (P=.056), respectively. L/M-ratio was three times higher in ACS patients compared to healthy controls (.06 ± .07 vs .023 ± .02, P = .014). There was no difference in the mean serum LPS or TMAO levels. Conclusion: ACS patients have dysbiosis and increased intestinal permeability. Further studies are required to find the role of dysbiosis in acute cardiac events.

Abstract: Arsenic is a worldwide environmental pollutant that is associated with skin and several types of internal cancers, such as liver, lung, kidney, urinary bladder, and prostate cancers. Recent reports revealed that organic arsenic metabolites, such as trimethylarsine oxide (TMAO), dimethylarsinic acid (DMA(V)), and monomethylarsonous acid (MMA(III)) could activate the toxic and carcinogenic potential of arsenic. Therefore, the objectives of the current dissertation were to: 1) Investigate the effect of TMAO on the activation of hepatic aryl hydrocarbon receptor (AhR)-regulated genes, and to investigate the underlying mechanisms in vitro. 2) Examine the effect of TMAO on modulation of AhR-regulated genes in vivo in extrahepatic tissues. 3) Investigate the effect of DMA(V) on the alteration of AhR-regulated genes in the hepatic and extrahepatic tissues. 4) Examine the effects of MMA(III) as compared to its parent compound, As(III), on the expression of prototypical AhR-regulated gene, CYP1A1, in vitro. Our in vivo results demonstrated that TMAO increased carcinogen activating enzymes Cyp1a1, Cyp1a2, and Cyp1b1, in addition to Nqo1, Gsta1, and Ho-1 at the mRNA level. Upon co-exposure to TMAO and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), TMAO potentiated the TCDD-mediated induction of Cyp1a1, Cyp1b1, and Nqo1 mRNA levels. Western blotting revealed that TMAO increased Cyp1a1, Cyp1a2, Nqo1, Gsta, and Ho-1 protein levels, and potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 at the protein levels. In addition, TMAO significantly increased Cyp1a1, Cyp1a2, Nqo1, Gst, and Ho-1 activities, and significantly potentiated the TCDD-mediated induction of Cyp1a1 activity. At the in vitro level, TMAO induced Cyp1a1 and potentiated the TCDD-mediated induction of Cyp1a1 at mRNA, protein and activity levels. In addition, TMAO increased the nuclear localization of AhR and AhR-dependent XRE-driven luciferase activity. With regard to the effect of TMAO on the extrahepatic tissues, TMAO increased Cyp1a1 and Cyp1b1 mRNA, protein, and activity in the lung. TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1a2 mRNA, protein and activity in the lung. In the kidney, TMAO increased Cyp1b1 mRNA and protein. TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 mRNA, protein and activity. In the heart, TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 mRNA. Moreover, TMAO induced Nqo1 mRNA in the lung, kidney and heart, with subsequent increase in Nqo1 protein and activity in the lung. TMAO increased Gsta1 mRNA in the heart, and increased Gsta protein and activity in the lung and kidney. TMAO increased Nqo1 mRNA compared to TCDD in the kidney and heart, and potentiated the TCDD-mediated induction of Gsta protein and activity in the kidney. As for the DMA(V), our results demonstrated that it has no significant effect on Cyp1a mRNA and protein expression levels or catalytic activity in the liver. On the other hand, DMA(V) significantly potentiated the TCDD-mediated induction of Cyp1a mRNA and protein expression levels, with a subsequent potentiation of catalytic activity in the lung. Moreover, DMA(V) significantly inhibited the TCDD-mediated induction of Cyp1a mRNA and protein expression levels with subsequent inhibition of catalytic activity in the kidney. Regarding phase II AhR-regulated genes, DMA(V) had no significant effect on Nqo1 mRNA and protein expression levels or activity in the liver, lung, or kidney. With regard to MMA(III) as compared to As(III), our in vitro results showed that MMA(III) and As(III) decreased CYP1A1 mRNA, protein, and catalytic activity levels, and inhibited the TCDD-mediated induction of CYP1A1 mRNA, protein, and catalytic activity levels. MMA(III) and As(III) significantly inhibited XRE-driven luciferase activity and inhibited the TCDD-mediated induction of XRE-driven luciferase activity, in addition both compounds showed inhibition of nuclear accumulation of the AhR transcription factor. MMA(III) and As(III) had no effect on CYP1A1 mRNA stability; however, MMA(III), but not As(III), decreased the protein stability of CYP1A1. As(III), but not MMA(III), induced HO-1 mRNA expression level. In addition, both MMA(III) and As(III) increased ROS production. In conclusion, the present work demonstrates for the first time that organic arsenic metabolites modulate phase I and phase II AhR-regulated genes in a tissue-, and enzyme-specific manner. This modulation could potentially participate in arsenic-induced toxicity and carcinogenicity. Our work opened new avenues for treatment of arsenic-induced carcinogenicity via using prodrugs that will be specifically activated inside tumor cells with CYP enzymes. The use of these rational therapies would decrease morbidity and mortality and hence diminish health care costs.

OBJECTIVE: Chapter 1: Investigate the impact that trimethylamine N-oxide (TMAO), dietary contribution of short chain fatty acids (SCFAs), and role of bile acids has on cardiovascular health and disease. Chapter 2: Evaluate the association between intakes of dietary protein from both animal and plant sources on lipid profile changes. METHODS: Chapter 1: Literature review using PubMed and EMBASE to search for published studies for dietary intake or supplementation impact on TMAO or its precursors and their role in the development or prevention of cardiovascular diseases. Chapter 2: Framingham Offspring Study, prospective cohort study using statistical methods to investigate the changes in lipid profiles with dietary animal and plant protein. PUBLISHED STUDIES/RESULTS: Chapter 1: The increased risk of cardiovascular diseases (CVD) correlates with increasing levels of circulating levels of TMAO. The risk of CVD in animal and human studies have shown to be distinct in groups with and without CVD, leading to either beneficial or adverse effects from the consumption of dietary phosphatidylcholine, choline, betaine, carnitine, or intact TMAO. A Western dietary approach has been linked with the development of dyslipidemia whereas, adherence to a Mediterranean diet reduces the risk of major CVD events. The dietary precursors involved in TMA production by the gut microbiota then respectively to TMAO through hepatic enzyme FMO3 provide both beneficial and detrimental effects. Mechanisms of action for TMAO on CVD risk involves changes associated with cholesterol and sterol metabolism leading to foam cell formation, and enhancement of scavenger receptors, CD36 and scavenger receptor-A, on macrophages affects the rate of cholesterol influx and efflux. Choline derived in a dose-dependent manner from eggs improves cardiometabolic biomarkers with no changes in fasting TMAO. Further, choline from eggs also increases the lipoprotein particle size for both HDL-cholesterol and LDL-cholesterol increasing the rate of reverse cholesterol transport (RCT). Betaine concentrations in humans are associated with health outcomes based on an individual’s overall systemic health at baseline. Supplementation with L-carnitine produced favorable effects in lean subjects compared to obese subjects, improved cardiometabolic status in patients with myocardial infarction, and improved lipid profiles among individuals with prevalent coronary heart disease (CAD). Fish consumption increases concentrations of TMAO due to its high levels of intact TMAO though, protective effects for CVD are obtained from fatty fish providing omega-3-fatty acids impacting positive changes in the lipid profiles. Antibiotic therapy suppresses the gut microbiota and eliminates the production of TMA from the dietary precursors that are required. Chapter 2: Men and women both showed a decreasing trend for LDL-cholesterol as the tertiles increased for animal protein intake. Plant protein intake showed a similar decreasing trend for LDL-cholesterol with increasing protein tertiles; however, men had inconsistency among the trend whereas women had a consistent decreasing trend. HDL-cholesterol content increases in males and females with both increasing tertiles for animal and plant protein, though plant protein presented much stronger effects when compared to animal protein. Log-transformed triglycerides were inversely associated with increasing animal protein intake, men revealing greater effects than females. Plant protein intake showed a stronger effect than animal protein intake in an increasing trend in the log of triglycerides over the 6 exams. Overall, total cholesterol content varied at each examination period, animal protein intake tertiles displayed decreased level of total cholesterol, there was a greater effect in men than women. Higher intake of plant protein had a similar trend to animal protein intake showing a decrease in the total cholesterol concentration. Women had a much greater effect in reducing total cholesterol with plant protein when compared to men. CONCLUSION: Chapter 1: Multiple human and animal trials addressed in the association between diet, dietary precursors, gut microbiota composition, and their derived metabolite TMAO on the presence or absence of CVD display contradictory results and identifies areas needing further study. Chapter 2: Regardless of the source of protein, the lipid profiles improved with the intake of either animal or plant protein as the protein intake was increased over the tertiles in each exam. The overall trend with increasing animal or plant protein intake led to decrease in LDL-cholesterol, log transformed triglycerides, and total cholesterol whereas, the HDL-cholesterol concentrations were increased. Men favored animal protein intake to show greater reductions in LDL-cholesterol and total cholesterol, whereas women favored plant protein. The increase in HDL-cholesterol concentration was stronger with the intake of plant protein in men and women. The changes in log transformed triglycerides were similar in men and women.

The overall goal of my thesis research is to further the understanding of the link among dietary trimethylamine-containing nutrients, intestinal microbiome, and cardiovascular disease (CVD) by testing the hypothesis that disease susceptibility is a transmissible trait via cecal microbial transplant. The positive correlation discovered between plasma TMAO and lesion in a panel of inbred mouse strains permitted the identification of atherosclerosis prone (high TMAO-producing, large lesion; C57BL/6J) and resistant mice (low TMAO-producing, small lesion; NZW/LacJ) to be used as donor strains for cecal microbial transplant into intestinal microbiota-suppressed recipient C57BL/6J Apoe-/- female mice. Recipients of C57BL/6J microbes on the choline diet initially had statistically significantly higher plasma TMA and TMAO as compared to choline diet NZW/LacJ recipients, and at the study endpoint, choline diet-dependent enhancement of atherosclerotic lesion area was observed in the recipients of C57BL/6J microbes but not in recipients of NZW/LacJ microbes. Examination of microbial communities from 16S rRNA bacterial gene sequencing analysis showed distinct donor communities faithfully transferred to the respective recipient mouse groups. Several donor strain-characteristic taxa were identified in recipient groups whose proportional abundances correlated with plasma TMAO and/or lesion area. The second set of studies explores thrombosis potential and platelet reactivity, the proximal events in heart attack and stroke, for a relationship with the microbiome. Plasma TMAO is associated with a 2-fold increased risk for thrombotic events in a large cohort (n=4007). TMAO enhances agonist dependent platelet responsiveness by increasing the release of intracellular calcium stores. In mice, provision of TMAO or dietary choline (solely in the setting of an intact microbial community) enhances platelet aggregation ex vivo and shortens the time to cessation of blood flow in vivo. Cecal microbial transplant study showed that germ-free recipients of the disease-prone donor microbes were transmitted bacterial communities, choline diet-dependent enhanced TMA lyase activity, elevated plasma TMAO, and prothrombotic phenotypes as compared to recipients of disease-resistant donors. Collectively, the results of these studies suggest that the body and its microbial inhabitants interact to promote health or disease. Targeting microbial communities could be an adjunct treatment for the management of cardiometabolic disease in the future.

The gut microbiota has critical roles in mammalian physiological processes andhas been increasingly recognized to be a culprit in disease pathogenesis. Werecently identified a pathway that links the consumption of dietaryphosphatidylcholine, the major dietary source of choline, the gut microbiota, andatherosclerosis. Choline, a trimethylamine compound, is metabolized by the gutmicrobiota to produce an intermediate compound known as trimethylamine(TMA). TMA is oxidized by hepatic flavin monooxygenase 3 (FMO3) to form theproatherogenic metabolite trimethyl amine N-oxide (TMAO). The recognition thatthe gut mediated metabolism of choline to TMAO promoted atherosclerosisraised the possibility that carnitine, another dietary trimethylamine found in redmeat, could contribute to TMAO formation. Studies in mice and humans confirmthat the formation of TMAO from carnitine is gut microbiota dependent.Interestingly, omnivorous subjects have a greater capacity to metabolize TMAOfrom carnitine than vegans/vegetarians and demonstrate significant differences ingut microbiota composition. Plasma TMAO levels are independently associatedwith prospective major adverse cardiovascular events (death, MI, stroke) and canpromote atherosclerosis by causing dysfunction in forward and reversecholesterol transport. Subsequent studies of carnitine metabolism by the gutmicrobiota demonstrate the production of two other gut microbiota metabolites, γ-butyrobetaine (γBB) and transcrotonobetaine (TC). γBB is the dominant gut microbiota metabolite of carnitine in mice and is an intermediate in the gutmicrobiota dependent metabolism of carnitine to TMAO. Remarkably, twoseparate bacterial taxa in the gut microbiota associate with the two stepmetabolism of carnitine to TMAO suggesting distinct populations in the gutmicrobiota working in cooperation. Although humans also have the capacity togenerate TMAO from carnitine in a gut microbiota dependent manner, γBB isproduced at a lesser amount than TMAO. Additionally, plasma γBB had noassociation with dietary status (omnivore vs. vegan/vegetarian). This datasuggests that in humans the direct metabolism of carnitine to TMA/TMAO is themajor gut microbiota mediated pathway of carnitine metabolism. Interestingly, aminor gut microbiota metabolite of carnitine, transcrotonobetaine, also promotesatherosclerosis in a gut microbiota dependent manner. Together these dataprovide a previously unrecognized link between the consumption of dietarytrimethylamines, the gut microbiota, and atherosclerosis.

Spectroscopic and kinetic studies of arsenite oxidase and DMSO reductase, both members of the DMSO reductase family of mononuclear molybdenum enzymes, have been undertaken to provide a better understanding of how the physical and electronic structure of the molybdenum center relates to the oxygen atom transfer chemistry catalyzed by each enzyme. Protein film voltammetry of arsenite oxidase shows that the molybdenum center displays highly concerted two-electron oxidation-reduction activity with a reduction potential of +254 mV vs. SHE and spectrophotometric reductive titrations of arsenite oxidase have revealed the heretofore unresolved reduction potentials of the [3Fe-4S] and [2Fe-2S] Rieske-type iron-sulfur centers of the enzyme. Stopped-flow kinetic studies of the reaction of oxidized R. sphaeroides DMSO reductase with the general reductant sodium dithionite is biphasic, with an intermediate species formed that is determined to be that of the catalytically relevant MoV enzyme form. UV-visible spectral deconvolution of enzyme-monitored turnover reactions using DMSO reductase with the oxidizing substrates (DMSO or TMAO) and the reductant sodium dithionite clearly reveals the mechanistic differences of catalytic oxygen atom abstraction between them where a stable Ered-substrate complex is only observed for reaction with DMSO. The pH-dependence of UV-visible spectra for DMSO reductase intermediates was also examined and only the spectrum of reduced enzyme is found to be particularly sensitive to pH. The spectrum of the high pH reduced enzyme implies dissociation of the Q pterin in reduced enzyme and further studies suggest that this Q pterin dissociation may stabilize an Eox•DMS intermediate which accumulates to an increasing degree as pH increases and is responsible for the observed pH-dependence of steady-state enzyme activity. Spectroscopic and kinetic analysis of the W116F mutant shows that, in contrast to previous studies, the functional form of the enzyme has a bis-dithiolene molybdenum center. Resonance Raman has also been employed to further characterize W116F DMSO reductase forms and shows that both oxidized and reduced species are vibrationally analogous to those of the wild-type enzyme. This provides further evidence of full bis-dithiolene coordination to the molybdenum in the mutant active center.