Notre travail a démontré l’implication du p-crésyl sulfate (PCS), de la zinc a2glycoprotéine (ZAG) et du triméthylamine-N-oxide (TMAO), représentants des 3 groupes de toxines urémiques décrits par le groupe EUTox, dans la survenue des complications métaboliques de la maladie rénale chronique (MRC). Nous avons démontré que l’augmentation du PCS induisait une insulino-résistance, une perte de masse grasse et une redistribution des lipides chez la souris, en montrant une altération de la voie de signalisation de l’insuline dans le muscle strié squelettique. Nous avons ensuite montré que la protéine ZAG était surexprimée par le tissu adipeux dans la MRC. Du fait de ses effets lipolytiques et anti-lipogeniques, l’élévation des concentrations tissulaires de cette adipokine pourrait être impliquée dans les phénomènes de dyslipidémies et dysfonction adipocytaire observés dans la MRC. Chez l’homme, notre étude a confirmé l’augmentation des concentrations de ZAG dans les stades 5 et 5D de la MRC uniquement, faiblement corrélée au débit de filtration glomérulaire (DFG). L’étude n’a en revanche pas permis de mettre en évidence de corrélation entre les taux circulants de ZAG et les critères de complications métaboliques, notamment la dénutrition. Enfin, en l’absence de données précises sur l’élimination rénale du TMAO et la physiopathologie de son augmentation dans la MRC, nous avons conduit une étude clinique mettant en évidence une forte corrélation entre les taux de TMAO et le DFG mesuré, excluant une contribution tubulaire à son excrétion. Les résultats d’une étude préliminaire chez l’animal suggèrent une possible implication du TMAO dans le développement de la fibrose rénale / Our work demonstrated the role of p-cresyl sulfate (PCS), zinc a2-glycoprotein (ZAG) and trimethylamine-N-oxide (TMAO) that belong to the 3 groups of uremic toxins described by the EUTox group, in the metabolic complications of chronic kidney disease (CKD). We demonstrated that the increase of PCS induced an insulin resistance, a loss of fat mass and a lipid redistribution in mice, showing an alteration of the insulin pathway in skeletal muscle. We showed a surexpression of ZAG by adipose tissue in CKD. Because of ZAG lipolytic and anti-lipogenic effects, the elevation of its tissu levels could be implicated in dyslipidemia and adipose dysfonction in CKD. In humans, our study confirmed the elevation of ZAG concentrations only in CKD stages 5 and 5D, with a poor correlation with the glomerular filtration rate (GFR). The study did not shown correlation between ZAG circulating levels and metabolic complications, as wasting. With the lack of data about renal elimination and physiopathology of TMAO, we conducted a clinical study that reported a strong correlation between TMAO levels and measured GFR, without proof of tubular excretion of TMAO. Results of a preliminary animal study suggests a possible implication of TMAO in the renal fibrosis

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Trimethylamine-N-oxide (TMAO) is an important biomarker of atherosclerosis. TMAO is the product of a hepatic conversion of trimethylamine (TMA). Releasing of TMA moieties is dependent on the adaptation of the gut microbiota to dietary TMA containing substrates such as phosphatidylcholine (PC), choline, and L-carnitine. A high-fat diet is an environmental risk factor that may increase TMAO production. However, it isn’t clear if the high dietary intake of TMA is sufficient to promote increased plasma TMAO or if a high-fat intake is also required. We hypothesized that TMAO would be increased after consuming a high-fat diet and a high PC diet independently, with greater increases when consumed together. Four groups of twelve mice each were maintained on different treatments that were either low or high-fat with or without PC over two weeks. Then, a meal containing 9.99 g of corn oil and 0.75 g soybean L-α-Lecithin per 1 kg body weight was provided to all mice to indirectly observe the adaptation of the microbiota to the altered diet. The results of circulating TMAO levels showed that fat appeared to suppress TMAO production, which is against previous evidence. The microbial adaptation to the different treatments wasn’t observed in the measurement of fecal TMA levels. As a result, our hypothesis was rejected. Future work addressing the impact of gene expressions of enzymes on the gut and the liver is needed. The use of another high TMA containing substrates such as choline and rats is recommended. / Master of Science in Life Sciences / Cardiovascular disease (CVD) is heart and blood vessel diseases - many of which are caused by atherosclerosis, a condition wherein fatty materials accumulate in the artery wall, reducing blood flow. The compound trimethylamine-N-oxide (TMAO) was found to be an important biomarker of atherosclerosis. TMAO levels increase in the body when gut microbiota releases trimethylamine (TMA) moieties from dietary phosphatidylcholine (PC), choline, and L-carnitine such as eggs and meat. A high-fat intake was believed to have an impact on increased levels of TMAO. However, it wasn’t clear if the dietary intake of high TMA containing substrates such as PC, is sufficient to promote TMAO formation or if a high-fat content is also required. We hypothesized that TMAO would be increased after consuming a high-fat diet and a high PC diet independently, with greater increases when consumed together. The results would suggest new dietary strategies to avoid CVD. Four groups of twelve mice each were maintained on different treatments that were either low or high-fat with or without PC over two weeks. Then, a meal containing corn oil and PC was provided to all mice to observe the adaptation of the microbiota to the altered diet. The results showed that fat reduces circulating TMAO production, which is against previous evidence. Fecal TMA levels showed that microbiota activities weren’t observed in the colon. As a results, no significant levels of TMA and its precursors were observed in feces.

Trimethylamine N-oxide (TMAO) is a metabolite derived from dietary choline, betaine, and carnitine via intestinal microbiota metabolism. In several recent studies, TMAO has been shown to directly induce inflammation and reactive oxygen species (ROS) generation in numerous cell types, resulting in cell dysfunction. However, whether TMAO will impact stem cell properties remains unknown. This project aims to explore the potential impact of TMAO on mouse embryonic stem cells (mESCs), which serve as an in vitro model of the early embryo and of other potent stem cell types. Briefly, mESCs were cultured in the absence (0mM) or presence of TMAO under two different sets of treatment conditions: long-term (21 days), low-dose (20µM, 200µM, and 1000µM) treatment or short-term (5 days), high-dose (5mM, 10mM, 15mM) treatment. Under these treatment conditions, mESC viability, proliferation, and stemness were analyzed. mESC properties were not negatively impacted under long-term, low-dose TMAO treatment; however, short-term, high-dose treatment resulted in significant reduction of mESC viability and proliferation. Additionally, mESC stemness was significantly reduced when high-dose treatment was extended to 21 days. To investigate an underlying cause for TMAO-induced loss in mESC stemness, metabolic activity of the mESCs under short-term, high-dose TMAO treatment was measured with a Seahorse XFe96 Analyzer. TMAO treatment significantly decreased the rate of glycolysis, and it increased the rate of compensatory glycolysis upon inhibition of oxidative phosphorylation (OxPHOS). It also significantly increased the rate of OxPHOS, maximal respiratory capacity, and respiratory reserve. These findings indicate that TMAO induced a metabolic switch of mESCs from high glycolytic activity to greater OxPHOS activity to promote mESC differentiation. Additionally, TMAO resulted in increased proton leak, indicating increased oxidative stress, and elucidating a potential underlying mechanism for TMAO-induced loss in mESC stemness. Altogether, these findings indicate that TMAO decreases stem cell potency potentially via modulation of metabolic activity. / Master of Science / Trimethylamine N-oxide (TMAO) is a metabolite that is produced by the bacteria in the gut after the consumption of specific dietary ingredients (e.g., choline, carnitine, betaine). These ingredients are commonly found in meat and dairy products, and thus make up a large part of the average American diet. Recently, it was discovered that high TMAO levels in the bloodstream put people at an increased risk for heart disease, neurodegenerative diseases (e.g., Alzheimer's Disease), diabetes, stroke, and chronic kidney disease. At the cellular level, there is evidence that TMAO increases inflammation and the production of oxygen radicals, which causes cells to lose their function and promotes the onset of disease. TMAO has been well studied in adult cell types; however, no one has investigated whether TMAO will impact cells of the early embryo. This project aims to explore the impact of TMAO on mouse embryonic stem cells (mESCs), which are cells that represent the early stage of embryonic development and are critical for proper development of the final offspring. In addition, mESCs may also help to provide insight into how TMAO impacts other stem cell types, some of which are present throughout the entire human lifespan and play an important role in the body's ability to repair itself and maintain overall health. My project demonstrated that TMAO does not impact the overall health of mESCs under normal conditions, which signifies that TMAO generated by a pregnant mother may not directly impact the early embryonic stage of development. Further studies should be conducted to determine the potential impact of TMAO on late stages of embryonic and fetal development. Next, to simulate diseased conditions, the mESCs were treated with extremely high concentrations of TMAO in order to determine what concentration of TMAO will negatively impact these cells. It was found that at 5mM TMAO, mESCs begin to lose their basic properties and become dysfunctional. They are impaired in their viability, growth, ability to become other cell types, and in their metabolic activity. These mESC properties are shared with several types of adult stem cells, and therefore, these findings help to provide insight into how TMAO may impact stem cells found in the adult body which are exposed to a lifetime of high TMAO levels. In the future, we would like to further explore the impact of TMAO on mESCs at the molecular level as well as examine the direct impact of TMAO on other stem cell types.

There is an association between the human microbiome and disease. Gut microbes metabolize dietary sources to release trimethylamine (TMA). TMA is absorbed and then oxidized by flavin monooxygenase 3 (FMO3) to form trimethylamine N-oxide (TMAO). Elevated TMAO is associated with increased risk of cardiovascular disease and type 2 diabetes; however, the causal nature is unclear. There is also limited evidence supporting the efficacy of strategies to reduce accumulation of TMAO. Therefore, the purpose of these studies is to determine the effects of increases in TMAO on cardiometabolic health. In study 1, healthy sedentary and endurance trained males consumed a high fat diet. Blood samples were obtained in a fasted state and every hour during a 4-hour high fat challenge. We hypothesized sedentary individuals would produce higher TMAO concentrations. In study 2, healthy sedentary individuals consumed an acute 1000 mg dose of choline (CHOL) and placebo (PLC). Fasted blood samples were collected, flow-mediated dilation (FMD) and oral glucose tolerance (OGT) were measured. In study 3, healthy sedentary individuals consumed 4-wks of CHOL and PLC. Fasted blood samples were collected, FMD and OGT were measured. We hypothesized acute and 4-wk choline supplementation would impair FMD and OGT. In study 1, neither fasting (1.49± 1.2 µM vs. 2.25 ± 1.4 µM, p>0.05) or postprandial TMAO changed significantly with the HFD in sedentary or endurance trained individuals even with the endurance group consuming more TMA dietary precursors. Study 2 found increased plasma TMAO concentrations after choline supplementation on day 1(PLC; 4.14 ± 2.6 μM vs. CHOL; 23.6 ± 33.8 μM, p=0.018) and day 2 (PLC; 5.13±4.9 μM vs. CHOL; 32.6±37.5 μM, p=0.082) however, there were no differences in OGT or FMD. Study 3 found no differences in FMD or OGT following 4-wks of choline consumption. In summary, there were no differences between sedentary and endurance trained individuals fasting or post-prandial TMAO. There was also no effect on acute or 4-wk supplementation of choline on FMD and OGT. More research is needed to understand effects of elevated TMAO on cardiometabolic health. / Doctor of Philosophy / For years, research has been performed to identify the health effects of eating large amounts of red meat on cardiovascular disease (CVD). Consuming red meat, fish, poultry and eggs increases a substance created during digestion and metabolism, called trimethylamine N-oxide (TMAO). Elevated TMAO has been associated with increased risk of CVD and type 2 diabetes but the direct causes are unknown. The purpose of these studies is to determine the effects of increases in TMAO on health in humans. Study 1 included healthy, sedentary and endurance trained males who consumed a high fat diet. Blood samples were collected to measure TMAO before and after a high fat meal. Study 2 included healthy, sedentary males and females who consumed 2 days of 1000 mg of choline, which is commonly found in red meat fish and eggs, and a placebo (carbohydrate) after subjects completed a series of tests to evaluate health. Study three included healthy, sedentary males and females who consumed 4-weeks of 1000 mg of choline per day and a placebo (carbohydrate). Following supplementation subjects underwent a series of tests to assess health. Overall, there were no differences found between sedentary and endurance trained individuals. Acute and 4-week supplementation of choline did not affect measures of blood sugar or blood vessel function.

Microbiome-derived trimethylamine N-oxide (TMAO) has been associated with platelet hyperreactivity and subsequent atherogenesis. Whether physiological TMAO-levels influence plateletderived lipid mediators remains unknown. Little is known about pre-analytic factors potentially influencing TMAO concentrations. We aimed at developing a quantitative LC-MS/MS method to investigate in-vivo and in-vitro pre-analytical factors in TMAO analysis to properly assess the proposed activating effect of TMAO on platelets. TMAO, betaine, carnitine, and choline were analyzed by HILIC-ESI-MS/MS within 6 min total run time. Method validation included investigation of reproducibility, recovery, sensitivity, and in-vitro pre-analytical factors. A 24-h monitoring experiment was performed, evaluating in-vivo pre-analytical factors like daytime or diet. Finally, the effects of different TMAO concentrations on platelet activation and corresponding alterations of plateletderived eicosanoid release were analyzed. The method showed high reproducibility (CVs 5.3%), good recovery rates (96–98%), and negligible in-vitro pre-analytical effects. The influence of in-vivo pre-analytical factors on TMAO levels was not observable within the applied experimental conditions. We did not find any correlation between TMAO levels and platelet activation at physiological TMAO concentrations, whereas platelet-derived eicosanoids presented activation of the cyclooxygenase and lipoxygenase pathways. In contrast to previously published results, we did not find any indications regarding diet dependency or circadian rhythmicity of TMAO levels. Our results do not support the hypothesis that TMAO increases platelet responsiveness via the release of lipid-mediators.

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Epidermolysis bullosa simplex (EBS) and epidermolytic ichthyosis (EI) are rare skin fragility diseases characterized by intra-epidermal blistering due to autosomal dominant-negative mutations in basal (KRT5 or KRT14) and suprabasal (KRT1 or KRT10) keratin genes,  respectively. Despite vast knowledge in the disease pathogenesis, the pathomechanisms are not fully understood, and no effective remedies exist. The purpose of this work was to search for keratin gene mutations in EBS patients, to develop in vitro models for studying EBS and EI, and to investigate novel pharmacological approaches for both diseases. We identified both novel and recurrent KRT5 mutations in all studied EBS patients but one which did not show any pathogenic keratin mutations. Using cultured primary keratinocytes from EBS patients, we reproduced a correlation between clinical severity and cytoskeletal instability in vitro. Immortalized keratinocyte cell lines were established from three EBS and three EI patients with different phenotypes using HPV16-E6E7. Only cell lines derived from severely affected patients exhibited spontaneous keratin aggregates under normal culture conditions. However, heat stress significantly induced keratin aggregates in all patient cell lines. This effect was more dramatic in cells from patients with a severe phenotype. In organotypic cultures, the immortalized cells were able to differentiate and form a multilayered epidermis reminiscent of those observed in vivo. Addition of two molecular chaperones, trimethylamine N-oxide dihydrate (TMAO) and sodium 4-phenylbutyrate (4-PBA), reduced the keratin aggregates in both stressed and unstressed EBS and EI keratinocytes, respectively. The mechanism of action of TMAO and 4-PBA was shown to involve the endogenous chaperone system (Heat shock proteins e.g. Hsp70). Besides, MAPK signaling pathways also seemed to be incriminated in the pathogenesis of EBS. Furthermore, depending on which type of keratin is mutated, 4-PBA up-regulated Hsp70 and KRT4 (possibly compensating for mutated KRT1/5), and down-regulated KRT1 and KRT10, which could further assist in protecting EBS and EI cells against stress. In conclusion, novel and recurrent pathogenic keratin mutations have been identified in EBS. Immortalized EBS and EI cell lines that functionally reflect the disease phenotype were established. Two pharmacologic agents, TMAO and 4-PBA, were shown to be promising candidates as novel treatment of heritable keratinopathies in this in vitro model.

Elevated serum levels of trimethylamine N-oxide (TMAO) were first associated with increased risk of cardiovascular disease (CVD) 10 years ago. Research has since defined that serum TMAO accumulation is controlled by the diet-microbiome-liver-kidney axis. Choline related nutrients are consumed in excess during over-nutrition from a Western diet. The resultant elevated serum TMAO is investigated across various chronic metabolic diseases and many tissue types. While TMAO is most clearly linked to CVD mechanisms in vascular tissue, its molecular effects on metabolic tissues are unclear. Here we report the current standing of TMAO research in metabolic disease context across relevant metabolic tissues including liver, kidney, brain, adipose, and muscle tissues. This review explores the variable TMAO effects in healthy and diseased conditions. Since impaired pancreatic β-cell function is a hallmark of metabolic disease pathogenesis which are largely unexplored in TMAO research, the following primary research results investigate TMAO effects on in vitro functional β-cell mass in relation to healthy and type 2 diabetes (T2D) conditions. Although we hypothesized that TMAO would aggravate functional β-cell mass, the data demonstrate that TMAO improves the T2D phenotype by increasing insulin secretion and production and reducing oxidative stress. Therefore, this work provides crucial support for the emerging context dependent molecular effects of TMAO during metabolic disease progression.