In the Ottawa Hospital Weight Management Clinic, we have previously identified subpopulations of patients in the upper and lower quintiles for rate of weight loss, and characterized them as ‘obese diet sensitive’ (ODS) and ‘obese diet resistant’ (ODR) patient groups, respectively. Skeletal muscle is a major contributor to basal metabolic rate and mitochondrial proton leak in skeletal muscle can account for up to 50 % of resting oxygen consumption. The overall aim of this research is to explore differences in mitochondrial function in human primary myotubes from ODS and ODR subjects. Subsets of ODS and ODR subjects (n = 9/group) who followed a hypocaloric clinical weight loss program at the Ottawa Weight Management Clinic consented to a muscle (vastus lateralis) biopsy. Human primary myoblasts obtained from biopsies were immunopurified and differentiated into myotubes. Mitochondrial function and distribution were compared in intact myotubes from ODS and ODR subjects. Mitochondrial proton leak was significantly lower (p< 0.05) in ODR myotubes compared to ODS myotubes, independent of whether cells were differentiated in low or high glucose medium. In addition, in low glucose medium, ODR myotubes had higher MnSOD protein levels compared to ODS myotubes (p< 0.05). However, there were no significant differences in mitochondrial content, mitochondrial membrane potential, cellular ROS levels or ATP content between ODS and ODR myotubes. Overall, our in vitro mitochondrial proton leak results are consistent with our previous ex vivo results. Future research should examine the possibility that differences in proton leak between ODS and ODR groups may be related to mechanisms of cellular ROS regulation.

In the Ottawa Hospital Weight Management Clinic, we have previously identified subpopulations of patients in the upper and lower quintiles for rate of weight loss, and characterized them as ‘obese diet sensitive’ (ODS) and ‘obese diet resistant’ (ODR) patient groups, respectively. Skeletal muscle is a major contributor to basal metabolic rate and mitochondrial proton leak in skeletal muscle can account for up to 50 % of resting oxygen consumption. The overall aim of this research is to explore differences in mitochondrial function in human primary myotubes from ODS and ODR subjects. Subsets of ODS and ODR subjects (n = 9/group) who followed a hypocaloric clinical weight loss program at the Ottawa Weight Management Clinic consented to a muscle (vastus lateralis) biopsy. Human primary myoblasts obtained from biopsies were immunopurified and differentiated into myotubes. Mitochondrial function and distribution were compared in intact myotubes from ODS and ODR subjects. Mitochondrial proton leak was significantly lower (p< 0.05) in ODR myotubes compared to ODS myotubes, independent of whether cells were differentiated in low or high glucose medium. In addition, in low glucose medium, ODR myotubes had higher MnSOD protein levels compared to ODS myotubes (p< 0.05). However, there were no significant differences in mitochondrial content, mitochondrial membrane potential, cellular ROS levels or ATP content between ODS and ODR myotubes. Overall, our in vitro mitochondrial proton leak results are consistent with our previous ex vivo results. Future research should examine the possibility that differences in proton leak between ODS and ODR groups may be related to mechanisms of cellular ROS regulation.

In the Ottawa Hospital Weight Management Clinic, we have previously identified subpopulations of patients in the upper and lower quintiles for rate of weight loss, and characterized them as ‘obese diet sensitive’ (ODS) and ‘obese diet resistant’ (ODR) patient groups, respectively. Skeletal muscle is a major contributor to basal metabolic rate and mitochondrial proton leak in skeletal muscle can account for up to 50 % of resting oxygen consumption. The overall aim of this research is to explore differences in mitochondrial function in human primary myotubes from ODS and ODR subjects. Subsets of ODS and ODR subjects (n = 9/group) who followed a hypocaloric clinical weight loss program at the Ottawa Weight Management Clinic consented to a muscle (vastus lateralis) biopsy. Human primary myoblasts obtained from biopsies were immunopurified and differentiated into myotubes. Mitochondrial function and distribution were compared in intact myotubes from ODS and ODR subjects. Mitochondrial proton leak was significantly lower (p< 0.05) in ODR myotubes compared to ODS myotubes, independent of whether cells were differentiated in low or high glucose medium. In addition, in low glucose medium, ODR myotubes had higher MnSOD protein levels compared to ODS myotubes (p< 0.05). However, there were no significant differences in mitochondrial content, mitochondrial membrane potential, cellular ROS levels or ATP content between ODS and ODR myotubes. Overall, our in vitro mitochondrial proton leak results are consistent with our previous ex vivo results. Future research should examine the possibility that differences in proton leak between ODS and ODR groups may be related to mechanisms of cellular ROS regulation.

In the Ottawa Hospital Weight Management Clinic, we have previously identified subpopulations of patients in the upper and lower quintiles for rate of weight loss, and characterized them as ‘obese diet sensitive’ (ODS) and ‘obese diet resistant’ (ODR) patient groups, respectively. Skeletal muscle is a major contributor to basal metabolic rate and mitochondrial proton leak in skeletal muscle can account for up to 50 % of resting oxygen consumption. The overall aim of this research is to explore differences in mitochondrial function in human primary myotubes from ODS and ODR subjects. Subsets of ODS and ODR subjects (n = 9/group) who followed a hypocaloric clinical weight loss program at the Ottawa Weight Management Clinic consented to a muscle (vastus lateralis) biopsy. Human primary myoblasts obtained from biopsies were immunopurified and differentiated into myotubes. Mitochondrial function and distribution were compared in intact myotubes from ODS and ODR subjects. Mitochondrial proton leak was significantly lower (p< 0.05) in ODR myotubes compared to ODS myotubes, independent of whether cells were differentiated in low or high glucose medium. In addition, in low glucose medium, ODR myotubes had higher MnSOD protein levels compared to ODS myotubes (p< 0.05). However, there were no significant differences in mitochondrial content, mitochondrial membrane potential, cellular ROS levels or ATP content between ODS and ODR myotubes. Overall, our in vitro mitochondrial proton leak results are consistent with our previous ex vivo results. Future research should examine the possibility that differences in proton leak between ODS and ODR groups may be related to mechanisms of cellular ROS regulation.

Pancreatic beta cells sense fluctuations in circulating nutrients and adjust the rate of insulin secretion to maintain glucose homeostasis. Mitochondria integrate changes in nutrient flux to the generation of signals that modulate insulin secretion via oxidative phosphorylation. Type 2 Diabetes (T2D) is characterized by beta cell mitochondrial dysfunction and impairment of insulin secretion. Early stage progression of this disease in obese and pre-diabetic subjects is characterized by basal hypersecretion of insulin and increased insulin resistance in peripheral tissues including muscle, liver and adipose tissue. Whether basal hypersecretion of insulin or insulin resistance is the primary defect in T2D progression is still debated. The molecular mechanism underlying basal insulin hypersecretion and how it may lead to beta cell failure are not understood. Herein, we optimize a model of glucolipotoxicity that results in increased basal and reduced stimulated insulin secretion response. Furthermore, we show that pancreatic islets exposed to excess nutrients in vitro or isolated from high fat diet fed animals, have a decreased bioenergetic efficiency, which is characterized by increased mitochondrial proton leak. Leak represents the fraction of oxygen consumed that is not coupled to ATP production. We show that leak is sufficient to induce insulin secretion at basal glucose levels and that nutrient-induced insulin secretion at basal glucose is leak-dependent. Finally, we identify the mitochondrial permeability transition pore (PTP) as the source of the leak. Our findings suggest the PTP may be a potential therapeutic target to prevent/delay the onset of hyperinsulinemia in pre-diabetic subjects.

UCP1 is a member of the mitochondrial transmembrane anion carrier protein superfamily and is required to mediate adaptive thermogenesis in brown adipose tissue (BAT). Once activated, UCP1 uncouples mitochondrial respiration from ATP synthesis, thereby wasting the protonmotive force formed across the mitochondrial inner membrane as heat. It is hypothesized that proton leaks through UCP1 could be a molecular target to combat certain forms of obesity. Although it is well established that UCP1 is regulated by allosteric mechanisms, alternative methods such as post-translational modification still remain to be explored. The aims of the present study were to confirm the phosphorylation of UCP1 and the physiological relevance of this modification. Using isoelectric focusing, we confirmed that UCP1 displayed acidic shifts consistent with phosphorylation in BAT mitochondria isolated from cold exposed versus warm acclimated mice. A mouse model that ectopically expressed UCP1 in skeletal muscle was used to explore the link between the mitochondrial redox status and UCP1 function. Our results show that the expression of UCP1 in skeletal muscle led to decreases in body and tissues weights. In contrast, glucose uptake into skeletal muscle, food intake and energy expenditure was increased with the expression of UCP1. Finally, proton leaks through UCP1 were determined to be increased in isolated mitochondria from transgenic versus wild-type mice. Taken together these results indicate a complex interplay between mitochondrial redox status, post-translational modification and UCP1 function. Elucidation of novel mechanisms regulating UCP1 offers alternatives strategies that can be explored in order to modulate BAT thermogenesis.

UCP1 is a member of the mitochondrial transmembrane anion carrier protein superfamily and is required to mediate adaptive thermogenesis in brown adipose tissue (BAT). Once activated, UCP1 uncouples mitochondrial respiration from ATP synthesis, thereby wasting the protonmotive force formed across the mitochondrial inner membrane as heat. It is hypothesized that proton leaks through UCP1 could be a molecular target to combat certain forms of obesity. Although it is well established that UCP1 is regulated by allosteric mechanisms, alternative methods such as post-translational modification still remain to be explored. The aims of the present study were to confirm the phosphorylation of UCP1 and the physiological relevance of this modification. Using isoelectric focusing, we confirmed that UCP1 displayed acidic shifts consistent with phosphorylation in BAT mitochondria isolated from cold exposed versus warm acclimated mice. A mouse model that ectopically expressed UCP1 in skeletal muscle was used to explore the link between the mitochondrial redox status and UCP1 function. Our results show that the expression of UCP1 in skeletal muscle led to decreases in body and tissues weights. In contrast, glucose uptake into skeletal muscle, food intake and energy expenditure was increased with the expression of UCP1. Finally, proton leaks through UCP1 were determined to be increased in isolated mitochondria from transgenic versus wild-type mice. Taken together these results indicate a complex interplay between mitochondrial redox status, post-translational modification and UCP1 function. Elucidation of novel mechanisms regulating UCP1 offers alternatives strategies that can be explored in order to modulate BAT thermogenesis.

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.

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