Thesis (M.S.)--Brock University, 2005. / Includes bibliographical references (leaves 68-85). Also available online (PDF file) by a subscription to the set or by purchasing the individual file.

The first aim of this dissertation was to monitor both rat and human responses to short-term perturbations in energy balance brought about through food energy restriction and refeeding, exercise training and the cessation of exercise training or surgical lipectomy. The second aim of this dissertation was to identify factors which might explain differences in food energy intake in weight-matched, weight-stable "large and small eaters". The final aim of this dissertation was to identify factors which might explain differences in resting energy expenditure in a large sample of weight-stable men and women, including exercising and non-exercising persons, and including persons who may be regarded as "restrained eaters".

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Recent studies showed that GPR30, a seven-transmembrane G protein-coupled receptor, is a novel estrogen receptor (ER) that mediates some biological events elicited by estrogen in several types of cancer cells. However, its physiological or pathological role in vivo is unclear. For the first project of my dissertation, I investigated the physiological role(s) of GPR30 in energy metabolism by using transgenic mouse model as well as immortalized cell lines and primary stromal cells. We discovered for the first time that GPR30 knockout (GPRKO) female mice were protected from high-fat diet (HFD)-induced obesity, glucose intolerance, and insulin resistance. The decreased body weight gain in GPRKO female mice is due to the reduction in body fat mass. These effects occurred in the absence of significant changes in food intake, intestinal fat absorption, or triglyceride metabolism. However, GPR30 had no significant metabolic effects in male mice fed the HFD and both sexes of mice fed a chow diet. Further, GPR30 expression levels in fat tissues of WT obese female mice greatly increased, whereas ERα/β expression was not altered. Deletion of GPR30 reduced adipogenic differentiation of adipose tissue-derived stromal cells. Conversely, activation of GPR30 enhanced adipogenic differentiation of 3T3-L1 preadipocytes. For the second project, I explored whether estrogen acts through GPR30 to affect adiposity in female mice. For this study, I generated and examined three independent transgenic mouse models, aromatase (Ar) knockout (ArKO) mice, GPRKO, and GPR30 and Ar double knockout (DKO) mice. We discovered that GPR30 deficiency had limited effects on energy metabolism in mice fed a standard chow diet (STD). However, deletion of GPR30 promoted metabolic flexibility in both genders fed a HFD regardless of the presence of estrogen, suggesting that GPR30 may not solely act as an ER. Consistent with our previous findings, GPRKO mice had higher body temperature, indicating that GPR30 deficiency may promote thermogenesis and energy metabolism, resulting in the reduced fat depots and enhanced metabolic flexibility. For the third project, I further explored whether GPR30 is involved in regulating browning of adipose tissue and thermogenesis in mice. The results show that the expression of UCP-1, the key regulator of thermogenic browning, was higher in the adipose tissue of HFD-fed GPRKO female mice as compared with that of WT mice. Consistently, deletion of GPR30 enhanced mitochondrial respiration in brown adipose tissue (BAT), suggesting that GPR30 deficiency at least partially suppressed the fat accumulation by promoting thermogenesis and dissipating energy. Ex vivo, the expression of thermogenic genes and UCP-1 protein level were upregulated in beige adipocytes differentiated from GPR30-deficient stromal vascular fraction (SVF) cells. These findings provide evidence for the first time that deletion of GPR30 reduces adiposity, promotes white adipose beigeing and thermogenesis, therefore preventing the development of obesity in female mice exposed to excess energy. Further investigations elucidating the underlying mechanism by which GPR30 promotes obesity in females could provide a novel therapeutic target to fight obesity in females. / Doctor of Philosophy / Estrogen can elicit pleiotropic genomic and rapid nongenomic cellular responses via a diversity of estrogen receptors (ERs). Unlike the genomic responses, where the classical nuclear ERα and ERβ initiate gene transcription in estrogen target tissues, the nongenomic cellular responses to estrogen are believed to start at the plasma membrane, leading to rapid activation of second messengers-triggered cytoplasmic signal transduction cascades. The recently acknowledged ER, GPR30, was discovered in human breast cancer cells two decades ago and subsequently in many other cells. Since its discovery, it has been claimed that estrogen, ER antagonist fulvestrant, as well as some estrogenic compounds can directly bind to GPR30, and therefore initiate the rapid nongenomic cellular responses. We are interested to investigate the physiological role(s) of GPR30 in energy metabolism by using transgenic mouse model as well as immortalized cell lines and primary stromal cells. We discovered for the first time that deletion of GPR30 protects female mice from high fat-diet (HFD)-induced obesity and the expression of GPR30 increased in fat tissues of wild type (WT) obese mice, while no alterations of classical ERα/β observed. Consistently, activation of GPR30 by the selective agonist G-1 promotes adipogenic differentiation of 3T3-L1 preadipocytes. ERα is known to exert a protective effect against excess fat accumulation whereas GPR30 may acts as an “obesity gene” and counteracts the classical ERα’s action in regulating fat metabolism. We speculated that there might be a “Yin-Yang” relationship between GPR30 and ERα regarding their actions in the development of obesity. Therefore, we generated three independent transgenic mouse models, GPR30 and aromatase (Ar) double knockout (DKO), GPR30 knockout (GPRKO), and Ar knockout (ArKO) to test our hypothesis that the excess fat accumulation in HFD-fed WT mice could be, or at least partially, caused by the enhanced estrogen-GPR30 signaling. Ar is the key enzyme that catalyzes the biosynthesis of C18 estrogens from C19 androgens in men and postmenopausal women, thereby the ArKO and DKO mouse models allowed us to investigate the role of GPR30 in the absence of endogenous estrogen. We discovered that GPR30 deficiency had limited effects on energy metabolism in young mice fed a standard chow diet (STD). However, deletion of GPR30 promoted metabolic flexibility in both genders fed a HFD regardless of the presence of estrogen, suggesting GPR30 may not solely acts via the ligation of estrogen. Interestingly, consistent with our previous findings, GPRKO mice had higher body temperature, indicating that GPR30 deficiency may promote thermogenesis and energy metabolism, resulting in the reduced fat depots and enhanced metabolic flexibility. Hence, we explored that deletion of GPR30 exerted thermo-promoting effect via upregulation of the mitochondrial uncoupling protein-1 (UCP-1) and enhanced mitochondrial respiration in brown adipose tissue (BAT). Further, the expression of thermogenic genes were significantly higher in the stromal cells-differentiated beige adipocytes, suggesting that GPR30 deficiency suppressed fat accumulation by promoting thremogenic browning of white adipose tissue (WAT) and dissipating excess energy as heat. In summary, my dissertation work provide valuable insight regarding the role of GPR30 in energy metabolism. Further investigations testing whether GPR30 acts as a pro-obesity gene would facilitate our understanding of obesity development and provide a novel therapeutic target to fight obesity.

The following conclusions were drawn from this activity study with one subject: 1. The Kofranyi-Michaelis apparatus may be successfully used with children if it is worn on the back of an adult beside the child. 2. More than one test should be used to establish the energy value for a particular activity with one child. 3. Basal metabolism tests should be made near the time that activity studies are to be made. 4. The actual protein content of the diet should be known and used in the Weir formula for accurate energy determinations. 5. Energy expenditure studies are expensive in terms of time, equipment, techniques to be mastered, and the number of personnel required. The conclusions that were drawn in developing the methods used for this study are: 1. It is desirable to have the stand designed for the Haldane-Henderson glassware from Arthur H. Thomas Company to permit proper alignment and closer readings from the carbon dioxide absorber. 2. That enough sampling bottles be available to hold duplicate samples from bladders made in two different activities it the Haldane-Henderson method or gas analysis is used. / Master of Science

Thesis (Ph. D.)--University of Wisconsin--Madison, 1940. / Typescript. Includes abstract and vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 180-182).

Call number: LD2668 .T4 1984 H365 / Master of Science

Alzheimer’s disease (AD) is a multifactorial, progressive, age-related neurodegenerative disease. Oxidative stress hypothesis is most prevalent and is gaining significant support. Inspite of the progress achieved on oxidative stress related damages in AD brain; the modification occurring on the various cellular antioxidant enzymes antioxidant has not been identified. Tyrosine nitration, a marker for peroxynitrite induced oxidative damage to protein is widespread in AD brain and Manganese superoxide dismutase (MnSOD), primary mitochondrial antioxidant enzyme is prone to peroxynitrite induced nitration and inactivation. Nitration of proteins involved in energy metabolism has been demonstrated in AD brain, which may explain the altered glucose metabolic status existing in AD brain. In the present study, we investigated the effect of tyrosine nitration of MnSOD on energy metabolism by the use of AD mouse model and cultured neuronal cells. The AD mouse model was generated from a double homozygous knock-in mouse, designated as APP/PS-1 mice, by incorporating the Swedish familial AD mutations in APP and P264L familial AD mutation in PS – 1. These animals develop age dependent increase in Aβ deposition beginning at 6 months along with an increase in insoluble Aβ1-40/Aβ1-42 levels. Genotype and age associated increase in nitration of MnSOD without any change in protein levels was also observed. MnSOD activity and mitochondrial respiration was decreased in APP/PS-1 mice. There was also concomitant increase in levels of lactate, an index of glycolytic activity in APP/PS-1 mice. To directly investigate the role of MnSOD inactivation in mitochondrial function and subsequent alteration in glycolytic activity, SH-SY5Y neuroblastoma cells line was used and treated with peroxynitrite. Enhanced nitration and reduction in the activity of MnSOD was observed upon peroxynitrite treatment. Peroxynitrite treatment also induced mitochondrial dysfunction, but MnSOD was inactivated at a concentration of peroxynitrite 10 times lower than that required to inhibit mitochondrial respiration. Mitochondrial dysfunction was alleviated by SOD mimetic and reproduced by MnSOD siRNA. The decline in mitochondrial function did not result in decreased ATP levels but was accompanied by an up-regulated glycolysis signified by high levels of lactate and lactate dehydrogenase activity but decreased activity of pyruvate dehydrogenase. These changes were prevented by SOD mimetic and were promoted by MnSOD siRNA. Specific reduction of MnSOD in MnSOD heterozygous knock-out mice resulted in decreased RCR and complex I activity with increased lactate levels. Taken together, these data demonstrate a critical role of MnSOD in influencing the mitochondrial function and thereby the switch in the energy metabolism switch that might occur under the pathological condition of MnSOD deficiency.