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Organisms are constantly balancing energy demand with an adequate supply of oxygen and substrates to sustain metabolic activity. Thermogenesis is an important metabolic process by which endotherms predominately burn lipids to regulate and maintain their body temperatures by balancing heat loss with heat production. Due to their high rates of heat loss, small winter-active mammals, like the North American deer mouse (Peromyscus maniculatus), are constantly challenged with thermogenesis. Deer mice are also native to high-altitude environments, conditions that further complicate the process of thermogenesis due to the inherent reduced oxygen availability. How metabolic substrates are used for fuelling and sustaining thermogenesis at high altitude remains unclear. The goal of my thesis was to examine how lipid metabolism has evolved to sustain heat production in animals living in high-altitude environments. This was achieved by using deer mice native to high- and low-altitudes acclimated to either standard lab conditions or simulated high altitude (cold hypoxia). I demonstrate that during thermogenic capacity (cold-induced V̇O2max), high-altitude deer mice have higher thermogenic lipid oxidation rates compared to their lowland counterparts, which is further increased after cold hypoxia acclimation. Interestingly, these high rates of lipid oxidation were associated with higher circulatory delivery rates of fatty acids and triglycerides to thermo-effector tissues. Specifically, I show that after a bout of cold-induced V̇O2max, fatty acid uptake occurs primarily in the skeletal muscle in control acclimated high-altitude deer mice, and then shifts to brown adipose tissue following acclimation to high altitude conditions. These findings clearly show that in high-altitude deer mice, maximal thermogenesis is reliant on elevated delivery of circulatory lipids to muscle and brown adipose tissue. This research further sheds light on the mechanistic underpinnings responsible for enhanced thermogenic capacity of high-altitude deer mice and capacity for the highest lipid oxidation rates observed in any mammal. / Thesis / Doctor of Philosophy (PhD) / Thermogenesis, the metabolic production of heat, allows endotherms to maintain stable body temperatures in cold environments. However, it was not yet understood how small mammals fuel and sustain heat production in the cold and low oxygen environment of high altitude. My thesis has uncovered how deer mice native to high altitudes have adapted to burning fats at high rates in hypoxia to sustain thermogenesis. My findings show that high delivery rates of fats to heat-generating tissues are responsible for the elevated rates of heat production in high altitude deer mice. My work contributes to our understanding of the inner workings of the fat pathways and how it has evolved to ensure survival in extreme environmental conditions.

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Obesity is a heterogeneous condition that can seriously impact the degree to which one is healthy and socially accepted. It is generally considered to be greatly influenced by genetic factors. Given that we cannot change our genes, it was the purpose of this study to try to further understand the variables related to obesity that are not genetic. Specifically, the purpose of this study was to investigate the impact of certain family systems variables and childhood feeding practices on Body Mass Index (BMI). The family variables of interest were intergenerational intimacy, intergenerational differentiation, intergenerational triangulation, spousal intimacy, spousal differentiation, nuclear family triangulation, and the relationship between eating and sex. The childhood feeding variables of interest related to the use of food as a reward, coercive use of food, parental disagreement about feeding, the expression of love through food, and feeding enmeshment (i.e., the perception of parental overcontrol in feeding). Because certain eating styles have been found to be related to obesity, further investigation revolved around the impact of the family and feeding variables on eating styles. The eating styles of interest were restrained eating, emotional eating, external eating, and binge eating. The variables were studied by surveying employees of a school system in southwest Virginia. Body Mass Index was found to be significantly positively correlated with feeding enmeshment, weight as a means of sexual avoidance, and eating in response to a lack of physical affection. Restrained eating was not found to be significantly related to any family or childhood feeding variable. Emotional eating, external eating, and binge eating were all significantly negatively correlated with intergenerational intimacy, spousal differentiation, and nuclear family triangulation health, significantly positively correlated with sexual avoidance and deprivation of affection. In addition, emotional eating and binge eating were significantly negatively correlated with intergenerational differentiation, while external eating and binge eating were significantly negatively correlated with spousal intimacy. Emotional eating was positively correlated with all of the childhood feeding practices, while external eating and binge eating were correlated with four and three, respectively, of the feeding practices. While there were many significant correlational findings, there were few significant coefficients in the hierarchical regression analyses, presumably because of the high intercorrelations between the predictor variables (the family and feeding variables). In general, it can be said that family functioning and childhood feeding behaviors are relevant to overeating and overweight. The family and feeding variables are better predictors of eating styles that can lead to obesity than of obesity per se. High levels of dysfunction in families and frequent use of food in non-nutritional ways are associated with high levels of emotional, external, and binge eating. These findings may have implications for physicians and therapists. / Ph. D.

The objective of these studies was to investigate the milk fat synthesis regulation by transcription factors. In the first study, bovine mammary epithelial (MAC-T) cells were treated with sterol regulatory element binding protein-1 (SREBP-1) specific siRNA. The mRNA and protein expression of SREBP-1 were decreased by more than 90% by siRNA. Fatty acid (FA) synthesis, uptake, and selected lipogenic enzyme expression were reduced in cells treated with SREBP-1 siRNA. Therefore, SREBP-1 plays an important role in integrated regulation of lipid synthesis in MAC-T cells through regulation of key enzymes. In the second study, MAC-T cells treated with hormones or FA were transfected with luciferase reporter constructs containing response elements for SREBP-1, peroxisome proliferator-activated receptor γ (PPARγ), or liver X receptor (LXR). The activation of PPARγ and SREBP-1 were stimulated by insulin and insulin combined with leptin, respectively. Trans-10, cis-12 conjugated linoleic acid (CLA) inhibited SREBP-1 activation, and this inhibition was not attenuated by insulin and leptin. Neither trans-10 nor cis-12 double bond inhibited SREBP-1 activation. Taken together, trans-10 and cis-12 double bonds need to be conjugated in CLA to reduce SREBP-1 activation and this inhibition cannot be overcome by insulin and leptin combination in MAC-T cells. In the third study, lactating dairy cows were intravenously infused with 0.625 g/h trans-10, cis-12 CLA for 14 h. We confirmed the appearance of trans-10, cis-12 CLA in the milk of CLA treated cows. Milk and component yield were not affected by the CLA treatment. The desaturation of stearic acid was reduced by CLA. The mRNA and protein expression of transcription factors or lipogenic enzymes were not affected by trans-10, cis-12 CLA. DNA-binding activities for PPARγ and LXR and the activation of SREBP-1 to its mature form were not changed by the treatment. The infusion time in this study was probably too short to induce any changes in transcription factors and lipogenic enzymes. We confirmed DNA-binding activities of PPARγ and LXR in bovine mammary gland. Overall, a prominent role for SREBP-1 in mammary epithelial cell lipid synthetic pathways was described and regulation of transcription factor activation by trans-10, cis-12 CLA was specific to SREBP-1. / Ph. D.

Consumer demand for milk fat has declined due to the increased risk of cardiovascular disease associated with consuming a high saturated fat diet. Milk fat synthesis is energetically expensive for the dairy cow, especially during early lactation or periods of poor nutrition. Thus, manipulating milk fat production and composition may promote the synthesis of more market-valuable milk components and improve energy utilization in dairy cows during periods of increased energy demand. Therefore, the objective of the present studies was to identify molecular proteins that regulate fatty acid synthesis in bovine mammary epithelial cells. The regulation of lipogenic genes including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) is controlled by transcription factors including sterol regulatory element binding protein-1 (SREBP1) and liver X receptor (LXR). In vivo, diet-induced milk fat depression or supplementing diets with polyunsaturated fatty acids inhibits milk fat synthesis by regulating SREBP1 expression. Results confirm that polyunsaturated fatty acids inhibit fatty acid synthesis in bovine mammary epithelial cells by regulating the expression of SREBP1. In hepatocytes, LXR can regulate the transcription of SREBP1 in addition to ACC and FAS. Results confirm that LXR activation enhanced synthesis of fatty acids in bovine mammary epithelial cells by promoting the transcription of FAS and SREBP1. Activation of LXR was unable to prevent the inhibitory effect of polyunsaturated fatty acids on fatty acid synthesis. In the lactating mammary gland, LXR may contribute to the synthesis of fatty acids by regulating the expression of SREBP1. In addition to modifying the expression of lipogenic genes, some enzymes can be phosphorylated by AMP-activated protein kinase (AMPK), an energy-sensing protein, inhibiting their activity. Presence of AMPK mRNA was identified in bovine mammary epithelial cells and activation of AMPK dramatically decreased fatty acid synthesis in bovine mammary epithelial cells. In the lactating mammary gland, AMPK may sense energy availability and regulate milk fat synthesis to control energy utilization. Identification of SREBP1, LXR, and AMPK as regulators of fatty acid synthesis in bovine mammary epithelial cells may lead to the development of technologies allowing dairy producers to modify milk fat production and composition to meet consumer demand and maximize profitability. / Ph. D.

Metabolic flexibility plays a significant role in energy homeostasis by regulating fuel selection in correspondence to energy demand. Obese and type II diabetic populations have displayed a hindered ability to properly transition from fat oxidation while in a fasted state to carbohydrate oxidation once fed, leading to a buildup of mitochondrial metabolites such as acylcarnitines. Carnitine, essential for fatty acyl-CoA transport through the inner and outer mitochondrial membranes, can be an indicator of mitochondrial distress as elevated levels tend to spill over into plasma suggesting a disruption in oxidation. The current study was designed to examine the effect of short term, high fat feeding on plasma acylcarnitine species diversity and levels and if acylcarnitines are associated with metabolic flexibility. 13 healthy, non-obese, sedentary males, aged 18-40 years participated in this study. Following a 12-hour overnight fast a biopsy was taken from the quadricep before and 4 hours after a high fat meal. Blood draws were obtained pre-biopsy while fasted and every hour for 4 hours post high fat meal consumption. Acylcarnitines from plasma were converted to their butyl esters and analyzed by electrospray ionization tandem mass spectrometry (MS/MS). Changes were observed in acetylcarntine (P=0.0125), glucose oxidation (P=0.0295), C16:1/C16:0 desaturation index (P= 0.0397), and C18:1/C18:0 desaturation index (P=0.0012). We did not find that individual changes in flexibility correlated with circulating acylcarnitine measurements in a fasted state / Master of Science