▼ The majority of women today work outside the home, and the fastest growing segment of working women is the working mother. Many women have retained responsibility for caring for their families and their households despite their fulltime employment. Little is known about the relationship between these multiple roles and her health behaviors. This study examined the relationships among attitudes toward and knowledge of nutrition, outcome values and expectancies, self-efficacy, social support, physical activity level, body image, self-esteem, body mass index, demographic characteristics and diet quality and dietary intake of calcium, iron, folate, total fat, fiber, and kilocalories in 356 fulltime working women 36.3 ± 6.1 years of age. Social cognitive factors were assessed using a Likert-type questionnaire and a food frequency questionnaire. Body mass index was calculated using self reported height and weight information. Diet quality was determined using the Healthy Eating Index. Qualitative interviewing was conducted in 35 subjects to identify perceptions working women have about their dietary and physical activity behaviors. There was no difference in diet quality or dietary intake between working women children and women without children. Women with children had significantly lower exercise index scores than women without children (p < .01). Multiple regression models that included sociodemographic covariates related to diet quality and dietary intake and social cognitive factors, explained between 21 and 23% of the variance in diet quality and between 7 and 19% of the variance in dietary intake measures. A model to explain the relationship among social cognitive factors and diet quality in working women is proposed. Results of the qualitative interview suggest that fulltime working women perceive that the multiple roles they have play a negative role in their dietary and physical activity behaviors. This study underscores the importance of taking attitudes toward and knowledge of nutrition, self-efficacy, physical activity, and sociodemographic factors into account when developing worksite nutrition programs for women. Advisors/Committee Members: Mitchell, Mary C.

▼ Oxidative and nitrative stress play pivotal roles in the etiology of chronic diseases such as cancer and heart disease. Therefore, investigations to determine the effects of oxidative and nitrative stress on dietary antioxidant utilization are critical for our understanding of chronic disease prevention. To date, the impact of these stressors on vitamin E utilization in humans is controversial. Therefore, for this dissertation, we hypothesized that oxidative and nitrative stress from cigarette smoking will alter vitamin E utilization such that smokers have higher dietary requirements for vitamin E than nonsmokers. Using novel analytical techniques in liquid chromatography/mass spectrometry, we determined that the increased reactive nitrogen species associated with cigarette smoking caused a doubling of plasma nitro-gamma-tocopherol. Furthermore, using deuterium labeled alpha-tocopherols, we observed that smokers, compared with nonsmokers, had a 13% faster plasma alpha-tocopherol disappearance, suggesting that alpha-tocopherol functions in vivo as an antioxidant. Further, smokers’ alpha-tocopherol disappearance rates correlated with plasma ascorbic acid concentrations, suggesting that higher plasma ascorbic acid concentrations could prevent the rapid alpha-tocopherol disappearance. Therefore, we conducted a double-blind, placebo-controlled, cross-over investigation in smokers and nonsmokers who were provided supplemental ascorbic acid (2-weeks, twice daily, 500 mg) or placebo prior to evaluating vitamin E disappearance kinetics. Plasma ascorbic acid concentrations doubled in both groups in response to the supplement compared to the placebo. In smokers during placebo treatment, vitamin E disappearance kinetics were again faster than in nonsmokers. However, during vitamin C supplementation, smokers’ vitamin E disappearance was normalized. Plasma F2alpha-isoprostanes, a marker of lipid peroxidation, remained 34% higher than nonsmokers and vitamin E metabolite production was unchanged in the smokers during supplementation. Taken together, these data suggest that in vivo plasma tocopherols and ascorbic acid interact during lipid peroxidation via the reduction of tocopheroxyl radicals to tocopherol by ascorbic acid. Collectively, these investigations have provided evidence that the oxidative stress from cigarette smoking increases vitamin E utilization, justifies higher dietary intakes of vitamins E and C among smokers, and warrants further research to determine if other reductive antioxidants can interact with vitamin E and further modulate vitamin E kinetics. Advisors/Committee Members: Bray, Tammy M.

▼ Epidemiological and experimental evidence suggests that increased consumption of soy can reduce the risk for developing prostate cancer (PCa). Isoflavones have been identified as one group of biologically active components in soy thought to be responsible, in part, for this anticancer activity. The 3 major isoflavones found in soy are the glycoside derivatives of genistein, daidzein, and glycitein. Several lines of evidence suggest that the isoflavones genistein and daidzein may protect against PCa. However, the anticancer activity of glycitein has not been extensively investigated.The objective of the first study was to examine the effects of soy isoflavones on the activation of the extracellular signal-regulated kinase (ERK1/2) pathway in noncancerous prostate epithelial cells (RWPE-1). The ERK1/2 cascade is involved in PCa progression and its activation may be associated with prevention of this disease. Data presented in the first study show that glycitein is the most potent activator of ERK1/2 signaling as compared with other soy isoflavones. Glycitein-induced ERK1/2 activation was sustained for at least 2 hours and decreased cellular proliferation by 40%. Sustained ERK1/2 activation is thought to induce cellular differentiation in the prostate epithelium. The sustained ERK1/2 responsed elicited by glycitein in the first study lead to the hypothesis that glycitein may induce differentiation of the RWPE-1 cell line. Loss of cellular differentiation of the two primary differentiated prostate epithelial cells, luminal and basal cells, contributes to the progression of PCa. Soy isoflavones have been shown to induce cellular differentiation in a number of tissues. However, glycitein-induced differentiation in the prostate has not been examined. Cellular differentiation was characterized by cytokeratin expression. Data presented in this study show that glycitein significantly reduced the expression of luminal cell markersyet maintained the expression of basal cell markers. These data suggest that glycitein may induce basal cell differentiation in prostate epithelial cells. The ability of glycitein to induce basal cell differentiation may represent a novel mechanism of preserving this cell population and reducing PCa risk. Data from the first two studies of this dissertation show that glycitein is a unique isoflavone that induces a robust ERK1/2 response and induces cellular differentiation of prostate epithelial cells. Structurally, glycitein is the only soy isoflavone with a methoxy group. The third hypothesis was that isoflavones with methoxy groups have greater antiproliferative and ERK1/2 responses in prostate cells as compared with nonmethylated isoflavones. To test this hypothesis, noncancerous, precancerous, and cancerous cells were treated with nonmethylated and methylated isoflavones. The results of this study show that although the red clover isoflavones biochanin A and formononetin are methylated, ERK1/2 activation is similar to that of genistein and daidzein. All isoflavones inhibited the proliferation of these cell lines with greater potency in the noncancerous and precancerous cells. However, glycitein-induced ERK1/2 activation was the only isoflavone with associated antiproliferative effects of early stage prostate cells. These results suggest that the position of the methoxy group may be important for the bioactivity of isoflavones. Furthermore, isoflavones may have greater anticancer activity during noncancerous and precancerous stages of PCa. Advisors/Committee Members: Bomser, Joshua.

▼ Adipose triglyceride lipase (ATGL) is a newly identified lipase that initiates triglyceride lipolysis. We characterized expression of ATGL in pig, cattle, and mice. In pig, we concentrated on hormonal and dietary regulation of ATGL expression. In cow, we examined whether ATGL transcription was upregulated by the increased energy requirements of early lactation. We cloned porcine and bovine ATGL genes and designed and validated real-time PCR primer sets. ATGL gene expression increases dramatically in subcutaneous adipose during development and maturation, as well as during in vitro adipogenesis. Treatment of differentiated primary pig preadipocytes with insulin, epinephrine and forskolin decreased ATGL gene expression, suggesting that PI3K and PKA signaling decrease ATGL gene expression. ATGL gene and protein levels were increased in perirenal and subcutaneous fat depots by two weeks of calorie restriction in gilts. We continued our research on the metabolic regulation of ATGL in brown adipose tissue (BAT) of mice as well as whole BAT lipid metabolism. BAT is a unique fat depot in mammals for oxidation of dietary fuel substrates without production of chemical energy. Pharmaceutical-induced nonshivering thermogenesis in BAT results in the prevention and treatment of obesity in rodents and shows promise in humans. Cold exposure led to a decrease in ATGL protein and gene expression in mouse BAT. To investigate how cold exposure-induced signaling affects ATGL expression, the beta3-adrenergic receptor agonist CL 316,243 was used. CL 316,243 potently activated PI3K/Akt, ERK 1/2, and p38 ex vivo and in cultures of primary brown adipocytes and results in rapid and significant decreases in ATGL and adipose fatty acid binding protein. CL 316,243 induced increased lipolysis, despite decreased ATGL protein. Our data suggest that ATGL is degraded by PKA signaling. However, the proteasome inhibitor MG 132 failed to rescue CL-mediated degradation, suggesting non-proteasomal degradation of ATGL via beta3-adrenergic signaling. Inhibition of CL 316,243-mediated PI3K/Akt signaling by LY294002, ERK 1/2 signaling by PD-98059, and p38 signaling by SB 203580, failed to rescue ATGL protein degradation. However, inhibition of PKA by H89, fully rescued ATGL gene and protein levels at 1 and 3 hours, further suggesting that ATGL is targeted by PKA signaling. Advisors/Committee Members: Lee, Kichoon.

▼ Valid measures of household food insecurity are critical to accurately evaluate the impact of food assistance programs in developing countries. The goal of this dissertation research was to assess the validity the Colombian Household Food Security Scale (CHFSS), used in the 2006 food supplement component evaluation of the Plan for Improving Food and Nutrition in Antioquia, Colombia (MANA). The twelve-item household CHFSS was applied to a cross-sectional stratified random sample of 2,784 low-income households with pre-school children receiving MANA food supplements. Internal validity of the CHFSS was established using Rasch Modeling to evaluate the psychometric characteristics of the items through measure and INFIT values. To assess the criterion validity of the CHFSS, households were characterized as food secure, mildly food insecure, moderately food insecure, and severely food insecure based on their survey score. Chi-square tests and ANOVA analyses were used to determine associations between the food security categories and demographic characteristics, socioeconomic characteristics and child health status. A multiple linear regression model was developed to determine coefficients of household food expenditure by food security status. Logistic regression models were further developed to assess the risk of child malnutrition by household food insecurity status in both bivariate and multiple regression models. Rasch Modeling revealed that most CHFSS items presented good fitness. Statistically significant differences were found between household food security status and household size, parental age, income, area of residence, gender head of household, consumption of MANA food supplements and household food expenditures (p<0.01). Statistically significant associations were found between household food insecurity and diagnoses of children's diarrhea, upper respiratory infection and parasitosis (p<0.0001). The risk for child stunting and underweight increased is a dose-response manner as food insecurity became more severe. These results indicate that the adapted CHFSS is a valid tool to assess household food security in participants of food assistance programs like MANA. This research provides agencies and institutions carrying nutrition interventions in similar areas and conditions with a valid tool that can be adapted to specific evaluation needs and describes the methodology to assess the validity of the proposed tool. Advisors/Committee Members: Melgar-Quinonez, Hugo.

▼ Tomato-based products and soy foods contain compounds that have been shown to beneficially impact health, however, adequate information on phytochemical in vivo biodistribution and bioavailability, as well as the effect of consumption of these foods on antioxidant/oxidative environment regulation and prostate biomarker modulation is not available. First, in a study of healthy individuals, the change in plasma lycopene and resistance of lipoproteins to ex vivo oxidative stress following variation in tomato-based product intake was assessed. Total plasma lycopene concentrations significantly decreased during a 7-day washout period and significantly increased after consumption of tomato-based foods for 15 days. A complex array of plasma lycopene isomers was also detected throughout the study. Additionally, dietary intervention with tomato-based products significantly enhanced the protection of lipoproteins to ex vivo oxidative stress. Secondly, a study was performed to determine if men with prostate cancer would consume tomato-based products or a soy protein supplement daily during the period between their diagnosis and surgery (i.e. a period of 2-4 weeks). In addition, this study was conducted to examine in vivo concentrations of phytochemicals from these foods and their effect on modulating hormone levels. All study participants were compliant. Tomato-based product or soy protein supplement intake significantly increased plasma and prostate tissue carotenoid concentrations or urinary and prostate tissue isoflavone levels, respectively. It appears that tomato sauce and tomato soup provide a more bioavailable form of lycopene than vegetable juice. In addition, this study showed that dietary intervention with tomato-based products and soy decreases serum PSA concentrations. A significant increase in lycopene and isoflavone concentrations following tomato and/or soy intake suggests a preferential uptake and requirement of these phytochemical-containing foods in biological processes related to reducing both oxidative stress and prostate cancer progression. Finally, using new technologies, an evaluation of stored samples from the first two studies was conducted to assess the influence 2-4 weeks of tomato-based product consumption had on additional markers of oxidative stress in healthy participants and prostate cancer patients. Tomato-based product intake significantly increased plasma lipid-soluble antioxidant capacity in each of the study populations, while decreasing urinary levels of 8-iso-PGF2a in prostate cancer patients. Advisors/Committee Members: Schwartz, Steven.

▼ Strict blood glucose control has been shown to be an effective method for delaying the onset and progression of diabetes related complications. Novel ingredients may be added to carbohydrate containing food products which may attenuate postprandial glycemia. Three studies were conducted, all with nondiabetic adults. The first explored whether a small dose of fructose administered before or simultaneously with a high glycemic index starchy food decreases postprandial glycemic response. The positive incremental area under the glucose curve was reduced 25 and 27% (P < 0.01) only when fructose was fed 60 or 30 min before the meal, respectively. In the second study, the glycemic, insulinemic and breath hydrogen responses to food starch esterified with 1-octenyl succinic anhydride (OSA) were compared to a glucose control solution. With the OSA beverage, peak plasma glucose concentration was reduced 8% (P < 0.03) and peak insulin concentration was decreased 28% (P < 0.004). Similarly, OSA reduced (P < 0.05) the area under the curve for both plasma glucose and insulin. The number of positive breath hydrogen (BH) tests with the OSA-substituted starch suggests that the lowered glycemic response is at least partially due to small intestinal malabsorption of OSA. In the third study, beverages containing 3 doses of salacinol herbal extract were administered (500 mg, 700 mg, and 1000 mg). Incremental area under the curve (AUC) was reduced for both glycemic (P = 0.03) and insulinemic (P = 0.002) responses postprandially with the beverage containing 1000 mg salacinol. The AUC for BH was greater for all 3 beverages compared to the control (0 mg) (P = 0.0006, P < 0.0001 and P < 0.0001 respectively). The beverage containing 1000 mg of salacinol produced an increase in reported abdominal distention (P = 0.020) and flatulence (P = 0.002). BH results and gastrointestinal intolerance suggest that the decreases in glycemia and insulinemia with the 1000 mg dose of salacinol are at least partially due to incomplete carbohydrate digestion. In summary, fructose, OSA and salacinol all decrease postprandial glycemia by different mechanisms and may have applications in food products. Advisors/Committee Members: Schiller, Mary Rosita.

▼ Manipulation of the fermentation of the feeds by the microorganisms in the fore stomach of cattle (rumen) offers a potential approach to minimize waste excretion. However, the conventional evaluation of digestibility of feeds does not describe the microbial mechanisms involved in ruminal digestion. Examining microbial diversity using culture-independent techniques allows greater understanding of the function of complex microbial ecosystems such as the rumen. For the first set of experiments, ruminal and omasal samples were obtained from cows supplemented with different sources of methionine (Met). The objectives in this set of experiments were to study how Met supplementation affected microbial populations in the rumen and if omasal sampling provided a representative sample of ruminal bacteria. Neither the protozoal counts nor the denaturing gradient gel electrophoresis (DGGE) banding patterns derived from protozoa were different among the dietary treatments or for ruminal vs. omasal samples. As revealed by DGGE, bacterial populations clustered by treatments in ruminal and in omasal samples. The next experiments were performed in dual flow continuous culture fermenters to study the interaction between ruminal protozoa, methanogens and eubacteria in a controlled environment. We used microbial inhibitors to selectively suppress different functional groups of microbes in the presence or absence of protozoa. The fermenters were fed either no additive, 5% animal-vegetable, monensin, or bromoethanesulfonate (BES, 250 ìM). For the defaunated sub-period, total N flow and digestibilities of NDF and OM were significantly higher and ammonia concentration was lower. Protozoal counts were not different between treatments. Defaunation did not affect total VFA production but decreased the acetate: propionate ratio. Biohydrogenation was impaired in the defaunated fermenters but the flow of cis-9 trans-11 conjugated linoleic acid was unaffected by defaunation or by treatments other than added fat. Defaunation and BES changed methanogen populations despite not decreasing methane production. Monensin did not affect methanogen populations but tended to decrease methane. Defaunation changed eubacterial populations, but the effect of treatments were harder to ascertain. In conclusion, the culture-independent methods allowed us assess the changes in microbial populations in the rumen and in continuous culture. Our modified continuous culture system allowed us. Advisors/Committee Members: Firkins, Jeffrey L.

▼ The glycemic index (GI) measures the magnitude of the postprandial increase in blood glucose caused by a test food compared with a reference food/beverage, containing the same amount of carbohydrate. The insulin index is determined in a similar manner of GI calculation, except that blood insulin AUC is used in place of blood glucose AUC. Low GI and insulin index foods are desirable because foods with low GI and insulin index result in gradual increase in postprandial glycemia/insulinemia and lower blood glucose/insulin fluctuations compared with foods with high GI and insulin index. Fructose has low glycemic and insulin index, and catalytic amounts of fructose lower the glycemic response to other carbohydrates. However, prefeeding of fructose is necessary to achieve this effect due to the slow intestinal absorption of fructose. The overall objective of this dissertation was to investigate the current interest of carbohydrate metabolism. The first goal was to determine a difference in carbohydrate metabolism in populations with different metabolic status. The second objective was to determine fructose absorption in the presence of erythritol in vivo and in vitro. The GI and insulin index of raisins were determined and compared in healthy sedentary young adults, endurance athletes, and people with impaired glucose tolerance. The GI of raisins was low in the healthy sedentary people and people with pre-diabetes and was moderate in the athletes, but there were no differences among the subject groups. The insulin index of raisins was not significantly different among the groups. When the simultaneous ingestion of an equimolar amount of erythritol and fructose (FE) was consumed in healthy subjects, breath hydrogen production with FE was 207% higher than that of a beverage of fructose (F), indicating greater carbohydrate malabsorption. Because of the considerable rise in serum erythritol and the decrease in serum fructose in the FE versus F groups, it appeared that erythritol was absorbed at the expense of fructose. The inhibitory effect of erythritol on fructose absorption that we observed in healthy humans was reproducible in a Caco-2 cell model at high doses of fructose and erythritol. Erythritol inhibited fructose absorption in a dose-dependent fashion. Advisors/Committee Members: Smith, Anne.

▼ Although various factors are involved in the wound healing process, malnutrition may be a major factor that can lead to tissue damage and delay wound healing. It is well known that malnutrition triggers compromised immune functions and decreased antioxidant defense in many studies. Moreover, immune response and antioxidant defense are critical facets of the inflammatory stage that can be the rate-limiting step of later stages of wound healing. Thus, nutritional modulation of immune function and antioxidant status may play a crucial role in the control and regulation of wound healing. Reactive oxygen species (ROS) and proinflammatory cytokines produced by immune cells during inflammation activate NFkB, a redox sensitive transcription factor that induces expression of immunoregulatory genes such as chemokines and cytokines during the inflammatory stage. NFkB activation and proinflammatory cytokine production play key roles in regulating wound healing. Notably, protein energy malnutrition (PEM) and zinc deficiency are well-known health problems associated with delayed wound healing. N-acetyl cysteine (NAC) supplementation in PEM may help wound healing by enhancing immune response and antioxidant defense. Zinc supplementation may also increase immune function and antioxidant defense. Therefore, this dissertation was focused upon the role of nutrition and ROS in wound closure and the effect of nutritional supplementation on immune response and antioxidant defense at cellular and molecular levels during the early inflammatory stage of cutaneous wound healing. We have hypothesized that malnutrition delays cutaneous wound closure due to decreased immune response and antioxidant defense. Furthermore, we propose that nutritional supplementation will restore immune function and antioxidant defense in the inflammatory stage during cutaneous wound healing. To test these hypotheses, we have: i) investigated the effects of PEM and the role of ROS using CuZnSOD transgenic mice to determine if malnutrition or ROS affect cutaneous wound healing; ii) examined the role of dietary supplementation of NAC on wound closure and gene expression of pro-inflammatory cytokines (IL-1b and TNF-a) and IkB (indirect measurement of NFkB activation) during the early inflammatory stage in PEM mice; and iii) investigated the role of dietary zinc on wound closure and gene expression of IL-1b, TNF-a and IkB. The results of these experiments demonstrated that PEM impaired wound healing, possibly due to delayed neutrophil infiltration and decreased gene expression of IkB, IL-1b and TNF-a. However, NAC supplementation restored neutrophil response and normalized gene expression of IkB, IL-1b and TNF-a in the early inflammatory stage of cutaneous wound healing. In addition, we found that zinc deficiency delayed wound closure. Notably, we also found that zinc supplementation at 500 ppm accelerated neutrophil infiltration, increased expression of IkB and enhanced wound closure. However, mega dose zinc supplementation at 1000 ppm (20 times higher than that of a control diet) delayed neutrophil infiltration, decreased IkB levels and delayed normal wound closure. In conclusion, this study provides further evidence of the critical role of nutrition in wound healing. More importantly, results from these experiments demonstrated that NAC supplementation provides an effective intervention strategy to enhance wound healing in PEM patients. However, we have also demonstrated that supplementation strategies must be approached judiciously, as our results show that whereas zinc supplementation at 500 ppm may enhance wound healing, zinc supplementation at 1000 ppm significantly delays wound healing. Advisors/Committee Members: Bray, Tammy M.

▼ Prostate cancer is a major cause of morbidity and mortality in the United States. Epidemiologic, in vitro, and animal studies support the hypothesis that dietary patterns and nutritional compounds can influence prostate carcinogenesis. Prostate cancer generally has a protracted time course, therefore, many opportunities exist for nutritional interventions to alter the course of disease. This thesis involves three studies that target men during: 1) primary prostate cancer prevention, 2) treatment for localized disease and 3) treatment for advanced disease. The first study quantifies the use of nutritional supplements in a subgroup of men participating in a large, nationwide chemoprevention trial. We found that a majority of men were consuming supplements which are hypothesized to influence prostate cancer risk and therefore, may impact the results of prevention studies. Our second study precisely defines nutritional supplement use among men undergoing radiation therapy for primary treatment of newly diagnosed, localized prostate cancer. There are very few reports describing supplement use among men actively undergoing treatment for prostate cancer, and how dietary supplements may influence radiation therapy is currently an area of controversy. We found a majority of men were consuming supplements, many of which have been hypothesized to influence the efficacy or toxicity of radiation therapy. Our third study is an intervention trial where we evaluated adherence, safety, and biological effects of a diet rich in tomatoes / tomato products and soy foods in men with advanced prostate cancer. A diet rich in tomatoes and soy foods was tolerable and did not result in significant toxicity. Additionally, we observed that a diet rich in tomatoes and soy protein reduced serum PSA in 30 to 40% of men and may prolong the interval before more aggressive cancer therapy is needed. These studies indicate that men at risk of prostate cancer or undergoing primary treatment consume supplements that may influence risk and response to therapy. This data provides information that will help investigators define future intervention trials. Among the dietary components worthy of study for prostate cancer prevention and as an adjunct for therapy, tomatoes and soy are a reasonable combination for intervention studies. Advisors/Committee Members: Clinton, Steven K.

▼ Dietary agents have important implications in the treatment of type 2 diabetes. My first objective was to determine the effectiveness of the dietary fat, conjugated linoleic acid (CLA) in the attenuation of hepatic steatosis in Wistar rats fed a CON diet or a diet containing CLA. CLA decreased hepatic triglycerides (TG) compared to the CON diet. The CLA mediated difference was associated with increases in the mRNA markers of lipid oxidation and decreased markers of lipogenesis. These effects were observed in the absence of decreases in adiposity, adipokines, body weight and food intake suggesting that CLA is protective against hepatic TG accumulation in rats by modulating hepatic lipid metabolism. CLA is associated with the development of hepatic steatosis and insulin resistance in mice along with rapid ablation of adipose tissue. Therefore, my second objective was to determine the role of adipokines in CLA mediated insulin resistance in male C57BL/6 and leptin deficient ob/ob mice that were fed a diet supplemented with or without CLA. CLA supplementation significantly decreased body fat, induced hepatic steatosis and insulin resistance in mice. Furthermore, these changes were associated with significant depletion of adiponectin, but not leptin. When adiponectin levels were restored by either removal of CLA from diet of C57BL/6 mice or administering rosiglitazone to ob/ob mice fed CLA, insulin resistance and hepatic steatosis were attenuated. Thus, we show that adiponectin is an important factor responsible for insulin resistance caused by CLA. Because increased hepatic glucose production (HGP) contributes to fasting hyperglycemia in type 2 diabetes, my third objective was to determine the effectiveness and elucidate the mechanisms by which dietary bioactive compounds, especially the citrus fruit flavonoid naringenin, exert glucose lowering effects in cultured hepatocytes. The aglycone naringenin significantly suppressed HGP from Fao hepatoma cells similar to insulin and metformin. Furthermore, naringenin, similar to metformin, decreased cellular ATP concentrations and increased p-AMPK, which when activated, suppresses hepatic glucose output. The suppressive effects of naringenin on HGP and similarity to the action of metformin are novel and intriguing and future studies using in vivo models are warranted. Advisors/Committee Members: Belury, Martha A.

▼ With the increasing environmental concern for N excretion in the environment, the nutrition of dairy cows meets new challenges to maintain milk production while decreasing dietary protein. Various strategies to improve efficiency of N utilization in milk production have been developed. Decreasing protozoal abundance and the corresponding improvement of microbial protein efficiency could improve this efficiency of dietary protein utilization. The feeding of fat usually decreases the abundance of protozoa but can have detrimental effect on ruminal fermentation and milk fat production, thus needing further study to ascertain the potential benefits relative to potential detriments. In a first study, the effects of the availability of dietary fatty acids from cottonseed oil on ruminal metabolism and milk fat production were investigated. In our study, feeding a mix of pelleted and delinted cottonseeds appeared to modify ruminal fatty acid metabolic processes, decreasing the risk of milk fat depression and tending to increase dry matter intake and milk production over time compared with conventional or pelleted cottonseeds. In a second study, the effects of feeding Rumensin (R) in combination with animal vegetable (AV) fat or coconut oil were fed to six rumen-cannulated dairy cows. Using omasal collection, I measured microbial protein efficiency and nutrient digestibility. By feeding R to control the extent of amino acid deamination and combined with fat to control protozoal abundance, the efficiency of microbial protein synthesis might be improved in dairy cows. Animal vegetable fat can be biohydrogenated in the rumen and decrease its effectiveness, but diets supplemented with coconut oil (CO; rich in medium chain FA) are more consistent in inhibiting protozoa. Total protozoal abundance was decreased by CO supplementation for all genera expect for Epidinium, which maintained its numbers but was decreased by AV+R. The low acetate to propionate ratio for CO was associated with a decreased ruminal NDF digestibility. There was no effect of diet on efficiency of microbial protein synthesis. DMI was 5 kg/d lower with CO. Milk production was lower when cows were fed CO than AV and when diets contained R. Milk fat depression (MFD) occurred with AV+R and CO. The decrease in protozoal abundance was not associated with an increase in microbial protein efficiency. The detrimental effect of CO on DMI affected the energy available for milk synthesis Omasal flows of FA were characterized by an increased percentage of trans 18:1 for AV and CO diets, a higher percentage of 12:0 and 14:0 for CO, and higher cis 18:1 for AV. Milk FA composition reflected the changes observed for omasal FA digesta flow. The de novo FA synthesis in the mammary gland was inhibited with R and F supplementation. Higher trans 18:1 FA in milk fat was also observed for AV and CO. The feeding of CO did not prevent MFD, and no interactions between R and S were detected. The feeding of CO did compromise ruminal biohydrogenation, with accumulation of trans 18:1 in the rumen and in milk fat. Advisors/Committee Members: Firkins, Jeffrey.

▼ Soy isoflavonoids are phytoestrogenic compounds that exist in three families defined by their parent aglycones daidzein, glycitein and genistein. Beta-, acetyl- and malonylglucosides of isoflavonoid aglycones predominate in non-fermented soy products, whereas fermented soy foods are rich in aglycones. Isoflavonoid glucosides must be converted to aglycones prior to absorption from the gut. In this study, digestive stability and bioaccessibility of isoflavonoids and the microbial metabolite equol were examined using simulated digestion, contributions of the small and large intestines in absorption of soy isoflavonoids and isoflavonoid metabolite production were examined using swine with ileal canulae, and Caco-2 human intestinal cells were used to characterize transport and metabolism of equol. Isoflavonoids and equol were stable during simulated digestion and partitioning of aglycones and acetylgenistin into the aqueous fraction of digesta was significantly affected by the concentration of bile present during the small intestinal phase of the procedure. Ileal effluent from swine contained only isoflavonoid aglycones, indicating that hydrolysis of isoflavone glucosides from the meal occurred. Similar amounts of isoflavonoid equivalents were present in pig urine during small intestinal digestion (canulae open) compared to complete digestion (canulae closed) 24 h after consumption of the soy meal. The quantities of the microbial metabolites dihydrodaidzein, dihydrogenistein and equol in urines after complete digestion markedly exceeded those of parent aglycones. Accumulation of cellular equol equivalents was proportional to the initial medium concentration and reached a maximum within 1 h. Free equol in the basolateral compartment was greatest at 1 h and represented 20% of its initial concentration in the apical medium. By 4 h, 85% of equol was present as phase II conjugates with 50 and 35% of the starting amount located in the apical and basolateral compartments, respectively. In summary, the small intestine has a critical role in generating the bioavailable form of dietary isoflavonoids. The bioavailability of isoflavonoids from foods containing fat and protein may exceed that of isoflavonoid supplements consumed without food due to enhanced bile secretion. Moreover, ingested equol has the potential to be absorbed and individuals classified as “non-producers” may actually efflux equol conjugates into the intestinal lumen with high efficiency. Advisors/Committee Members: Failla, Mark L.

▼ We hypothesize that energy intake / obesity may increase the risk of prostate cancer, in part, via increased IGF-I activity. Furthermore, we propose that dietary components in tomato and soy may inhibit IGF-I activity and subsequently reduce prostate carcinogenesis. The first study describes the characterization of an in vitro serum free media (SFM) system in which rat AT6.3 prostate cancer cell proliferation and survival are dependent on IGF-I. Using this model, we show that IGF-I stimulates cell proliferation and survival via intracellular signaling pathways including AKT and ERK1/2. Polyphenols from tomato and soy, such as genistein, biochanin A, daidzein, quercetin, kaempferol and rutin, demonstrate unique abilities to inhibit intracellular signaling pathways involving tyrosine kinase activity thereby reducing proliferation and enhancing apoptosis. We next examined gene expression profiles of AT6.3 cells treated with SFM with 10% fetal bovine serum (FBS), SFM alone, and SFM plus 50 ng/ml IGF-I using Affymetrix microarray technology. Our results demonstrate that IGF-I stimulates specific patterns of gene expression involving cell cycle control, proliferation, metabolism, apoptosis, angiogenesis, invasion and metastasis. Our results show that the ability of IGF-I to upregulate glucose uptake and glycolysis is essential for proliferation. We next examined the ability of diet restriction to alter gene expression profiles in the rat prostate. Affymetrix U34A Genechips and microarray technology were employed and our results demonstrate that dietary restriction modulates expression of many genes related to hormone receptors, cytokines, growth factors, nutrient metabolism, and xenobiotic metabolism. Our results suggest that DR modulates gene expression profiles of normal prostate tissue in a manner that may decrease carcinogenesis, in part, via reduced serum IGF-I. In summary, our studies suggest that IGF-I stimulates prostate cancer progression and that dietary factors increasing IGF-I activity, such as energy intake, may stimulate carcinogenesis, whereas dietary components such as polyphenols may reduce IGF-I activity and inhibit carcinogenesis. Advisors/Committee Members: Clinton, Steven K.

▼ Overexpression of cyclooxgenase-2 (COX-2) and prostaglandin E2 (PGE2) have been demonstrated to play a significant role in the tumorigenesis of several human cancers. Inconsistent and controversial reports on the expression and activity of COX-2 in prostate cancer raised the question of whether COX-2 plays a pivotal role in prostate carcinogenesis. In contrast, there is more consistent support for a positive link between COX-2 overexpression and bladder carcinogenesis. However, the exact role of COX-2 overexpression and how changes in gene regulation alter in urinary bladder carcinogenesis has not been defined. I first examined the effects of COX-2 inhibition on prostate tumorigenesis in the transgenic adenocarcinoma mouse prostate (TRAMP) model by either feeding TRAMP mice a diet containing non-steroidal anti-inflammatory drugs (NSAIDs) or by genetic disruption of the expression of the COX-2 gene in TRAMP mice. Our results demonstrate that neither NSAIDs nor genetic disruption of COX-2 inhibited tumorigenesis of TRAMP prostate. Interestingly, I found that the expression of COX-1 and COX-2 mRNA, protein, and activity (ability to synthesize PGE2) dramatically decreased during TRAMP prostate carcinogenesis. I also used a gene expression array analysis to determine the effects of COX-2 overexpression on gene expression profile in urinary bladder of a COX-2 overexpression transgenic mouse model (BK5.COX-2). My results revealed that genes associated with Immune/Stress Response and Cell Cycle/Proliferation systems were significantly overexpressed in the BK5.COX-2 mouse bladders. Upregulated Cell Cycle/Proliferation genes included growth factors (Ereg and IGF-1) and cell cycle genes (Mki67, Cdc2a, and Top2a). Relevant downregulated genes included three transforming growth factor beta related genes (Tgfb2, Tgfb3, Tgfbi), and the anti-angiogenic gene thrombospondin 2 (Thbs2). The growth factor epiregulin was the most highly induced gene among those validated by qRT-PCR. I further demonstrated that epiregulin mRNA was directly induced by PGE2 treatment in the wild type mouse bladders. In addition, epiregulin increased cell proliferation and activated MAPK/Erk activity in bladder cancer cells. In summary, my results suggest that COX-2 may not play a significant role during prostate carcinogenesis in the prostate TRAMP model. However, overexpression of COX-2 may play a pivotal role in murine bladder carcinogenesis. Advisors/Committee Members: Clinton, Steve K.

▼ Conjugated linoleic acid (CLA), a group of dietary fatty acids, decreases body weight primarily through the reduction of adipose mass and is reported to modulate insulin resistance. The mechanisms by which CLA depletes adipose and the roles of adipokines in CLA-mediated insulin resistance are not completely understood. The first objective of this research was to determine the effects of CLA on lipid metabolism and fatty acid composition in the livers of Zucker diabetic fatty (fa/fa; ZDF) rats compared to a thiazolidinedione (TZD). CLA reduced adipose mass while improving glucose tolerance and hepatic steatosis in ZDF rats. The effects of CLA and TZD on hepatic lipid composition suggest that the effects of these two agents on glucose tolerance may be associated with a reduction in stearoyl-CoA desaturase-1. In mice, CLA rapidly reduces adipose mass and worsens insulin resistance and hepatic steatosis, effects which are preceded by the significant depletion of the adipokines leptin and adiponectin. Therefore, the second objective was to determine whether effects of CLA on insulin resistance, hepatic steatosis, and inflammation depend on the depletion of leptin by CLA. Recombinant leptin was co-administered with dietary CLA in ob/ob mice to, in effect, negate the leptin depletion effect of CLA. In both the absence and presence of leptin, CLA significantly reduced adipose mass and depleted adiponectin. In the absence of leptin, CLA worsened insulin resistance without evidence of inflammation in adipose tissue or hepatic steatosis. In the presence of leptin, CLA failed to worsen insulin resistance, but induced hyperinsulinemia and hepatic steatosis in ob/ob mice. The significant depletion of adipose in mice by CLA may contribute to the lipodystrophic-like effects that accompany. The final objective of this research was to determine mechanisms by which CLA reduces adipose mass. The depletion of adipose tissue in ob/ob mice by CLA was accompanied by the acquirement of brown adipose-like characteristics, such as increased CPT-1b, PGC-1α, and UCP-1, in the white adipose of CLA-fed mice. This alteration may facilitate the reduction of adipose mass by increasing mitochondrial oxidation and energy dissipation. However, it appears that CLA does not increase UCP-1 through β3AR signaling. Advisors/Committee Members: Belury, Martha A.