EFFECTS OF PERFLUORINATED COMPOUNDS (PFCs) ON METABOLIC TISSUES AND THE BENEFITS OF CALORIC RESTRICTION

The CDC states that there has been a dramatic increase in obesity from 1990 to 2010. Type-II diabetes and obesity prevalence are increasing worldwide. Often, obesity and Type-II diabetes are concurrent, and predispose individuals to development of fatty liver disease, referred to as Non-alcoholic fatty liver disease (NAFLD). Perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) are two commonly studied perfluorinated compounds (PFC’s) that are considered environmental toxicants that have the potential to elicit diabetic and NAFLD phenotypes. This dissertation presents novel findings of gaps within the literature to date. Traditionally, diabetes and obesity have been discussed in regard with genetics, diet and old age. Now, risk factors that also need to be considered are environmental chemicals. We found that PFOS elicits an insulin-resistant phenotype in adult mice, where they were not utilizing glucose as readily compared to the controls. The effect of PFOS on therapeutic management interventions has not previously been looked at. Here, we show that PFOS interferes with the Metformin-induced decrease in glucose. We also found a vast increase in the hepatic triglycerides with PFOS exposure. In this thesis, PFOS was administered in a sub-chronic low dose (100μg/kg) daily to mice fed ad libitum or placed on caloric restriction (CR) for five weeks. In the cohort we generated, we observed that PFOS exposure increased hepatic lipid content in mature male mice fed ad libitum and dampened the observed CR-induced decrease in hepatic lipids. PFOS administration did not affect glucose tolerance in ad libitum fed mice, but did interfere with CR-induced improvement of glucose tolerance. This was further associated with suppression of IRS-1 mRNA expression in liver. As hepatic lipid content is closely tied to insulin and glucagon signaling for hepatic glucose production, it was determined whether the observed effects in vivo were due to PFOSstimulated hepatic glucose production. Using cultured mouse hepatocytes under low glucose conditions, it was evaluated whether PFOS could enhance glucagonstimulated glucose production. PFOS stimulated hepatocyte glucose production and also enhanced glucagon-induced glucose production. Furthermore, in HEPG2 cells, PFOS exposure (25 and 250 μM) significantly increased glucose output and AiCar, which suppresses glucose production, was ineffective in the presence of PFOS. These findings provide a mechanistic explanation for the decreased glucose tolerance in our in vivo mouse cohort. PFOS increases glucose output from the in vitro models, even when challenged with metformin or AiCar, as well as, decreases the glucose utilization in the in vivo mouse study suggesting that it has a glucagon-like effect. PFOA is a perfluorinated carboxylic acid also commonly found in the environment. According to the EPA, low levels of PFOA are widely distributed in environmental media (Houde et al., 2011; Gewertz et al., 2013) and in the blood of the general United States population. The EPA states that PFOA is a known liver toxicant, development toxicant, and carcinogen in rodents. It has been detected in human liver, kidney and adipose tissue ranging from 0.3 to 3.8ng/g with the highest concentrations within the liver (Maestri et al., 2006). In another study, PFOA is considered an obesogen to mid-aged mice where insulin and leptin levels were altered at a very low concentration (Hines et al., 2009). PFOA is a Potent activator of peroxisomeproliferator activated receptor-alpha (Ppar-α) contributing to oxidative stress and activation of fatty acid oxidation pathways in hepatocytes. Given its persistence, the purpose of this study was to evaluate whether PFOA treatment affects fatty acid oxidation, lipid synthesis, and antioxidant response gene expression in adipose tissue in adult male mice. Utilizing an exposure paradigm characterized by the Environmental Protection Agency, adult male mice were treated with 1.0 or 3.0 mg PFOA ammonium salt/kg for 7 days. Adipose tissue was collected and total RNA was isolated. Analysis of mRNA was completed by quantitative PCR. For the most part, gene expression in adipose tissue from vehicleand PFOA-treated mice was similar. Literature lacks data on PFOA in adipose tissue and in human health, which continue to be discovered. Given its persistence, longer exposure periods and protein expression changes should be examined.

v ACKNOWLEDGMENTS I would like to thank my major professor, Dr. Angela L. Slitt, for giving me this opportunity to perform such meritorious research. Dr. Slitt has always helped to organize my thoughts and formulate cohesive presentations and papers that flowed smoothly. She has willingly written many letters of recommendations for me throughout the years and supported me through my research here at the University of Rhode Island. She has mentored me through this five-year journey and taught me how to be the best scientist I can be and for that, I am thankful.
I would like to thank my committee member, Dr. Ruitang Deng, for his input throughout each seminar and committee meeting, always helping me think outside the box into deeper research.
I would like to thank my committee member, Dr. Matthew Delmonico, for continually asking questions throughout committee meetings that truly engaged my mind to think about and how our work relates to human research. I would also like to thank him for meeting with me, boosting confidence in myself before major presentations. He is always willing to help in any way that he can and is always readily available to meet, and for that, I am extremely appreciative.
I would like to thank my committee member, Dr. Bongsup Cho, for his encouraging words throughout the five years of being in this program. I always loved speaking the little Korean that I knew to Dr. Cho and him responding so kindly to it.
He always knew how to uplift and motivate me to be better and work hard each and everyday.
vi I would like to thank my committee member and also chair, Dr. Brenton DeBoef, for comedic relief in each committee meeting by asking me to speak to the audience as if I am speaking to a fourth grader about my research. It is always stressful being in front of a group of very intelligent PhD's in various fields, but somehow he always found a way to alleviate that stress.
I would like to thank the Biomedical and Pharmaceutical Sciences Chair, Dr.
Bingfang Yan, for the motivational morning walks and talks. It was always a pleasure seeing someone else in the building in the early mornings from day to day. He would come in a talk to me about anything from research to personal life, and for that, I will always be thankful. He always believed in me and it came through when he would bring prospective professors or visiting speakers through our lab and always referred to me as one of the best students at URI when passing through. He had a peculiar way of motivating me by telling me to work harder and comparing my work ethic with his own and showing me his dissertation, telling me that I am not working hard enough, jokingly of course. From day one, he is one person that has influenced me and propelled me to move forward from the classroom to research, always leaving a positive vibe.
I would like to thank the Chemistry Department Chair, Dr. William Euler, who has provided opportunities to receive a teacher assistantship with the Chemistry Department in teaching General Chemistry and Organic Chemistry labs. He will never know how truly thankful I am for these opportunities, for I would not be able to be here without the support of the Chemistry Department.
vii I would like to thank my undergraduate advisor, Dr. Steven B. Symington, who has been a huge supporter of me since 2008. From his hard exams to writing grants in his Neuroscience class, he always pushed me to be the best student and scientist I could be. He was the first person who gave me a chance to perform research in the undergraduate research program of Rhode Island IDeA Network for Excellence in Biomedical Research (INBRE). The one email that changed my life in the best possible way was when I was assigned to him to perform research in his laboratory. It was also one of the happiest days of my life. Dr. Symington is a man who is extremely easygoing when it comes to golf and non-academic activities, however, when it came to class work and research, he was one of the toughest professors I can remember and I am extremely thankful for those few times he made me cry during presentations. He always made students stronger with his comments and his fantastic ways of teaching. He made sure I was fully prepared for graduate school and took me from being a terrified and nervous public speaker to a confident and well-rounded young scientist. Dr. Symington and I have had extremely inspiring discussions and he has always been, and will always be, a truly great mentor. Dr. Symington and the Salve Regina University Biology and Chemistry labs were truly the reason my love for research grew so rapidly. I thank you, Dr. Symington, from the terrifying Summer putting up with all the science talk that I brought you to for SOT in Phoenix, Arizona and to Dr. Symington's backyard pumpkin carving party! You have taught me so much that I will forever be thankful for and continue to be the best mother a daughter could ask for with the most positive attitude given any situation. You are the most caring person and the most phenomenal Registered Nurse the world has seen, thank you. Dad, you are and will forever be the smartest man I have ever known. I never take our talks for granted and always remember what you say. Your input to anything I do means so much to me and your words will forever positively impact my life.
From performing surgery and being the best Veterinarian for years in the greater Boston area to cooking the best meals and growing the most amazing roses, there is nothing you can't conquer. I have learned so much from watching you as I've grown up and continue to learn from you everyday. As I've grown up, you and mom have taught me that nothing is impossible. You two have overcome some of the hardest Sure enough, at 80-years of age, it's what she wanted to do and we all got tattoos! It was an experience I will never forget during a very hard time in my studies. Her and my mother even came to a seminar that I was giving for school. Thank you, Grandmama, for everything.
I would like to thank my three older brothers, Adam Lawrence Salter, Andrew Mitchell Salter and Daniel Justin Salter. They have all taught me life lessons that I still live by to this day. Adam, I know it has not been the easiest track for you, but watching you grow through this part of life that has been so challenging for you, is one of the most inspiring stories I will forever hold to my heart. Your determination and persistent willpower has been a huge factor in my graduate career. About eight years ago, I found out about your disease, which has overtaken your life and has inhibited you from many of life's natural activities such as college or a career. You always had high hopes of becoming a very wealthy person and have always been extremely smart x and I truly believe you will be hugely successful in the future. You are doing so well in your recovery and the past eight years, even though they have been undoubtedly difficult for all of us, you have continued to show us that you are not willing to give up. Your persistence to excel in life is nothing short of amazing. You have been an inspiration and continue to amaze me everyday; I love you and don't give up.
Andrew, ever since I could walk, I always tried to keep up with you. Your athletic ability (which was and still is astounding!) and intelligence were things I longed for early in our childhood. As far back as I can remember you were the person I always aspired to be like and to this day, show me how great of a person you are. You  would always make me take the time to sit down and eat whatever she made for the day to ensure I was getting proper nutrients. She was always looking out for me and helping through daily obstacles. We have had the best and worst of times when it came to funding and school-related situations, and I am so grateful to have had her in the same building to talk to through these years. We have conquered so much together and I hope you the very best in finishing your graduate career.
Lastly, I would like to thank Ian Lester for putting up with all of the technical difficulties from the old to the new building. He has been to the rescue in many presentation and seminar talks from students to faculty. He does the entire behind the scenes work that needs to be recognized because we wouldn't be able to operate properly without computers when they get viruses or the help of IT when things go wrong. I cannot thank him enough for lending me chargers and adapters when I needed them at the last minute, not to mention the amount of keycards he has granted me after misplacing them. I am extremely appreciative of him printing our posters for the 2015 SOT conference. Ian, thank you very much for all that you have helped with along the way to make this journey a successful one.
xiii DEDICATION To my parents: my stability, strength, and support. It is to them that I dedicate the following dissertation, for their endless support, motivation and encouragement.

Marcia Wood Salter
Wilbur Mitchell Salter II xiv

PREFACE
The following thesis project is in Manuscript Format. Based on our previous lab findings, the initial goals of this thesis project were to evaluate different models for changes and regulation of Nrf2, a transcription factor. However, after interesting observations with perfluorinated compounds (PFC's), my thesis evolved into a more focused and mechanistic study on Perfluorooctanesulfonic acid (PFOS), a major PFC.
The bulk of the thesis work has been to understand whether PFOS exposure can interfere with the benefits of caloric restriction in vivo or in vitro. Epidemiology studies associating PFOS exposure with altered metabolics has further provided justification for studying PFOS effects.
Because the thesis is manuscript format, there have been experiments not included in the manuscript, but still essential to key observations of the thesis work.
These have been included. Appendix material will also present findings for PFOS and PFOA with regard to adipose tissue and gene expression, along with work performed regarding the Nrf2 activating compounds, oleanolic acid (OA) and butylated hydroxyanisole (BHA), on interaction with a lithogenic diet that I generated early in my graduate career. xv

Non-alcoholic fatty liver disease (NAFLD) statistics and etiology
NAFLD is a common chronic liver disease that is increasingly growing within the   AMPK activation is also decreased resulting in Srebp1c activation and lipid synthesis via Acc1 and Fas within the liver eliciting a fatty liver (Kohjima et al., 2008) ( Figure   C1-2).

Therapeutic strategies for NAFLD Treatment
The recommended therapeutic intervention to treat NAFLD is with diet and exercise, which have the ability to reverse hepatic lipid accumulation (Chalasani et al., 2012).

Molecular Mechanisms by which CR decreases Liver Fat
The response to CR hinges upon a cellular metabolic shift in which AMPK is a central mediator. CR induces phosphorylation of AMP-activated protein kinase (AMPK) upon redox status (high AMP:ATP ratio) ( Figure C1-3). AMPK is a regulator of hepatic metabolism in energy balance by promoting catabolic pathways and inhibiting ATP-consuming pathways and is a good target for the treatment of T2D (Viollet et al., 2006). AMPK activation is implicated for the benefits of glucose and lipid metabolism with exercise, weight loss, and use of anti-diabetes drugs (Towler et al., 2007).   Levels of PFOA and PFOS were detected in non-occupationally exposed general population humans of liver, kidney, adipose tissue, brain, basal ganglia, hypophysis, thyroid, gonads, pancreas, lung, skeletal muscle and blood and found PFOA ranging from 0.3 to 3.8ng/g and PFOS ranging from 1.0 to 13.6ng/g with the highest concentrations within the liver (Maestri et al., 2006). The overall goal of this project was to determine whether PFOS exposure interferes with recommended therapies to decrease hepatic lipid content that are used to treat NAFLD. It was hypothesized that PFOS administration could interfere with the benefits of CR and metformin potentially through targeting AMPK phosphorylation. Overall, we present novel findings illustrating that PFOS administration concurrent with a modest reduction in caloric intake thwarted CR-induced decline in hepatic lipids and improvement in glucose tolerance and interfered with metformin-induced glucose lowering effects in vitro.

Results
Effect of CR and PFOS on body weight, liver weight, and serum chemistry. 21-week old male C57BL/6 mice were fed ad libitum or 25% calorically restricted for 5 weeks. In each group, mice were administered vehicle (VEH) or 100 µg/kg PFOS daily. In Figures 1a and b

PFOS interfered with CR-induced improvement of glucose utilization.
CR improves glucose tolerance and increases insulin sensitivity (Colman et al., 2009;Fontana, 2009). The effect of PFOS on this CR-induced benefit was evaluated. CR decreased glucose levels over time (Figures 2a and b). Overall, CR decreased glucose load by 57.9% compared to AL fed mice (Figure 2c).
In AL fed mice, PFOS administration did not significantly affect response to glucose challenge, although the glucose load tended to increase, but did not reach statistical significance (p=0.8938) (Figures 2a-c). Interestingly, PFOS administration did affect the response to glucose challenge in mice that were placed on CR, with glucose clearance being decreased in CR mice administered PFOS (Figure 2a and b). Compared to CR mice administered VEH, glucose load was 74.3% higher in CR mice administered PFOS ( Figure   2c). In CR and AL fed mice, PFOS significantly increased the glucose load Protein levels in all groups were compared (Figure 4b).
Cluster of differentiation (CD36), also known as fatty acid translocase (FAT), and fatty acid synthase (Fas) both increased significantly upon PFOS exposure with CR fed mice by over 2-fold and 2.5-fold, respectively, compared to CR control ( Figure 4d). Astoundingly, P-ampk significantly decreased over 7-fold in mice that underwent CR with PFOS administration compared to CR control ( Figure   4d).

PFOS significantly increased glucose within the media of Primary
Hepatocytes and HepG2 cells.
Based on our GTT observations, and changes in P-AMPK in liver, the effect of PFOS on glucose production in hepatocytes was measured. Primary          Eighteen-week old C57Bl/6 mice underwent treatment for approximately six weeks of either water (VEH) or Perfluorooctanesulfonic acid (PFOS) administration (0.1mg/kg/day) and fed either ad libitum (AL) or underwent 25% kCal caloric restriction (CR). After euthanization, these parameters were assayed and analyzed. *, p<0.05, CR-VEH compared to AL-VEH control. $, p<0.05, AL-PFOS compared to AL-VEH control. #, p<0.05, CR-PFOS compared to AL-VEH control. §, p<0.05, CR-PFOS compared to CR-VEH control. Peroxisome proliferator-activated receptor alpha (PPAR-alpha) is found predominantly in the liver and is involved in lipid and lipoprotein metabolism reducing triglyceride levels as well as maintaining energy homeostasis regulating obesity (Tyagi, et al. 2011). PPAR-gamma targets LPL and is involved in lipid metabolism and lipid uptake into the adipocytes and is involved in adipocyte differentiation and hypertrophy (Tyagi, et al. 2011).
Ppar-y is highly expressed within adipose tissue and may be an unrecognized In this study, adult male mice were treated with 1.0 mg PFOA/kg or 3.0 mg PFOA/kg in corn oil for 7 days. We hypothesized that PFOA would alter gene expression in adipose tissue of mice in regard to fatty acid oxidation, lipid synthesis and antioxidant response.

Animals and treatment paradigm
Animal treatment and PFOA dosing we previously used to investigate Louis, MO) was administered once per day at a dose volume of 5ml/kg for seven days. For 1 mg/kg dose, 2 mg PFOA was dissolved in 1 ml deionized (DI) water. This stock was vortexed and 9 ml of DI water was added to make a 2mg/10 ml solution that was filter sterilized. For 3mg/kg dose, 6 mg PFOA was dissolved in 1 ml DI water and vortexed. Then 9 ml DI water was added to make a 6mg/10 ml solution, which was filter sterilized. Organs and tissues were collected and snap frozen and stored in -80 until RNA analysis.

PCR Assay
Total RNA was isolated from the collected adipose tissue by phenol-

Statistical Analysis
Statistical analyses of differences were performed by Student's t test. P < 0.05 was considered statistically significant. Unless otherwise stated, all data were presented as mean ± SE of five animals.

RESULTS
PFOA treatment did not affect body weight, however, did increase liver weight.
PFOA has been suggested to induce body weight gain in mice, however, in our study, the mice did not have and increased average body weight compared to the vehicle in either 1.0 mg/kg or 3.0 mg/kg PFOA which is illustrated in Figure 1. It is suggested that mice with PFOA administration have increased liver weights due to an accumulation of hepatic triglycerides.
In our study, there was a statistical increase that is dose-dependent. The white adipose tissue weight was unchanged between groups. Kidney tissue weights were increased with a treatment of 1.0 mg/kg PFOA compared to vehicle, but not in the 3.0 mg/kg PFOA treatment.

PFOA treatment did not affect lipid synthesis and accumulation gene expression in white adipose tissue.
Srebp1c is the regulator of lipid synthesis and acts on its target genes, Scd1 and Acc1. As shown in Figure 2

PFOA treatment did not affect oxidative stress gene expression in adipose tissue
Oxidative stress mRNA expression is illustrated in Figure 3. Also, it is suggested that prenatal PFOA exposure is associated with the increased risk of obesity and metabolic hormone differences in 20-year old women (Post, et al. 2012).
The increase in body weight of mice in a PFOA-dependent manner could be due to increases of triglycerides, phospholipids, and cholesterol accumulation in the liver, which induced fatty liver in mice caused by PFOA administration (Post, et al. 2012).
Other studies report findings primarily based on liver tissue. Steenland et al suggest there is a significant, positive correlation of PFOA and cholesterol levels (Steenland, et al. 2010). Although the mode of action is not well understood, PFOA's action is suggested to be part PPAR-alphadependent and part PPAR-alpha-independent. PPAR-alpha independent is thought to be because the branched isomers of PFOA increased liver weight but was less effective in activating PPAR-alpha (Post, et al. 2012 (Post, et al. 2012). Literature is abundant with effects of PFOA in the liver but lacks data in adipose tissue. This preliminary study is one of the first to explore the mechanism of PFOA in adipose tissue that may be attributable to its toxic effects.   OA administration significantly increased expression of nrf2 and its target genes in wild-type mice including nqo-1, gclc, and ho-1, however, not in  OA significantly decreased total cholesterol when given a HFD and OA compared to the HFD alone (de Melo, 2010).

Study Hypothesis
The purpose of this study was to demonstrate that an nrf2 activator, oleanolic acid, decreased hepatic cholesterol synthesis.

The effect of oleanolic acid on serum and hepatic cholesterol levels in
mice. OA has been used in Chinese medicine for the treatment of liver disorders protecting against oxidative and electrophilic stress and activates the nuclear factor erythroid 2-related factor 2/ kelch-like ECH-associated protein 1 pathway inducing expression of cytoprotectant genes (Resiman, 2010).

The effect of oleanolic acid feeding on lithogenic diet-induced cholesterol uptake, synthesis, and transport gene expression in liver.
Abcg5/abcg8 is a heterodimer responsible for cholesterol excretion from liver to the bile. Abca1 resides in the basolateral membrane transporting cholesterol back into the blood to lipid-poor lipoproteins. Ldlr is responsible for the uptake of cholesterol into the liver and hmg-coar is the rate limiting enzyme in which cholesterol is synthesized. The lithogenic diet alone increased abcg5/abcg8 mRNA expression 4-fold. OA administration with the lithogenic diet increased abcg5/abcg8 mRNA expression 3.5-fold. Abca1 mRNA expression was increased 2.5-fold on the lithogenic diet alone and 2fold with OA on the lithogenic diet. Ldlr mRNA expression was slightly decreased on the lithogenic diet and more decreased with OA administration on the lithogenic diet. Hmg-coaR was decreased 3-fold and 5-fold on the lithogenic diet and lithogenic diet with OA respectively.

The effect of oleanolic acid feeding on lithogenic diet-induced bile acid uptake, synthesis, and transport gene expression in liver.
Cyp7a1 is the rate-limiting enzyme in which cholesterol is converted to bile acids. Bsep is the bile salt export pumped from the liver into the bile. The lithogenic diet alone increased bsep mRNA expression slightly, and even more in the lithogenic diet with OA administration.

The effect of oleanolic acid feeding on Nrf2 and Nrf2-target gene expression in liver.
OA administration significantly increased expression of nrf2 and its target genes in wild-type mice including nqo-1, gclc, and ho-1, however, not in nrf2-null mice (Reisman, 2010).

Conclusion
OA and BHA are two compounds that are considered to be Nrf2-activators. In In this study, adult male C57BL/6 mice were pair-wise fed ad libitum i) standard diet (CONT), ii) lithogenic diet (LD, 15% fat, 1.25% CH, 0.5% sodium cholate), iii) standard diet with oleanolic acid (0.1% w/w), iv) LD with oleanolic acid (OA, 0.1% w/w) for six weeks. BHA reduced body weight, however increased the liver weights of mice. BHA treatment doubled serum cholesterol levels in mice fed the lithogenic diet compared to mice that received lithogenic diet alone. Mice treated with OA in their diets have similar serum cholesterol levels to untreated mice. However, the overall finding of this study was that Oleanolic acid decreased hepatic cholesterol content.