University of Nevada, Reno Metabolic Effects of a Grape Seed Procyanidin Extract and its Relation to Bile Acid Homeostasis A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cell and Molecular Biology By Rebecca M. Heidker Dr. Marie-Louise Ricketts/Dissertation Advisor May, 2016 Copyright by Rebecca M. Heidker 2016 All rights reserved THE GRADUATE SCHOOL We recommend that the dissertation prepared under our supervision by REBECCA HEIDKER Entitled Metabolic Effects Of Grape Seed Procyanidin Extract On Risk Factors Of Cardiovascular Disease be accepted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Marie-Louise Ricketts, Advisor Patricia Berinsone, Committee Member Patricia Ellison, Committee Member Cynthia Mastick, Committee Member Thomas Kidd, Graduate School Representative David W. Zeh, Ph. D., Dean, Graduate School May, 2016 i Abstract Bile acid (BA) recirculation and synthesis are tightly regulated via communication along the gut-liver axis and assist in the regulation of triglyceride (TG) and cholesterol homeostasis. Serum TGs and cholesterol are considered to be treatable risk factors for cardiovascular disease, which is the leading cause of death both globally and in the United States. While pharmaceuticals are common treatment strategies, nearly one-third of the population use complementary and alternative (CAM) therapy alone or in conjunction with medications, consequently it is important that we understand the mechanisms by which CAM treatments function at the molecular level. It was previously demonstrated that one such CAM therapy, namely a grape seed procyanidin extract (GSPE), reduces serum TGs via the farnesoid X receptor (Fxr). GSPE treatment also induces the expression of hepatic cholesterol 7α-hydroxylase (Cyp7a1), the rate limiting enzyme for de novo BA synthesis. Herein, we demonstrate that both gene and protein expression of Cyp7a1 is increased due to the fact that GSPE selectively regulates intestinal Fxr target genes involved in BA uptake and transport. Apical sodium dependent bile acid transporter (Asbt) expression is decreased with a concomitant reduction in fibroblast growth factor 15 (Fgf15), leading to a lack of repression on hepatic Cyp7a1. The subsequent 47% decrease in serum BAs and 69% increase in fecal BA excretion results in a significant reduction in serum TG and cholesterol. These Fxr dependent effects are lost in Fxr-/- mice, clearly demonstrating the critical role of this nuclear receptor. In a subsequent study we confirm that GSPE represses Asbt expression, while the BA sequestrant cholestyramine (CHY) induces expression. Treatment with either GSPE or CHY increases expression of Cyp7a1, with co-administration augmenting the increase. In the liver, GSPE and CHY independently induce expression of genes regulating cholesterol and lipid synthesis; however, when ii combined the expression of cholesterogenic and lipogenic genes induced by CHY is attenuated. Taken together these data indicate that GSPE has the potential for use either alone or as a complementary therapy in the treatment of hypertriglyceridemia and hypercholesterolemia. These findings, combined with the ability of low molecular weight procyanidins (LMW-PCNs) to modify intracellular proteins and signaling pathways led us to optimize a protocol for isolating LMW-PCNs from the seeds of grapes grown at the University of Nevada, Reno (UNR) vineyard. An ethyl acetate based extraction process utilizing whole seeds was found to be both time and cost effective, while preserving the anti-oxidant properties of the procyanidin-rich extract. This protocol will provide the basis for further extractions in order to conduct in vitro and in vivo testing, potentially allowing for the development of a value added product from the UNR vineyards. iii Dedication To my son, Andrew, for providing me with the inspiration to start this journey. To my family and friends who believed in me and supported me throughout this process. iv Acknowledgement I would like to thank my advisor and mentor, Marie-Louise Ricketts for fostering independence in her students. She has encouraged me to think critically and see my projects through to completion. She has also reminded me on multiple occasions that graduate school is always an up and down journey, and she has stayed with me throughout those ups and downs. I want to thank my labmates for their assistance, friendship and support. I’m lucky to have met and gotten to know each of you, and I’ve learned so much from being around you. I would also like to thank my committee members, Patricia Bernisone, Thomas Kidd, Patricia Ellison, and Cynthia Mastick. They have provided me with excellent guidance and suggestions along the way. I appreciate their support more than words can say. In addition, I need to thank my family for their support. My father, James Heidker, has cooked countless meals for me and my son when I’ve been too busy to keep up. He has always had faith in my ability to complete this process, and has answered more chemistry questions than I count. My mother, Jean Heidker, answered questions about basic assays and the science behind them while I got my feet under my as a research scientist. And last, but not least, my sister, Moira Kolada, taught me the ropes of graduate school at UNR. v List of Tables………………………………………………………………………….…….…..ix List of Figures…………………………………………………………………………...….…..ix Chapter 1: Introduction…………………………………………………………………....…1 1.1. Grape seed procyanidin extract and health .............................................................. 2 1.1.1. Human Health Relevance ................................................................................. 2 1.1.2. Procyanidins ..................................................................................................... 2 1.1.3. Grape seed procyanidin extract ......................................................................... 4 1.1.4. Bioavailability .................................................................................................... 5 1.1.5. GSPE alleviates cardiovascular disease risk factors ......................................... 6 1.1.5.1. The impact of procyandins on atherosclerosis ............................................ 7 1.1.5.2. Effects of grape seed extracts on lipogenesis and diabetes mellitus ........... 8 1.1.5.3. Regulation of dyslipidemia .........................................................................10 1.2. Grape seed procyanidin extract and nuclear receptors ...........................................11 1.2.1 Nuclear receptors ..............................................................................................12 1.2.2. Structure of nuclear receptors ..........................................................................14 1.2.3. Classification of nuclear receptors ....................................................................16 1.2.3.1. Class I nuclear receptors ...........................................................................17 1.2.4. Class II nuclear receptors .................................................................................18 1.2.4.1. Farnesoid X receptor .................................................................................19 1.2.5. Class III nuclear receptors ................................................................................21 1.2.5.1. Small heterodimer partner .........................................................................21 1.3. Bile acid synthesis ..................................................................................................24 1.3.1. Classical bile acid synthesis .............................................................................28 vi 1.3.2. Alternative bile acid synthesis ..........................................................................29 1.4. Enterohepatic recirculation .....................................................................................30 1.4.1. Bile acid transport in the small intestine ...........................................................32 1.4.2. Hepatic bile acid transport ................................................................................33 1.5. Cholesterol and triglyceride synthesis and transport ...............................................34 1.5.1. Cholesterol synthesis .......................................................................................34 1.5.2. Hepatic export of cholesterol and triglycerides .................................................36 1.5.3. Intestinal transport of triglycerides and cholesterol ...........................................37 1.5.4. Excretion of cholesterol ....................................................................................40 1.6. Summary ................................................................................................................40 Chapter 2: Dietary Procyanidins Selectively Modulate Intestinal Farnesoid X Receptor-regulated Gene Expression to Alter Enterohepatic Bile Acid Recirculation: Elucidation of a Novel Mechanism to Reduce Triglyceridemia…....45 2.1. Abstract ..................................................................................................................47 2.2. Introduction .............................................................................................................49 2.3. Materials and Methods ...........................................................................................51 2.4. Results