The Impact of Bioactive Phytosterol, Stigmasterol, on Cholesterol Elimination Pathways in Mice

University of Kentucky UKnowledge

Theses and Dissertations--Pharmacy College of Pharmacy

2018

THE IMPACT OF BIOACTIVE PHYTOSTEROL, STIGMASTEROL, ON CHOLESTEROL ELIMINATION PATHWAYS IN MICE

Hannah C. Lifsey University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/ETD.2018.189

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Recommended Citation Lifsey, Hannah C., "THE IMPACT OF BIOACTIVE PHYTOSTEROL, STIGMASTEROL, ON CHOLESTEROL ELIMINATION PATHWAYS IN MICE" (2018). Theses and Dissertations--Pharmacy. 88. https://uknowledge.uky.edu/pharmacy_etds/88

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The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above.

Hannah C. Lifsey, Student

Dr. Gregory A. Graf, Major Professor

Dr. David Feola, Director of Graduate Studies THE IMPACT OF BIOACTIVE PHYTOSTEROL, STIGMASTEROL, ON CHOLESTEROL ELIMINATION PATHWAYS IN MICE

THESIS

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the College of Pharmacy at the University of Kentucky

Hannah Carlene Lifsey

Lexington, Kentucky

Director: Dr. Gregory A. Graf, Professor of Pharmaceutical Sciences

Lexington, Kentucky

2018

ABSTRACT OF THESIS

THE IMPACT OF BIOACTIVE PHYTOSTEROL, STIGMASTEROL, ON CHOLESTEROL ELIMINATION PATHWAYS IN MICE

Despite advances in healthcare, cardiovascular disease (CVD) remains the leading cause of death in the United States. Elevated levels of plasma cholesterol are highly predictive of CVD and stroke and are the principal driver of atherosclerosis. Unfortunately, current cholesterol lowering agents, such as statins, are not known to reverse atherosclerotic disease once it has been established. In preclinical models, agonists of nuclear receptor, LXR, have been shown to reduce and reverse atherosclerosis. Phytosterols are bioactive non-cholesterol sterols that act as LXR agonists and regulate cholesterol metabolism and transport. We hypothesize that stigmasterol would act as an LXR agonist and alter intestinal cholesterol secretion to promote cholesterol elimination. Mice were fed a control diet, or a diet supplemented with stigmasterol (0.3% w/w) or T0901317 (0.015% w/w), a known LXR agonist. In this experiment we analyzed the sterol content of bile, intestinal perfusate, plasma, and feces. Additionally, the liver and small intestine were analyzed for relative levels of transcripts known to be regulated by LXR. We observed that T0901317 robustly promoted cholesterol elimination and acted as a strong LXR agonist. Stigmasterol also promoted cholesterol elimination but did not alter LXR-dependent gene expression. Stigmasterol promoted transintestinal cholesterol secretion through an LXR-independent pathway.

KEYWORDS: Cardiovascular Disease, Transintestinal Cholesterol Excretion, Phytosterol, Stigmasterol, Liver X Receptor, T0901317

Hannah Carlene Lifsey

April 27, 2018

THE IMPACT OF BIOACTIVE PHYTOSTEROL, STIGMASTEROL, ON CHOLESTEROL ELIMINATION PATHWAYS IN MICE

Hannah Carlene Lifsey

Dr. Gregory A. Graf

Director of Thesis

Dr. David Feola

Director of Graduate Studies

April 27, 2018

ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. Gregory A. Graf for his mentorship, guidance, and understanding throughout my graduate studies. Additionally, I would like to acknowledge my lab group members Lisa Bennett, Bradley Thompson, Rupinder Kaur, and Rui Liu for their support, assistance, and friendship. Bradly Thompson and Lisa Bennett were invaluable in their expertise in executing the mouse surgeries and tissue collections, and I am thankful for their help, time, and expertise. I would like to thank Sierra Schlicht for her help with the GC-FID analysis of my samples, and I appreciate Rupinder Kaur’s work in determining the dose response data for T0901317 and stigmasterol. I would also like to thank Dr. Jianing Li for the development of the simultaneous biliary and intestinal cholesterol secretion surgery protocol. I am very appreciative of the assistance and perspective I was given by committee members Dr. Gregory A. Graf, Dr. Esther Black, and Dr. Tracy Macaulay. Furthermore, I am grateful to the University of Kentucky College of Pharmacy and Department of Pharmaceutical Sciences and all of the incredible people I met during my time here.

Finally, I would like to thank my friends and family for their love and support over the course of my graduate and professional studies.

iii TABLE OF CONTENTS

Acknowledgements ...... iii

List of Figures ...... v

Chapter One: Introduction 1.1 Cardiovascular Disease Treatment ...... 1 1.2. Atherosclerosis Pathophysiology ...... 2 1.3. Reverse Cholesterol Transport ...... 5 1.4. Transintestinal Cholesterol Excretion ...... 7 1.5 Liver X Receptor ...... 10 1.6 Stigmasterol ...... 13

Chapter Two: Materials and Methods 2.1. Animals and Treatments ...... 14 2.2. Tissue and Plasma Collection ...... 14 2.3. Surgical Assessment of Biliary and Intestinal Cholesterol Secretion ...... 15 2.4. Intestinal Perfusate Preparation ...... 16 2.5. Lipid Analysis ...... 18 2.6. Phospholipid Assay ...... 19 2.7. Bile Acid Assay ...... 19 2.8. Fecal Bile Acid Assay ...... 20 2.9. Biliary Bile Acid Assay ...... 20 2.10. Dose Response Assay ...... 20 2.11. RT PCR ...... 21 2.12. Statistical Analysis ...... 22

Chapter Three: Results 3.1. Stigmasterol does not affect plasma cholesterol levels or fecal cholesterol loss ..... 23 3.2. Stigmasterol does not have an effect on basal bile flow or composition ...... 25 3.3. Stigmasterol promotes biliary and intestinal cholesterol secretion ...... 27 3.4. Stigmasterol did not increase gene expression of LXR targets ...... 29 3.5. Stigmasterol is a weak LXR agonist compared to T0901317 ...... 32

Chapter Four: Discussion ...... 34

References ...... 39

Vita ...... 42

iv LIST OF FIGURES

Figure 1.1. Atherosclerosis Progression ...... 4 Figure 1.2. Reverse Cholesterol Transport ...... 6 Figure 1.3. Cholesterol Movement in Duodenum ...... 9 Figure 2.1. Surgical Assessment of Biliary and Intestinal Cholesterol Secretion ...... 17 Figure 3.1. Plasma and Fecal Neutral and Acidic Sterol Concentrations ...... 24 Figure 3.2. Basal Bile Flow and Composition ...... 26 Figure 3.3. Biliary and Intestinal Cholesterol Secretion ...... 28 Figure 3.4. Hepatic and Intestinal Gene Expression ...... 30 Figure 3.5. Treatment and Sex Interaction in Gene Expression ...... 31 Figure 3.6. Stigmasterol and T0901317 Dose Response Curve ...... 33

v Chapter 1: Introduction

1.1. Cardiovascular Disease Treatment

In the United States, cardiovascular disease (CVD) remains the number one cause of death [1]. It is estimated that one in three deaths each year are due to CVD with an average of 1 death occurring every 40 seconds from a myocardial infarction. There are a variety of elements that contribute to the development of CVD including poor dietary habits, physical inactivity, race, age, sex, genetic factors, and comorbid disease states such as diabetes. Some of these factors are modifiable with lifestyle interventions and evidence-based pharmacologic therapies. Currently, there are multiple drugs used in the treatment of CVD, many of which work by managing risk factors, most notably, hypercholesterolemia.

Current guidelines endorse statins as the first-line option for both primary and secondary prevention of atherosclerotic cardiovascular disease (ASCVD) events [2].

Principally, statins are recommended in patients with clinical ASCVD, patients with a primary elevation of LDL cholesterol of 190 mg/dL or higher, patients with diabetes

(type 1 or 2) aged 40 to 75 years old with an LDL cholesterol of 70 to 189 mg/dL and without clinical ASCVD, and in patients without diabetes or clinical ASCVD who have an estimated 10-year ASCVD risk score of 7.5% or greater. However, statins have an adverse effect profile that can make these agents intolerable to patients and lead to low compliance. Specifically, many patients report muscle-related complaints such as myalgia, muscle aches, or discomfort though it is unclear if statins are the primary cause for these symptoms. With reports of 50% or more of patients discontinuing statin use in the first year of treatment initiation, a better tolerated alternative to statins is needed in

1 the management of CVD [3]. Additionally, statins have been implicated in the risk of new-onset diabetes (NOD). Trials assessing the relationship between NOD and statins have produced controversial results with some claiming that there is no effect and others showing an increased risk of NOD ranging from 9% to 13% in randomized clinical trials

[4]. A meta-analysis of observational studies found that there was a 44% increased risk of

NOD with statin-users compared to non-users. While statins remain a pillar in the treatment of ASCVD, issues of adherence and undesirable adverse effects have resulted in their underuse and many patients going without treatment. Because of these issues with statins, there is room for improvement in the management of cardiovascular disease.

Specifically, the investigation of alternative pathways involved in ASCVD could lead to more effective and better tolerated agents. However, before investigating alternative pharmacologic options, an understanding of the pathology of ASCVD and which of these pathways may be targetable is needed.

1.2. Atherosclerosis Pathophysiology

Cardiovascular disease is a complex condition with multiple risk factors. Even though advances have been made in its management, it continues to be a major source of illness in the United States [1]. As researchers investigated the cause of ASCVD, they developed the lipid hypothesis that brought to light the central role of hypercholesterolemia in the development of atherosclerotic plaques [5]. Atherosclerosis, a chronic immunoinflammatory disease, is characterized by the buildup of cholesterol and lipoproteins beneath the endothelium of large and medium-sized arteries [6]. This lesion formation is further characterized by the involvement of other players including

2 foam cells, macrophages that have internalized lipoproteins and cholesterol, that contribute to the pro-inflammatory nature of atherosclerosis. As the plaque grows, it becomes problematic not only due to the narrowing of the vessel lumen, but also due to the risk of plaque rupture. When a plaque ruptures, it can lead to a myocardial infarction or stroke and can ultimately be fatal if not treated appropriately [6,7]. The formation and progression of an atherosclerotic plaque is described in Figure 1.1. By targeting cholesterol excretion and promoting reverse cholesterol transport (RCT), excess cholesterol can be removed from peripheral tissues and potentially both treat and prevent the formation of these atherosclerotic plaques.

Figure 1.1. In a dysfunctional endothelium, cholesterol and lipoproteins infiltrate the subendothelial layer and become oxidized [6]. These modified molecules lead to a proinflammatory, proatherosclerotic state within the intima. As the plaque begins to form and grow, immune cells are recruited to the site of inflammation. Circulating monocytes extravasate into the intima where they differentiate into macrophages. Scavenger receptor proteins expressed on the surface of macrophages facilitate receptor-mediated endocytosis of oxidized LDL particles. Unlike the LDL receptor, scavenger receptor proteins do not have a negative feedback loop that internalizes the receptor in the presence of high levels of cholesterol. Therefore, the macrophage continues to take in oxidized LDL and forms lipid droplets in its cytoplasm. At this point, the macrophage is classified as a foam cell. Eventually the foam cells die through either apoptosis or necrosis leading to further inflammation and the formation of a lipid rich core within the plaque. Plaque progression is also characterized by a fibroproliferative response mediated by surrounding intimal smooth muscle cells. As more collagen is produced, a fibrous cap is formed to provide stability to the plaque and to prevent rupture.

4 1.3. Reverse Cholesterol Transport

Reverse cholesterol transport is the process through which excess cholesterol is removed from peripheral tissues and transported to the liver for elimination [8,9]. As shown in Figure 1.2, cholesterol is transported through the body in high density lipoproteins or HDL, primarily in the form of cholesteryl esters. HDL is then delivered to the liver where it binds to SRB1 which facilitates the clearance of plasma HDL. Another pathway through which HDL transports cholesterol to the liver is through the transfer of cholesteryl esters to LDL in exchange for triacylglycerols via enzyme CETP [9]. LDL can then bind to the LDL receptor and promote cholesterol uptake into the liver. Once in the liver, cholesterol is then either converted into bile salts using cytochrome P450 enzymes such as CYP7a1 or it is transported into the bile via sterol transporter

ABCG5/G8 [8, 10, 11]. From there, cholesterol moves into the intestine where it is either eliminated in the feces or reabsorbed at the apical surface of enterocytes through transporter NPC1L1 and then reincorporated into HDL via ABCA1 at the basolateral surface or into chylomicrons. Another pathway for cholesterol elimination is thought to occur independent of the liver. It is understood that this pathway takes place in the small intestine and is known as TICE, or transintestinal cholesterol excretion; however, the mechanisms of this pathway are currently unknown [9].

Figure 1.2. Reverse cholesterol transport is the movement of cholesterol from peripheral tissues to the liver for elimination [8,9]. Once in the liver, cholesterol is either converted to bile salts or transported into the bile via sterol transporter ABCG5/G8. Once in the bile, cholesterol is then moved into the intestine where it is either reabsorbed or eliminated in the feces. An alternative pathway for cholesterol elimination that is independent of the liver is known as TICE or transintestinal cholesterol excretion. The mechanisms of this pathway are currently unknown.

6 1.4. Transintestinal Cholesterol Excretion

As shown in Figure 1.3, TICE is a pathway in which cholesterol is eliminated through the small intestine. Though the mechanism of TICE is unknown, several groups have shown evidence of its existence [9]. A study carried out by van der Velde et al. in

2007 observed no change in fecal neutral sterol production in ABCG8 deficient mice that were fed a standard chow diet compared to wild-type (WT) mice [12]. Additionally, in

ABCG8 knockout mice fed a western diet high in cholesterol, there was no difference in the rate of intestinal cholesterol secretion compared to WT mice. A western diet is known to increase the expression of transporter ABCG5/G8, and because there was no difference in intestinal cholesterol excretion rates between WT and ABCG8 KO mice, other players in the TICE pathway were upregulated and contributed to cholesterol secretion in the small intestine of the ABCG8 deficient mice. These findings suggest that regardless of

ABCG8 expression and in the absence of biliary cholesterol secretion, a compensatory pathway is present for cholesterol elimination. Furthermore, a study done in ABCG5/G8 knockout mice found that there may be sex differences in cholesterol secretion.

Previously, studies in TICE have primarily utilized male mice. However, Li et al. found that each sex demonstrated a preferred pathway for cholesterol excretion [13]. In male mice, they found that cholesterol secretion favored the intestinal pathway, and in female mice, the biliary pathway for cholesterol secretion was preferred. This is not altogether surprising considering that previous studies have shown a positive association between estrogen and increased levels of biliary cholesterol secretion [14]. Interestingly, in the Li et al. study, they also found that the sum of biliary and intestinal cholesterol secretion in each sex was approximately the same demonstrating that these two pathways work

7 together to maintain whole body cholesterol elimination [13]. Further experiments by this group strengthened the argument that ABCG5/G8 is not fully responsible for TICE. In female ABCG5/G8 KO mice, they saw an increase in intestinal cholesterol secretion compared to WT mice indicating that these knockout mice had developed an adaptation to bypass biliary cholesterol secretion. Altogether, these studies reinforce the concept of

TICE as an alternative route for cholesterol excretion that cannot be fully explained by the presence of ABCG5/G8.

Observations by van der Velde et al. have indicated where along the tract of the small intestine this phenomenon of TICE may be taking place [12]. In WT mice, van der

Velde et al. performed a surgical procedure to cannulate the bile duct in order to divert and exhaust endogenous biliary cholesterol excretion and collected intestinal perfusate to measure intestinal cholesterol excretion. In this experiment, they found that the highest rate of cholesterol secretion occurred in the proximal 10 cm of the small intestine which happens to also be the primary region where cholesterol is absorbed. Because of this study, experiments exploring TICE have primarily focused on the proximal small intestine.

Figure 1.3. TICE or transintestinal cholesterol excretion is an alternative pathway for cholesterol elimination that occurs in the small intestine [9]. The small intestine is also the site of absorption for sterols including both cholesterol and phytosterols, non- cholesterol plant sterols [8, 19]. Transporter NPC1L1 promotes sterol absorption and sterol transporter ABCG5/G8 promotes sterol elimination.

9 1.5. Liver X Receptor

In terms of TICE regulation on the molecular level, liver X receptor (LXR) has been shown to be involved [15]. LXR are ligand-activated transcription factors that regulate the expression of a variety of genes that control cholesterol metabolism and transport. LXR transcription factors can act through two different mechanisms [16]. The first is through direct activation where LXR forms an obligate heterodimer with retinoid x receptor, another transcription factor, and binds to LXR-responsive elements (LXREs) to promote the transcription of genes. In the absence of a ligand, LXR binds to LXREs in complex with corepressors and results in a non-active state. However, when a ligand binds, it results in a conformational change in LXR that releases the corepressors and recruits coactivators for direct activation of gene transcription. In the second mechanism,

LXR acts through transrepression by inhibiting the transcription of proinflammatory genes. Specifically, upon ligand binding, LXR changes conformation and binds to

Nuclear Receptor Corepressor (N-CoR complex). This results in the dissociation of the

N-CoR complex from NF-kB and prevents transcription of proinflammatory genes. There are two types of LXRs, LXRa and LXRb [15,16]. LXRa is predominantly expressed in the liver, small intestine, kidney, macrophages, and in adipose tissue, whereas LXRb is expressed ubiquitously. LXRs act as cholesterol sensors by regulating genes that defend cells from cholesterol overload. These transcription factors control cholesterol and bile acid synthesis as well as reverse cholesterol transport and cholesterol movement in the intestine. When cholesterol levels are too high, LXR works to decrease cholesterol absorption and increase fecal sterol excretion. LXR does this by downregulating NPC1L1 expression and increasing ABCG5/G8 expression in the intestine. Additionally, LXR has

10 been shown to regulate genes associated with reverse cholesterol transport including

ABCA1, ABCG1, and APOE, all of which are involved in the formation and maturation of

HDL, a key player in the movement of cholesterol from peripheral tissues to the liver.

Finally, it has been shown that LXR activation leads to increased fecal neutral sterol loss with recent studies indicating the intestine playing a major part in this process. A study done by Kruit et al. found that exposure to synthetic LXR agonist GW3965 in MDR2 knockout mice with dramatically reduced biliary cholesterol secretion resulted in a two- fold increase in fecal neutral sterol loss, similar to that seen in WT mice [17]. One group took these findings a step further by discovering that LXR activation by synthetic ligand

T0901317 activated TICE by promoting blood-derived cholesterol flux into the feces through the intestine [18]. In their experiment, treatment with T0901317 was shown to triple cholesterol loss in the feces compared to the control treatment group in WT mice.

These findings indicate that manipulation of LXR can increase TICE and promote blood derived cholesterol movement to the intestine for elimination.

Activators of LXR include oxysterols, oxidized cholesterol derivatives, which act as endogenous ligands [15]. These oxysterols bind to LXR with varying affinity with 24

(S), 25-epoxycholesterol being the most potent agonist. Interestingly, cholesterol itself is not an LXR agonist. In addition to endogenous ligands, there are other agonists of LXR including phytosterols and synthetic agonists such as T0901317 and GW3965. Although these agents have been protective against atherosclerosis in preclinical models, synthetic

LXR agonists have also resulted in triglyceride accumulation and hepatic steatosis; therefore, they are not good drug candidates for the treatment of ASCVD. Phytosterols are bioactive plant sterols that have exhibited cholesterol lowering capabilities [19].

11 These agents are structurally related to cholesterol and can be found in a variety of foods including vegetables and vegetable oils. Like cholesterol, transporter NPC1L1 facilitates phytosterol absorption and ABCG5/G8 promotes their excretion. Studies have shown that

2 grams of phytosterols a day can reduce cholesterol absorption and lower plasma LDL- cholesterol by 10%. Additionally, researchers found that the addition of phytosterols to statin treatment resulted in a greater lowering of LDL-cholesterol (10-15%) compared to doubling the dose of a statin (6%). Though phytosterols have been shown to be beneficial in lowering cholesterol, their role in the treatment of and protection against atherosclerosis is still unknown. Investigations of the effects of these phytosterols in mouse models of atherosclerosis have reported mixed results with some showing benefit and others harm. In one study, researchers reported reductions in arterial lipid accumulation as well as the inhibition of atherosclerotic plaque formation and progression in mice with one copy of the LDL receptor treated with a statin plus plant sterols (or stanols) compared to statin alone [20]. However, another group found that exposure to plant sterol esters reduced endothelial vasorelaxation and increased atherosclerotic lesion formation [21]. Many of these studies investigating the effects of phytosterols have used commercially available mixtures of plant sterols; however, it is clear that individually, the biological activity of these agents can be both cell-type and sterol specific [22].

12 1.6. Stigmasterol

Stigmasterol, a bioactive phytosterol, has been shown to have in vivo activity by decreasing intestinal cholesterol absorption and reducing hepatic cholesterol content in rats [23]. It has also been demonstrated to promote ABCA1 expression in the adrenal glands of ABCG5/G8 knockout mice as well as increase the expression of ABCA1 and other LXR dependent genes in macrophages [22, 24]. These findings contribute to the idea that phytosterols, specifically stigmasterol, play an active role in cholesterol metabolism and transport with evidence suggesting that stigmasterol promotes reverse cholesterol transport. The goal of our experiment was to test the viability of stigmasterol as an agent that would promote reverse cholesterol transport through the intestine in mice. It was our objective to explore TICE as a new targetable pathway and the potential of stigmasterol as a cholesterol-lowering agent that could be used in the treatment of hypercholesterolemia. In our experiment, we hypothesized that stigmasterol would act as an intestinal-specific LXR agonist, due to the activity of sterol transporter ABCG5/G8, to promote cholesterol elimination in mice.

13 Chapter 2: Methods and Materials

2.1. Animals and Treatments

C57BL/6J male and female mice were purchased from Jackson Laboratory (Bar Harbor,

ME). Mice at 8 weeks of age were individually housed in ventilated cages and had free access to water and treatment diet. Treatments were pelleted diets made from control diet

(Research Diets Inc, AIN-76A) with 0.3% (w/w) stigmasterol (Sigma-Aldrich, Product

Number S2424) or 0.015% (w/w) T0901317 (Cayman Chemical, Product Code 71810).

Mice were fed either the control diet, stigmasterol supplemented diet, or T0901317 supplemented diet for the experiment. First, mice were fed the control diet for three days.

On day four, mice were randomized to receive either control diet, 0.3% stigmasterol supplemented diet, or 0.015% T0901317 supplemented diet for a total treatment period of four days. On day 8, mice were either euthanized for plasma and tissue collection (n=16) or anesthetized for a surgical procedure whereby bile and intestinal perfusate were collected (n=15-17). Two independent cohorts of mice were used for plasma and tissue collection and the surgical procedure. Both cohorts were balanced containing approximately the same number of male and female mice in each group.

2.2. Tissue and Plasma Collection

Mice were euthanized by carbon dioxide and exsanguination at termination. Liver and small intestine sections were dissected, snap frozen in liquid nitrogen, and stored in -

80°C. The small intestine was divided into three equal sections at dissection representing the duodenum, jejunum, and ileum. Blood was collected in EDTA coated tubes and then centrifuged at 2000xg for 15 minutes at 4°C. Plasma was then stored in -80°C.

14 2.3. Surgical Assessment of Biliary and Intestinal Cholesterol Secretion

Mice were anesthetized using 2% isoflurane (Henry Schein, Product Number 050033) and placed on a temperature-controlled heating pad system (Protech International Inc,

TC-1000 Temperature Controller) to maintain a body temperature of 37°C. Mice were supplied with oxygen (Scott Gross, Item Number 421) and isoflurane using a nose cone.

A longitudinal cut was made along the midline from the lower abdomen to the sternum.

Two forceps were then used expose the peritoneum where an incision is made to expose the abdominal organs. Using a sterile cotton tipped applicator that was moistened with warm PBS (Sigma-Aldrich, P3813), the lobes of the liver were then carefully lifted to expose the common bile duct and gallbladder. The common bile duct was then ligated using a 3cm silk suture, and the gallbladder was cannulated with 10cm of PE tubing

(Braintree Scientific Inc, PE10 tubing). The cannula was then secured to the bile duct using another suture. Basal bile was collected for the first 30 minutes with collections occurring at 15 minute intervals thereafter for 120 minutes total. Collected bile was kept on ice. After basal bile collection, the tail vein was fitted with an inflow tail vein catheter

(Braintree Scientific Inc, Item Number MTV-01) and infused with 20mM sodium taurocholate (Sigma, 86339) at a rate of 100 nmol/min using a syringe pump (Kent

Scientific, model Genie Plus) to maintain biliary cholesterol secretion for the duration of the procedure. For the intestinal perfusate collection, a perfusate inflow catheter was inserted into the proximal small intestine just below the fundus of the stomach. This catheter was connected to a peristaltic pump (Bio-Rad, Econo Pump Model EP1) and facilitated the movement of perfusate at approximately 150 nmol/min. Perfusate was a mixture of Krebs-Henseleit buffer (Teknova, Product Number K235206), 10mM sodium

15 taurocholate hydrate (Sigma-Aldrich, Catalogue Number 86339), and 2mM L-a- phosphatidylcholine (Sigma-Aldrich, Catalogue Number P3556). Below is the preparation of the intestinal perfusate. A collection cannula was inserted about 10cm distal to the inflow catheter. Sutures were used to secure the inflow catheter and outflow cannula to the small intestine. Prior to collections beginning, the small intestine was flushed with warm PBS to remove the luminal contents. Collections were done over a 90 minute period with fractions collected every 15 minutes and stored on ice. Bile and intestinal perfusate samples were then stored in -80°C. A cartoon version of the surgery is shown below in Figure 2.1.

2.4. Intestinal Perfusate Preparation

Intestinal perfusate was composed of a modified Krebs-Henseleit buffer (Fisher,

NC0881992) with sodium taurocholate and L-a-phosphatidylcholine. For each surgery

20mL of intestinal perfusate were made. First, sodium taurocholate was dissolved in methanol (Fischer Scientific, Product Number A4564), and L-a-phosphatidylcholine was dissolved in chloroform (Fischer Scientific, Product Number C6064). The mixtures were then placed under a nitrogen dryer at 45°C. After the evaporation films have been lyophilized, they are stored at -20°C. On the day of the surgery, the taurocholate and phophatidylcholine films are reconstituted using the Krebs-Henseleit buffer and combined to make the modified Krebs-Henseleit buffer. The final buffer concentration is

10mM sodium taurocholate and 2mM L-a-phosphatidylcholine.

Figure 2.1. A) The common bile duct is ligated. B) An outflow catheter is inserted into the gallbladder to facilitate bile collection. C) An inflow catheter is inserted into the tail vein and bile is infused at a rate of 5uL/min to maintain bile flow. D) The proximal small intestine is fitted with an in-flow perfusion catheter and perfusate is infused at a rate of

14uL/min. E) The small intestine is ligated 10cm distal to the in-flow catheter and fitted with an out-flow catheter to collect intestinal perfusate. Drawing courtesy of Dr. Gregory

A. Graf.

17 2.5. Lipid Analysis

Total and unesterified cholesterol of plasma, bile, and intestinal perfusate were measured using a commercial enzymatic, colorimetric assay (Fisher Scientific). In this assay, there were two control serums (Wako Diagnostic, 41600102) used as well as a standard (Fisher

Scientific, 23666198) to create a standard dilution curve. To determine the cholesterol concentration, different volumes of sample were used depending on the type of sample.

For both bile and plasma, 2uL of sample were used. For intestinal perfusate, 25uL of sample were used. Then, either 98uL or 75uL of 0.9% normal saline (NaCl, Sigma,

SLBG2141V) depending on the sample type to make a total volume of 100uL. Next,

100uL of total cholesterol reagent (Fischer Scientific, 23666201) are added to the plate.

The plate was then incubated at 37°C for 45 minutes. The absorbance was then measured at 492nm using the Biotek Synergy H1 Hybrid plate reader.

Additionally, cholesterol and non-cholesterol sterols were extracted from plasma and feces. For plasma, sterols were extracted using a chloroform/methanol mixture. 50% potassium hydroxide was then added to the bottom phase of the extraction to facilitate hydrolysis and this mixture was incubated at 65°C for 60 minutes. After the mixture had cooled to room temperature, hexane and water were added for the final step of the extraction. After the samples were centrifuged, the organic (top) layer was removed, dried, resuspended in hexane, and analyzed using gas chromatography with flame ionization detector (GC-FID). A similar protocol was used for the extraction of sterols from the feces, however, the fecal samples were incubated for 3 hours at 65°C. 5 alpha- cholestane (Sigma Aldrich, Product Number C8003) was used as the internal standard

18 during preparation of these samples. This protocol was adapted from a previously published protocol for sterol extraction [25].

2.6. Phospholipid Assay

Phospholipid concentration was measured in the basal bile using a commercial enzymatic, colorimetric assay (Sigma Aldrich, MAK122). In this assay, a 200uM standard working solution of phosphatidylcholine (Sigma, MAK122C) was used to generate a standard dilution curve. After adding the reaction mixture to 2uL of basal bile that had been diluted in 1800uL of 0.5% Triton X-100 buffer, the plate was incubated at room temperature for 30 minutes. The reaction mixture was composed of an assay buffer

(Sigma, MAK122A), enzyme mix (Sigma, MAK122B), PLD enzyme (Sigma,

MAK122D), and dye reagent (Sigma, MAK122E). The absorbance was then measured at

570nm using a plate reader.

2.7. Bile Acid Assay

Bile acid concentration was measured in the feces and basal bile using a commercial enzymatic, colorimetric assay. The assay used sodium taurocholate (Fischer Scientific,

0877253) as the standard as well as a bile acid assay buffer that contained glycine

(Sigma, G8898), hydrazine sulfate salt (Sigma, 216046), disodium EDTA (Sigma,

2726046), b-nicotinamide dinucleotide (Sigma, N1511), and hydroxysteroid dehydrogenase (Worthington Biochemical, LS004910). The individual procedures used for fecal and biliary bile acid assay preparation are described below.

19 2.8. Fecal Bile Acid Assay

Approximately 200mg of ground feces was added to 100% ethanol and 10mM NaOH.

This mixture was then incubated for 2 hours in a 70°C water bath where samples were vortexed every 15 minutes. The samples were then centrifuged for 10 minutes at 1300g.

The supernatant was then transferred to a new tube, sealed, and refrigerated overnight at

4°C. For the bile acid assay, 10uL of extract (or the sodium taurocholate standard) were added to 200uL of assay buffer. The plate was then incubated at room temperature for 1 hour. The absorbance was then measured using a plate reader at 340nm.

2.9. Biliary Bile Acid Assay

Both the sodium taurocholate standard and bile samples were diluted 1:10 in methanol.

Then 10uL of bile or standard was added to 200uL of assay buffer. The plate was incubated at room temperature for 1 hour and the absorbance was measured using a plate reader at 340nm.

2.10. Dose Response Assay

LS174T human adenocarcinoma colon cancer cells (ATCC, 70003535) were cultured in eagle’s minimal essential medium (EMEM) (ATCC, 00831) with 10% fetal bovine serum

(Atlanta Biologicals) and a penicillin/streptomycin solution (Sigma, 087M4871V) at

37°C and 5% carbon dioxide. Cells were seeded with 100,000 cells per well. They were incubated for 48 hours total with the culture media replaced at 24 hours. After 48 hours, the cells were treated with EMEM that contained 0.2% fatty acid free bovine serum albumin (Sigma, SLBT1197) and one of three treatments. One treatment was with

20 increasing concentrations of stigmasterol to a final concentration of 40mM. The other treatment was with T0901317 with increasing concentrations to a final concentration of

10mM. The control treatment was ethanol. Cells were exposed to treatment media for 24 hours. They were then washed with PBS (Sigma, SLBR6772V) and lysed with RNA

STAT-60 to isolate RNA. RNA and cDNA were then prepared as described below to determine the relative abundance of gene expression. Human ABCA1 and GAPDH

Taqman probes were used during RT PCR. A dose response curve was then made plotting the changes in ABCA1 gene expression compared to the concentration of treatment used. The EC50 was determined using GraphPad Prism (Version 7.04).

2.11. RT PCR

RNA was isolated from duodenum and liver samples using RNA STAT-60 (Tel-Test,

Inc.) and the RNeasy Mini Kit (Qiagen). RNA was quantified using the Nanodrop One

(ThermoScienfitic) then diluted to 50ng/uL concentration. The iScript cDNA Synthesis

Kit (Bio-Rad) was used to convert RNA to 20uL of cDNA . Quantitative real-time PCR was used to measure changes in gene expression. The relative abundance was determined using SYBR Green primers and the Quant Studio 7 Flex Real-Time PCR System

(Thermo Fischer Scientific). GAPDH was used for normalization of the transcript abundance. Data was expressed relative to control mice with delta delta CT calculations.

21 2.12. Statistical Analysis

All data were presented as mean ± SEM with statistical significance defined as p<0.05.

Data were analyzed using linear regression, unpaired t-test, one-way ANOVA, and two- way ANOVA where indicated. A Dunnett’s post hoc test was used to compare treatment groups with the control group. Statistical analyses were performed using GraphPad Prism

(Version 7.04).

22 Chapter 3: Results

3.1. Stigmasterol does not affect plasma cholesterol levels or fecal sterol loss

Synthetic LXR agonists increase cholesterol plasma and fecal neutral sterol loss by promoting reverse cholesterol transport [26]. Therefore, we first determined the effect of stigmasterol on plasma and fecal sterol levels. Mice treated with synthetic LXR agonist

T0901317 had an increase in plasma cholesterol, fecal cholesterol, and fecal acidic sterol concentrations (Figure 3.1). Fecal cholesterol loss was increased by 75% with T0901317 treatment compared to control. Alternatively, in mice fed stigmasterol, there was no change in plasma cholesterol levels compared to the control treatment group.

Additionally, in the stigmasterol treatment group, fecal cholesterol loss was increased by

38% compared to control, and fecal acidic sterol loss trended to increase but was not statistically significant. When analyzing the plasma of mice challenged with stigmasterol, stigmasterol was not detected using GC-FID. However, due to the presence of stigmasterol in the feces of the stigmasterol treatment group, we confirmed that the mice were consuming the treatment diet. To ensure that the changes observed were due to treatment and not sex, a two-way ANOVA analysis was done to determine if there was an interaction between treatment and sex. When comparing male and female stigmasterol levels in the feces, we found a statistically significant interaction between sex and treatment (p<0.0001). However, this effect was determined to be due to differences in the magnitude of response to stigmasterol between the two sexes and not an observance of one sex responding without the other.

Figure 3.1. Stigmasterol increased fecal cholesterol loss but not plasma cholesterol.

Plasma and feces were collected and analyzed using GC-FID to detect sterol levels (A-

C). To measure acidic sterols in the feces, a commercial enzymatic assay was used (D).

Values reported as mean ± SEM. N=16. *=p<0.05; **=p<0.01; ****=p<0.0001.

24 3.2. Stigmasterol does not have an effect on basal bile flow or composition

Basal bile was characterized to determine the effect of treatment on basal bile composition and flow (Figure 3.2). Basal bile was defined as the bile collected during the first 30 minutes of surgery as described in “Methods.” Bile flow did not differ between treatment groups, but there was an increase in basal biliary cholesterol secretion in mice challenged with T0901317. T0901317 treatment also resulted in a decrease in basal biliary bile acid and basal biliary phospholipid concentrations. Stigmasterol did not alter basal bile composition with basal biliary cholesterol, basal biliary bile acid, and basal biliary phospholipid concentrations not differing from the control treatment group.

Figure 3.2. Basal bile composition and flow was unaffected by stigmasterol. Basal bile was collected for 30 minutes. Basal biliary cholesterol, basal biliary bile acid, and basal biliary phospholipid concentrations were measured using a commercial colorimetric, enzymatic assay. Values reported as mean ± SEM. N=15-17. **=p<0.01; ***=p<0.001.

26 3.3. Stigmasterol promotes biliary and intestinal cholesterol secretion

The synthetic LXR agonist, T0901317, promotes biliary cholesterol secretion [26]. Bile and intestinal perfusate were collected at 15 minute intervals to determine the rate of cholesterol secretion in the bile and intestine (Figure 3.3). Mice challenged with

T0901317 had a robust response in both biliary and intestinal cholesterol secretion

(biliary slope: slope: 0.004627±0.0001142; intestinal slope: 0.002446±0.0002127) compared to control (biliary slope: 0.002573±0.0001042; intestinal slope:

0.001823±0.000121). Mice treated with stigmasterol saw a modest effect on biliary cholesterol secretion (slope: 0.002907±5.014e-005) compared to control. Although this difference reached statistical significance, the increase in biliary cholesterol secretion was modest and a significant biological impact is unlikely. Alternatively, stigmasterol promoted a significant increase in intestinal cholesterol secretion (slope:

0.002467±8.17e-005). Sum cholesterol secretion was calculated by adding biliary and intestinal cholesterol secretion rates to represent total body cholesterol secretion. Both

T0901317 and stigmasterol showed robust increases in cholesterol secretion compared to control (stigmasterol slope: 0.00544±9.584e-005; T0901317 slope: 0.007164±0.0002772; control slope: 0.00443±0.0002348). Bile flow was also calculated to determine if treatment would have an effect on the rate of bile secretion. T0901317 treated mice had an increase in the rate of bile flow (slope: 0.005657±0.0001151) compared to control

(slope: 0.004253±0.0001372). The bile flow of mice challenged with stigmasterol did not differ from control (stigmasterol slope: 0.003944±8.347e-005); however, it appeared to did trend toward decreasing it.

Figure 3.3. Stigmasterol promoted biliary and intestinal cholesterol secretion. A surgical method was used to simultaneously collect bile and intestinal perfusate. Bile and intestinal perfusate were then analyzed using a commercial colorimetric, enzymatic kit to determine cholesterol concentration (A, B). Sum cholesterol secretion was calculated by adding biliary and intestinal cholesterol secretion rates (C). Values reported as mean ±

SEM. N=15-17.

28 3.4. Stigmasterol did not increase gene expression of LXR targets

Agonists of the transcription factor LXR are known to regulate LXR-dependent gene expression [26, 27]. Many of these genes are responsible for cholesterol transport and metabolism. To determine if LXR agonism would occur in the liver and small intestine, the relative abundance of LXR-responsive genes was measured. Mice treated with

T0901317 demonstrated a robust change in LXR-target genes (Figure 3.4). Alternatively,

LXR-responsive gene expression did not change with stigmasterol treatment compared to control. To ensure that the changes observed were due to treatment and not sex, a two- way ANOVA analysis was done to determine if there was an interaction between treatment and sex. When comparing male and female changes in LXR-dependent gene expression, we found a statistically significant interaction between sex and treatment

(p<0.0001) in ABCA1 expression in the duodenum and SREBP-1c expression in the liver

(Figure 3.5). However, this effect was determined to be due to differences in the magnitude of response to T0901317 between the two sexes and not an observance of one sex responding without the other. Additionally, NPC1L1 expression in the duodenum was found to have a significant interaction between sex and treatment; however, unlike

ABCA1 and SREBP-1c, the change in gene expression was not due to a difference in the magnitude of response. With NPC1L1 expression, male mice responded to T0901317 treatment, but female mice did not.

Figure 3.4. Stigmasterol had no effect on LXR target genes. Mean ± SEM of relative mRNA expression of genes involved in cholesterol transport and metabolism. RNA was extracted from liver and small intestine tissue samples. Relative expression was normalized to GAPDH. N=16. ** =p<0.01; **** =p<0.0001.

Figure 3.5. The interaction between sex and T0901317 treatment was found to be statistically significant in ABCA1, SREBP1-c, and NPC1L1. Mean ± SEM of relative mRNA expression of genes involved in cholesterol transport and metabolism. RNA was extracted from liver and small intestine tissue samples. Relative expression was normalized to GAPDH. N=16. ** =p<0.01; **** =p<0.0001.

31 3.5. Stigmasterol is a weak LXR agonist compared to T0901317

The absence of LXR response with stigmasterol was surprising given previous studies that have shown LXR activity in cultured macrophages and adrenocortical Y1-BS1 cells

[22,24]. Therefore, we confirmed LXR activity in a human intestinal cell line. A dose response curve was generated for both stigmasterol and T0901317 in human adenocarcinoma LS174T colon cancer cells (Figure 3.6). The LXR-responsive gene used to determine the EC50 was ABCA1. T0901317 had a calculated EC50 of 70nM, and stigmasterol had an EC50 of 200uM.

Figure 3.6. Dose response curve of stigmasterol and T0901317. Curve was generated measuring the dose response change in LXR-dependent gene ABCA1 gene expression with increasing treatment concentrations. Relative expression was normalized to

GAPDH.

33 Chapter 4: Discussion

Despite advances in medicine, cardiovascular disease remains the leading cause of death in the United States [1]. Current therapies that target risk factors of CVD include statins, a medication used to lower cholesterol [2]. However, the literature shows that there are high rates of noncompliance associated with statin use resulting in patients going without the treatment needed to manage their CVD [3]. Many patients report not taking statins due to the adverse effects associated with the medication including muscle pain and myopathies. Other concerns regarding the use of statins include the potential to develop new-onset diabetes [4]. Due to these complications, many patients are currently undertreated for their CVD creating a gap in therapy. Phytosterols are plant-based, non- cholesterol sterols that have been shown to reduce cholesterol levels by 10-15%; however, the benefit of these agents in CVD treatment and prevention is currently unknown [19]. Commercially, phytosterol supplements are available as a mixture including a variety of phytosterols such as stigmasterol, sitosterol, and campesterol among others [22]. Studies in vivo and in vitro have demonstrated that, individually, phytosterols possess biological activity that is sterol-specific [22-24]. Specifically, stigmasterol has been reported as an LXR agonist that has cholesterol lowering properties.

In our study, we fed mice either a control diet, a diet supplemented with stigmasterol, or a diet supplemented with T0901317, a known LXR agonist, to determine if stigmasterol would promote reverse cholesterol transport and, in turn, intestinal cholesterol secretion. We hypothesized that due to the activity of sterol transporter

ABCG5/G8, stigmasterol would not be systemically absorbed, but would instead act

34 locally in the intestine to regulate the expression of LXR-dependent genes and promote intestinal cholesterol secretion. In our experiment, we observed robust responses in mice challenged with T0901317 with increased plasma cholesterol concentrations and fecal cholesterol loss. The increase in fecal cholesterol concentration observed was further supported by T0901317 robustly promoting both biliary and intestinal cholesterol secretion. T0901317 was also noted to alter basal bile composition by increasing basal biliary cholesterol and bile acid concentrations while lowering basal biliary phospholipid concentrations. Finally, T0901317 exerted LXR-agonist activity in the changes in gene expression that we observed in LXR-target genes. With an EC50 of 70nM, T0901317 increased the gene expression of hepatic and intestinal sterol transporter ABCG5 and

ABCG8 as well as downregulated other genes involved in cholesterol uptake including

NPC1L1 in the duodenum. T0901317 also upregulated the expression of transcription factor SREBP-1c, a protein involved in the transcription of genes associated with lipogenesis [28]. The changes we observed in mice challenged with T0901317 were similar to those reported in the literature [26-27, 29-30].

Mice treated with stigmasterol resulted in a moderate increase in fecal cholesterol loss (38%) with no change in plasma cholesterol levels compared to control. Fecal acidic sterols trended toward an increase similar to that seen with T0901317 treatment but did not reach statistical significance. Additionally, in the GC-FID analysis of the plasma of mice challenged with stigmasterol, we did not observe the presence of stigmasterol suggesting that it was not systemically absorbed. This observation was further supported by the effect of stigmasterol on biliary and intestinal cholesterol secretion. Stigmasterol was observed to have a robust effect on intestinal cholesterol secretion but only a mild

35 effect on biliary cholesterol secretion. Additionally, stigmasterol treatment did not seem to have an effect on basal bile composition or flow. Altogether, these results indicate that stigmasterol was not systematically absorbed and, therefore, exerted a local effect in the duodenum to promote intestinal cholesterol secretion that was reflected in the modest increase in fecal cholesterol loss. A potential explanation for why we did not detect stigmasterol in the plasma could be due to the activity of sterol transporter ABCG5/G8.

Because of the efficiency of this transporter, ABCG5/G8 prevented the systemic absorption of stigmasterol. To explore the validity of this possibility, an intestinal specific ABCG5/G8 KO mouse model could be used where the mice are fed stigmasterol.

If we observed stigmasterol in the plasma as well as more robust responses with LXR- dependent genes, then we could conclude that the activity of ABCG5/G8 prevented the systemic absorption and subsequent LXR activity of stigmasterol in our experiment.

At the molecular level, we did not observe a robust response with stigmasterol treatment. The dose response curve with stigmasterol revealed an EC50 of approximately

200uM indicating that stigmasterol has low potency as an LXR agonist compared to

T0901317. This could, in part, explain the lack of response seen in gene expression where treatment with stigmasterol did not have an effect on LXR-target gene expression.

Although stigmasterol did not alter gene expression compared to the control treatment group, it did increase fecal cholesterol loss and promote biliary and intestinal cholesterol secretion. Considering these events, there seems to be an alternative mechanism occurring in the small intestine that is promoting TICE, transintestinal cholesterol excretion, independent of LXR. Though the mechanisms of TICE are currently unknown,

36 it is possible that stigmasterol promoted this pathway to increase intestinal cholesterol secretion.

Another potential reason for why we did not see LXR activity with stigmasterol could be that it was not systemically absorbed at a high enough concentration. Assuming a density of 1g/mL and that the diet contained 0.3% stigmasterol, the concentration of stigmasterol present in the intestinal lumen is approximately 7mM. This concentration far exceeds the needed 200uM for our calculated EC50. However, when considering the amount of chyme and bile needed to digest and facilitate the absorption of stigmasterol, it is unclear how much of that 7mM stigmasterol was actually absorbed. To determine this, we would need to collect the intestinal contents of a mouse treated with stigmasterol, centrifuge the contents, and then determine how much stigmasterol was solubilized and, therefore, capable of absorption. By determining this, we would be able to ascertain if enough stigmasterol was able to be systemically absorbed and achieve LXR activity.

The findings of our experiment expand the current knowledge of what role phytosterols, specifically stigmasterol, play in the regulation of cholesterol elimination.

Despite the lack of stigmasterol treatment effect on the expression of LXR-dependent genes, we still observed stigmasterol act as a bioactive phytosterol to promote cholesterol elimination. In our experiment, we demonstrated that mice challenged with stigmasterol had an increase in fecal cholesterol loss that was further supported by the promotion of biliary and intestinal cholesterol secretion. In the future, further studies that elucidate the mechanism of TICE as well as how stigmasterol is potentially promoting this pathway could lead to new therapeutic targets for lowering plasma cholesterol. Additionally, further investigation into compounds that are structurally similar to stigmasterol but with

37 stronger LXR agonistic properties are warranted and could lead to the development of an intestinal specific LXR agonist that promotes intestinal cholesterol secretion. By investigating these bioactive compounds, we could bring a new therapeutic agent to market that would be used in the treatment of ASCVD by promoting reverse cholesterol transport.

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41 VITA

Hannah Carlene Lifsey

EDUCATION

Doctor of Pharmacy Anticipated May 2018 University of Kentucky College of Pharmacy Lexington, Kentucky Magna Cum Laude

Master of Science, Pharmaceutical Sciences Anticipated May 2018 University of Kentucky College of Pharmacy Lexington, Kentucky Magna Cum Laude

Bachelor of Science, Biochemistry and Molecular Biology August 2010 – May2014 Centre College Danville, Kentucky Cum Laude

PROFESSIONAL EXPERIENCE

Graduate Student Researcher March 2016 – May 2018 University of Kentucky College of Pharmacy Lexington, Kentucky Principal Investigator: Greg Graf, PhD

Pharmacy Intern April 2015 – October Kroger Pharmacy Store 347 2015 Lexington, Kentucky Preceptor: Patti Taluskie, PharmD, Pharmacy Manager

Undergraduate Student Researcher April 2011 – May 2012 Centre College Danville, Kentucky Principal Investigator: Joe Workman, PhD

ACADEMIC HONORS

Dean’s List, 6 semesters August 2014 – May 2017 University of Kentucky College of Pharmacy Lexington, Kentucky