Role of PXR Signaling in Mediating the Cardioprotective Effects of  -3 Fatty Acids Saraswathi Viswanathan, Ph.D. Assistant Professor Department of Internal Medicine/DEM University of Nebraska Medical Center-Omaha

Abdominal obesity Atherogenic dyslipidemia Insulin resistance Elevated blood pressure Pro-inflammatory state Metabolic Syndrome and CVD

Fish Oil and  -3 PUFAs EPA-Eicosapentaenoic acid DHA-Docosahexaenoic acid Fish Oil and  -3 Fatty Acids

30 g of fish per week reduced caronary artery disease (Kromhout DBE, 1985). EPA&DHA reduced plasma TG and non- HDL cholesterol in patients with type 2 diabetes and dyslipidemia (De Luis, DA 2009).  -3 fatty acids reduced plasma TG, total cholesterol without altering glycemic index (Sirtori CR, 1998). Clinical Evidence for the Beneficial Effects of Fish Oil

Mechanisms TG-Lowering Effect Reduced TG secretion Increased TG clearance Increased  -oxidation

Mechanisms Mediating the Cholesterol-Lowering Effects Cholesterol Bile acid CYP 7A1 CYP 27A1 CYP 11A CYP 3A Bile acid Detoxification Sulfotransferases Glutathione S transferases Cholesterol hydroxylation CYP3A Sult1e1 Sult2a1 Sult3e1 Gsta1 Gsta2 PXR

Interference with arachidonic acid metabolism COX-derived 3-series eicosanoids LOX-derived resolvins Cytochrome P450-derived epoxides Mechanisms Mediating the Anti- Inflammatory Effects of  -3s

PXR and Inflammation PXR CYP 2C and CYP 3A  -3 epoxides  -3 FAs Anti- inflammatory

PXR Drug detoxification Bile acid homeostasis Cholesterol metabolism Reduce inflammation

Effect of Fish Oil on Plasma Lipids n=13-15 per group; ^P<0.001 vs OO OO-Olive Oil, FO-Fish Oil Preliminary Data

Effect of Fish Oil on Hepatic Steatosis n=4-10 per group; ^P<0.001 vs OO OO-Olive Oil, FO-Fish Oil

Effect of Fish Oil on Inflammatory Genes in Liver n=5-6 per group; ^P<0.001 and # P<0.01 vs OO OO-Olive Oil, FO-Fish Oil

Genes Upregulated in Liver upon Fish Oil Feeding-Microarray Analysis GenesFold Increase CYP3A441.6 CYP2C681.6 Sult 1e12.2 Sult 1b12.0 Sult 3a11.8 GSTA12.2 GSTA21.6

Effect of Fish Oil on PXR and CYP3A in Liver OO,Olive Oil; FO, Fish Oil, n=5-6 samples per group, # P<0.01 vs OO

Anti-inflammatory Effects Cholesterol- lowering Effects  -3 Fatty Acids (EPA & DHA) Cardioprotective Effects Hypothesis PXR Overall Hypothesis: The cholesterol-lowering and anti- inflammatory effects of  -3 fatty acids are mediated via PXR.

Specific Aims Specific Aim 1: To determine the role of PXR in mediating the cholesterol-lowering and anti-inflammatory effects of  -3 fatty acids in a model of diet-induced obesity and dyslipidemia. Specific Aim 2: To determine the role of PXR in mediating the cholesterol-lowering and anti-atherosclerotic effects of  -3 fatty acids in a model of genetic dyslipidemia. Specific Aim 3: To determine whether the  -3 fatty acids modulate PXR signaling in cultured hepatocytes.

Mutant-PXR-/- WT-PXR+/+ Experimental Design-Specific Aim 1 High Fat Diet 45% Fat (energy) 1% Cholesterol 0.56%  -3s Chow Diet 0.56% Oleic Acid 0.56%  -3s Chow Diet 0.56% Oleic Acid Chow Diet Study Groups Experimental Diets

Proposed Experiments-Specific Aim 1 Lipid profiles in plasma and liver Expression of genes/proteins involved in cholesterol/bile acid metabolism and inflammatory response Analysis of gall bladder bile for cholesterol and phospholipids Levels of  -3 epoxide metabolites in liver

LDLR;PXR-/- LDLR-/- Experimental Design-Specific Aim 2 High Fat Diet 40% Fat (energy) 0.5% Cholesterol 0.56%  -3s Chow Diet 0.56% Oleic Acid 0.56%  -3s Chow Diet 0.56% Oleic Acid Chow Diet Study Groups Experimental Diets

Proposed Experiments-Specific Aim 2 Lipid profiles in plasma and liver Genes involved in cholesterol/bile acid metabolism and inflammatory response Analysis of gall bladder bile for cholesterol and phospholipids Atherosclerotic lesion area

Inflammation Apoptosis Bile Acid Detoxification Bile Acid Detoxification PXR Signaling LCA+  -3s LCA Experimental Design-Specific Aim 3 Primary Hepatocytes from WT and PXR-/- Mice

Apoptosis  Experimental Design-Specific Aim 3 LCA Human HepG2 Cell Line  -3s Scrambled siRNA for PXR siRNA for PXR Inflammation  Apoptosis  Inflammation 

Summary  -3 Fatty Acids (EPA & DHA) PXR Signaling CYP 2C & CYP 3A Cholesterol & Bile Acid Metabolism  3-Epoxides Lipid-lowering Effects Anti-inflammatory Effects Anti-atherosclerotic Effects Genetic Dyslipidemia Dyslipidemia in Obesity Liver Hepatocyte Inflammation & Apoptosis SA1 SA2 SA3

Impact Identification of novel molecular mechanisms by which  - 3 fatty acids mediate their cholesterol-lowering and anti- inflammatory effects. The findings will be critical to target PXR using dietary factors to efficiently prevent/treat dyslipidemia in humans without adverse side effects.