Role of PXR Signaling in Mediating the Cardioprotective Effects of -3 Fatty Acids

Saraswathi Viswanathan, Ph.D.

Assistant ProfessorDepartment of Internal Medicine/DEMUniversity 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 acidDHA-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 7A1CYP 27A1CYP 11ACYP 3A

Bile acid Detoxification

SulfotransferasesGlutathione S transferases

Cholesterol hydroxylation

CYP3ASult1e1Sult2a1Sult3e1Gsta1Gsta2

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

TC-Plasma

To

tal C

ho

lest

ero

l(m

g/d

L)

OO FO0

100

200

300

400

500

600

^

TG-Plasma

Tri

gly

ceri

des

(mg

/dL

)

OO FO0

50

100

150

200

250

^

FFA-Plasma

Fre

e F

atty

Aci

ds

(mE

q/L

)

OO FO0.0

0.2

0.4

0.6

0.8

1.0

^

Effect of Fish Oil on Plasma Lipids

n=13-15 per group; ^P<0.001 vs OOOO-Olive Oil, FO-Fish Oil

Preliminary Data

Effect of Fish Oil on Hepatic Steatosis

CE

OO FO0

5

10

15

20

25

30

^

Ch

ole

ster

ol e

ster

(m

g/g

) TG

OO FO0

15

30

45

60

75

90

^

Tri

gly

ceri

des

(m

g/g

)

FC

OO FO0

1

2

3

4

Fre

e ch

ole

ster

ol (

mg

/g) FFA

OO FO0.0

0.2

0.4

0.6

0.8

1.0

1.2

Fre

e fa

tty

acid

s (m

g/g

)

A

C

B

D

n=4-10 per group; ^P<0.001 vs OOOO-Olive Oil, FO-Fish Oil

MCP-1

OO FO0.0

0.5

1.0

1.5

^

MC

P-1

mR

NA

(Rel

ativ

e E

xpre

ssio

n)

MIP-1

OO FO0.0

0.5

1.0

1.5

^

MIP

-1

mR

NA

(Rel

ativ

e E

xpre

ssio

n) MMP-12

OO FO0.0

0.5

1.0

1.5

^MM

P-1

2 m

RN

A(R

elat

ive

Exp

ress

ion

)

IL-1

OO FO0.0

0.5

1.0

1.5

^

IL-1

m

RN

A(R

elat

ive

Exp

ress

ion

) TNF

OO FO0.0

0.5

1.0

1.5

2.0

#

TN

F

mR

NA

(Rel

ativ

e E

xpre

ssio

n) SAA-1

OO FO0.0

0.4

0.8

1.2

#

SA

A-1

mR

NA

(R

elat

ive

Exp

ress

ion

)

A B C

D E F

Effect of Fish Oil on Inflammatory Genes in Liver

n=5-6 per group; ^P<0.001 and #P<0.01 vs OOOO-Olive Oil, FO-Fish Oil

Genes Upregulated in Liver upon Fish Oil Feeding-Microarray Analysis

Genes Fold Increase

CYP3A44 1.6

CYP2C68 1.6

Sult 1e1 2.2

Sult 1b1 2.0

Sult 3a1 1.8

GSTA1 2.2

GSTA2 1.6

PXR/GAPDH

PX

R/G

AP

DH

(Arb

itrar

y U

nits

)

OO FO0.0

0.2

0.4

0.6#

CYP3A/GAPDH

CY

P3A

/GA

PD

H(A

rbitr

ary

Un

its)

OO FO0.0

0.2

0.4

0.6

0.8#

PXR

GAPDH

CYP3A

GAPDH

OO FO OO FO

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)

CardioprotectiveEffects

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 Diet45% Fat (energy)1% Cholesterol

0.56% -3s

Chow Diet0.56%

Oleic Acid0.56% -3s

Chow Diet0.56%

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 Diet40% Fat (energy)0.5% Cholesterol

0.56% -3s

Chow Diet0.56%

Oleic Acid0.56% -3s

Chow Diet0.56%

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

InflammationInflammation

ApoptosisApoptosis

Bile Acid Detoxification

Bile AcidDetoxification

PXRSignaling

LCA+-3sLCA

Experimental Design-Specific Aim 3

Primary Hepatocytes from WT and PXR-/- Mice

Apoptosis

Experimental Design-Specific Aim 3

LCA

Human HepG2 Cell Line

-3s -3s

Scrambled siRNA for PXR siRNA for PXR

Inflammation

Apoptosis

Inflammation

Summary -3 Fatty Acids

(EPA & DHA)

PXR Signaling

CYP 2C & CYP 3A

Cholesterol & BileAcid Metabolism

3-Epoxides

Lipid-lowering Effects

Anti-inflammatory Effects

Anti-atherosclerotic Effects

Genetic Dyslipidemia

Dyslipidemia in Obesity

Liver

HepatocyteInflammation &

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.