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

Tota

l Cho

lest

erol

(mg/

dL)

OO FO0

100200300400500600

^

TG-Plasma

Trig

lyce

rides

(mg/

dL)

OO FO0

50

100

150

200

250

^

FFA-Plasma

Free

Fat

ty A

cids

(mEq

/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

^

Cho

lest

erol

est

er (m

g/g)

TG

OO FO0

15

30

45

60

75

90

^

Trig

lyce

rides

(mg/

g)

FC

OO FO0

1

2

3

4

Free

cho

lest

erol

(mg/

g)

FFA

OO FO0.0

0.2

0.4

0.6

0.8

1.0

1.2

Free

fatty

aci

ds (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 Ex

pres

sion

) MIP-1

OO FO0.0

0.5

1.0

1.5

^

MIP

-1

mRN

A(R

elat

ive

Expr

essi

on) MMP-12

OO FO0.0

0.5

1.0

1.5

^MM

P-12

mR

NA

(Rel

ativ

e Ex

pres

sion

)

IL-1

OO FO0.0

0.5

1.0

1.5

^

IL-1

m

RNA

(Rel

ativ

e Ex

pres

sion

) TNF

OO FO0.0

0.5

1.0

1.5

2.0

#

TNF

mRN

A(R

elat

ive

Expr

essi

on) SAA-1

OO FO0.0

0.4

0.8

1.2

#

SAA-

1 m

RN

A (R

elat

ive

Expr

essi

on)

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 IncreaseCYP3A44 1.6CYP2C68 1.6Sult 1e1 2.2Sult 1b1 2.0Sult 3a1 1.8GSTA1 2.2GSTA2 1.6

PXR/GAPDH

PXR

/GAP

DH

(Arb

itrar

y U

nits

)

OO FO0.0

0.2

0.4

0.6#

CYP3A/GAPDH

CYP

3A/G

APD

H(A

rbitr

ary

Uni

ts)

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% -3sChow Diet 0.56%

Oleic Acid0.56% -3sChow 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 Diet40% Fat (energy)0.5% Cholesterol

0.56% -3sChow Diet 0.56%

Oleic Acid0.56% -3sChow 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

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.