PXR

Report
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
Metabolic Syndrome and CVD
•
•
•
•
•
Abdominal obesity
Atherogenic dyslipidemia
Insulin resistance
Elevated blood pressure
Pro-inflammatory state
Fish
and
PUFAs
Fish
OilOil
and
-3-3
Fatty
Acids
EPA-Eicosapentaenoic acid
DHA-Docosahexaenoic acid
Clinical Evidence for the Beneficial
Effects of Fish Oil
• 30 g of fish per week reduced caronary
artery disease (Kromhout DBE, 1985).
• EPA&DHA reduced plasma TG and nonHDL 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).
Mechanisms
TG-Lowering Effect
•
•
•
Reduced TG secretion
Increased TG clearance
Increased -oxidation
Mechanisms Mediating the
Cholesterol-Lowering Effects
Cholesterol
CYP 7A1
CYP 27A1
CYP 11A
CYP 3A
Cholesterol
hydroxylation
Bile acid
Sulfotransferases
Glutathione S transferases
CYP3A
Sult1e1
Sult2a1
Sult3e1
Gsta1
Gsta2
Bile acid
Detoxification
PXR
Mechanisms Mediating the AntiInflammatory Effects of -3s
•
•
•
•
Interference with arachidonic acid metabolism
COX-derived 3-series eicosanoids
LOX-derived resolvins
Cytochrome P450-derived epoxides
PXR and Inflammation
PXR
CYP 2C and
CYP 3A
-3 FAs
-3 epoxides
Antiinflammatory
PXR
•Drug detoxification
•Bile acid homeostasis
•Cholesterol metabolism
•Reduce inflammation
Preliminary Data
Effect of Fish Oil on Plasma Lipids
1.0
250
500
^
400
300
200
100
OO
FO
200
150
^
100
50
0
OO
FO
n=13-15 per group; ^P<0.001 vs OO
OO-Olive Oil, FO-Fish Oil
Free Fatty Acids
(mEq/L)
600
0
FFA-Plasma
TG-Plasma
Triglycerides
(mg/dL)
Total Cholesterol
(mg/dL)
TC-Plasma
0.8
0.6
^
0.4
0.2
0.0
OO
FO
Effect of Fish Oil on Hepatic Steatosis
B
CE
25
20
15
^
10
5
0
75
60
45
^
30
15
FO
O
O
FO
0
D
Free fatty acids (mg/g)
3
2
1
FO
O
O
0
FFA
1.2
1.0
0.8
0.6
0.4
0.2
0.0
n=4-10 per group; ^P<0.001 vs OO
OO-Olive Oil, FO-Fish Oil
FO
FC
4
O
O
C
Free cholesterol (mg/g)
TG
90
Triglycerides (mg/g)
30
O
O
Cholesterol ester (mg/g)
A
Effect of Fish Oil on Inflammatory
Genes in Liver
0.0
OO
1.0
^
0.5
0.0
FO
OO
IL-1
1.0
^
0.5
0.0
OO
FO
MMP-12
1.5
1.0
0.5
^
0.0
FO
E
1.5
MMP-12 mRNA
(Relative Expression)
^
0.5
MIP-1
1.5
OO
FO
F
TNF
2.0
1.5
1.0
#
0.5
0.0
OO
FO
SAA-1 mRNA
(Relative Expression)
1.0
MIP-1 mRNA
(Relative Expression)
MCP-1
1.5
D
IL-1 mRNA
(Relative Expression)
C
B
TNF mRNA
(Relative Expression)
MCP-1 mRNA
(Relative Expression)
A
SAA-1
1.2
0.8
#
0.4
0.0
OO
n=5-6 per group; ^P<0.001 and #P<0.01 vs OO
OO-Olive Oil, FO-Fish Oil
FO
Genes Upregulated in Liver upon Fish
Oil Feeding-Microarray Analysis
Genes
CYP3A44
CYP2C68
Sult 1e1
Sult 1b1
Sult 3a1
GSTA1
GSTA2
Fold Increase
1.6
1.6
2.2
2.0
1.8
2.2
1.6
Effect of Fish Oil on PXR and CYP3A
in Liver
OO
PXR
CYP3A
GAPDH
GAPDH
PXR/GAPDH
CYP3A/GAPDH
#
0.4
0.2
0.0
OO
FO
0.8
CYP3A/GAPDH
(Arbitrary Units)
PXR/GAPDH
(Arbitrary Units)
0.6
FO
OO
FO
#
0.6
0.4
0.2
0.0
OO
FO
OO,Olive Oil; FO, Fish Oil, n=5-6 samples per group, #P<0.01 vs OO
Hypothesis
Cholesterollowering Effects
-3 Fatty Acids
(EPA & DHA)
PXR
Cardioprotective
Effects
Anti-inflammatory
Effects
Overall Hypothesis: The cholesterol-lowering and antiinflammatory 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.
Experimental Design-Specific Aim 1
Experimental Diets
High Fat Diet
45% Fat (energy)
1% Cholesterol
Chow
Diet
Study Groups
Mutant-PXR-/-
WT-PXR+/+
Chow Diet
0.56%
Oleic Acid
0.56%
-3s
Chow Diet
0.56%
Oleic Acid
0.56%
-3s
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
Experimental Design-Specific Aim 2
Experimental Diets
High Fat Diet
40% Fat (energy)
0.5% Cholesterol
Chow
Diet
Study Groups
LDLR;PXR-/-
LDLR-/-
Chow Diet
0.56%
Oleic Acid
0.56%
-3s
Chow Diet
0.56%
Oleic Acid
0.56%
-3s
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
Experimental Design-Specific Aim 3
Primary Hepatocytes from WT and PXR-/- Mice
PXR
Signaling
LCA
Bile Acid
Detoxification
Inflammation
Apoptosis
LCA+
-3s
Bile Acid
Detoxification
Inflammation
Apoptosis
Experimental Design-Specific Aim 3
Human HepG2 Cell Line
Scrambled
siRNA for PXR
siRNA for PXR
Inflammation 
Inflammation 
-3s
-3s
LCA
Apoptosis 
Apoptosis 
Summary
Dyslipidemia
in Obesity
Genetic
Dyslipidemia
-3 Fatty Acids
(EPA & DHA)
SA2
SA1
Hepatocyte
Inflammation &
Apoptosis
PXR Signaling
SA3
CYP 2C & CYP 3A
Cholesterol & Bile
Acid Metabolism
3-Epoxides
Liver
Lipid-lowering
Effects
Anti-inflammatory
Effects
Anti-atherosclerotic
Effects
Impact
•
Identification of novel molecular mechanisms by which 3 fatty acids mediate their cholesterol-lowering and antiinflammatory effects.
•
The findings will be critical to target PXR using dietary
factors to efficiently prevent/treat dyslipidemia in humans
without adverse side effects.

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