1. CETP transgenic female mice on high-fat diet are obese but insulin sensitive
Analysis of mRNA expression (normalized to ppia)
CETP Augments Estrogen Signaling to Promote Insulin Sensitivity in Obese Female Mice
Laura Abell1,2, Brian Palmisano1, David Cappel1, John Stafford1
AKT – phosphorylated vs. total
ERK 1/2 – phosphorylated vs. total
Mice are an ideal model organism for studying disease mechanisms...
BUT, lipid profiles differ between mice and humans
Solution:
Transgenic CETP mouse is a better
model of human lipid metabolism
CETP: A Mouse Model of Atherosclerosis
20 40 60
0
1
2
3
4
5
HDLLDLVLDL
CETP Mice
Fraction
Cholesterol(mg/fraction)
20 40 60
0
1
2
3
4
5 HDLLDLVLDL
C57 Mice
Fraction #
Cholesterol(mg/fraction)
20 40 60
0
1
2
3
4
5
HDLLDLVLDL
HUMAN
Fraction
Cholesterol(mg/fraction)
A B C
• Mice have high HDL (“good”
cholesterol) & very little LDL or VLDL
• Mice don’t express CETP
Genetically uniform Easy access to tissue samples
Normal mouse HumanCETP transgenic mouse
Diabetes increases risk of CHD
• The leading cause of death in patients
with diabetes is heart disease.
• CHD accounts for 65% of all deaths in
individuals with diabetes
• Lifetime risk of cardiovascular events is
2-4 times greater in diabetics vs.
Those without diabetes
Female gender reduces risk of CHD
• Women have a delayed onset of CHD
compared to men of the same age and
BMI
Diabetes, Heart Disease, and the Gender Gap
11.5
6.3
29.9
19.2
0
5
10
15
20
25
30
35
Men Women
Yearlydeathsper1000people
Heart Disease
No diabetes
Diabetes
35 45 55 65
0
5
10
15
Women
Men
FirstMI
(per1000persons)
About
Half
Delayed Onset
25 35 45 55 65
0
2
4
6
8
FirstMI
(per1000persons)
Presence of
Estrogen?
Pre Menopause Post Menopause
• After menopause, instance of heart attack
and other cardiovascular events rises
• Is estrogen involved?
Plasma
Triglyceride
Production Storage
C
B100
mature
VLDL
TG
B100
LDL
CE CETP TGCholesterol
Delivery to liver
(RCT)
CETP CETP
CE CETP TG
Under high fat diet, CETP may protect against development of insulin
resistance by promoting TG storage in adipose and reducing liver fat
≈
“Healthy” Obesity: Paradox or Possibility?
Menopause as a model of a metabolic
transition between low and high risk for
cardiovascular disease.
Sample
Vein Artery
Insulin
Glucose
RBCs
[3-3H] Glucose
[14C] Glycerol
Hyperinsulinemic-euglycemic
clamp is used to measure insulin
sensitivity
• Insulin is infused at a constant
rate
• Glucose is infused at a rate
necessary to maintain
euglycemia (~150 mg/dl)
• More glucose infused
indicates greater insulin
sensitivity
Male Female Male Female
Non-Transgenic LittermatesCETP Transgenic
Four
groups
W
T
M
ale
C
ETP
M
ale
W
T
Fem
ale
C
ETP
Fem
ale
0
50
100
150
200
* *
CETPActivity
(pmolTransfered)
W
T
M
ale
C
ETP
M
ale
W
T
Fem
ale
C
ETP
Fem
ale
0
50
100
150
200
FastingBloodGlucose
(mg/dL)
W
T
M
ale
C
ETP
M
ale
W
T
Fem
ale
C
ETP
Fem
ale
0.0
0.5
1.0
1.5
2.0 *
*
*
FastingInsulin(ng/mL)
C D
0
10
20
30 * ***
High Fat Diet - + - + - + - +
*
CETP
Female
WT
Female
CETP
Male
WT
Male
Adiposity
(%BodyFat)
A B
W
T
M
ale
C
ETP
M
ale
W
T
Fem
ale
C
ETP
Fem
ale
0
50
100
150
200
* *
CETPActivity
(pmolTransfered)
W
T
M
ale
C
ETP
M
ale
W
T
Fem
ale
C
ETP
Fem
ale
0
50
100
150
200
FastingBloodGlucose
(mg/dL)
W
T
M
ale
C
ETP
M
ale
W
T
Fem
ale
C
ETP
Fem
ale
0.0
0.5
1.0
1.5
2.0 *
*
*
FastingInsulin(ng/mL)
C D
0
10
20
30 * ***
High Fat Diet - + - + - + - +
*
CETP
Female
WT
Female
CETP
Male
WT
Male
Adiposity
(%BodyFat)
A B

Low fasting
insulin
% body fat: low-fat vs. high fat diet
All groups gained fat
Fasting insulin during high-fat diet
Low fasting insulin in CETP females
W
T
M
aleC
ETP
M
aleW
T
Fem
aleC
ETP
Fem
ale
0
200
400
600
800
*
*
*
InsulinSensitivityIndex
mg·ml/(kg·min·mU)
0 50 100
0
10
20
30
40
50
*
*
*
*
*
*
* * * *
WT Male
CETP Male
WT Female
CETP Female
* *
Time (min)
GIR(mg/kg/min)
0 50 100
0
50
100
150
200
WT Male
CETP Male
WT Female
CETP Female
Time (min)
BloodGlucose(mg/dL)
Glucoseto 125-150mg/dL
Insulin4mU/kg/min
Donor erythrocytes
3-3
H-Glucose Primed constant infusion
1200-90
Hyperinsulinemic-Euglycemic ProtocolA B
C D

High rate of
glucose infusion

High insulin
sensitivity
Clamp studies confirmed that
CETP female mice on HFD were
more insulin-sensitive than either
CETP males or WT mice
CETP augments estrogen-mediated changes in mRNA expressionHypothesis:
Why do the effects of
CETP differ with gender?
Estrogen-induced mRNA changes in
ovariectomized female mice
CETP
Wild Type
CETP requires estrogen to enhance insulin signaling in males
Gpat2 (ppia)
0 hr 24 hr 0 hr 24 hr
0.0
0.5
1.0
1.5
WT
CETP*
#, &, %SHP (ppia)
0 hr 24 hr 0 hr 24 hr
0.0
0.5
1.0
1.5
2.0
2.5
WT
CETP
*
CETP (ppia)
0
H
r
24
H
r
0.0
0.5
1.0
1.5
FoldChange
Insulin-induced signaling effects in
estrogen-treated male mice
Hypothesis:
Our Model
I would like to thank the Vanderbilt Summer Science Academy and its leadership for their
support, as well as the Stafford lab for facilitating my research experience this summer.
This work was supported by the Department of Veterans Affairs Merit Award (BX002223).
Acknowledgements
1Department of Medicine, Division of Diabetes Endocrinology, Vanderbilt University, Nashville, TN.
2Auburn University
CETP expression remained
constant at 24 hours post-
injection in CETP+ mice.
CETP
In CETP+ mice, SHP expression
expression increased over 24 hours
in response to estrogen injection.
SHP GPAT2
Baseline GPAT 2 expression was
significantly decreased in CETP vs.
WT mice.
Is estrogen responsible for
CETP’s gender-specific protection?
Wild Type
CETP
P-AKT
AKT
CETP transfers triglycerides
from VLDL to HDL & moves
cholesteryl esters from HDL to
VLDL
Cholesteryl esters in VLDL can be
taken up by the liver and
converted to bile acids
Bile acids promote insulin
sensitivity
WT saline WT insulin CETP saline CETP insulin
Results: AKT phosphorylation increased in response to insulin, but was not
enhanced in CETP mice vs. WT
P-ERK 1/2
ERK 1/2
WT saline WT insulin CETP saline CETP insulin
Results: ERK 1/2 phosphorylation increased in response to insulin, but only in
CETP mice
We hypothesize that CETP enhances insulin
sensitivity by:
• Enhancing estrogen signaling through Erα,
activating SHP and downregulating
gluconeogenesis
• Improving uptake of cholesteryl esters for
conversion to bile acids
5 weeks high fat diet
E2 injection every 4 days
Final E2 injection
6 hr fast
Insulin
Saline
Liver tissue harvested 15 minutes after saline or insulin injection
1 week
Subcutaneous
E2 injection
0 hr tissue
harvest
24 hr tissue
harvest
Ovariectomy
Bile Acids
Cholesteryl
Ester
Fxr
Shp
Gluconeogenic Genes
•G6pc
•Pck1
CETP
+
ERα
E2
+
High GIR in CETP females High insulin sensitivity index
in CETP females
P-Akt
- + - +
0
1
2
3
4
insulin
WT CETP
FoldChange
P-Erk
- + - +
0
1
2
3
FoldChange
insulin
WT CETP