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CETP Expression Prevents Diet-Induced Atherosclerosis in Diabetic Mice
1. Cholesteryl Ester Transfer Protein
(CETP) Expression Prevents Diet-
Induced Atherosclerotic Lesions
in Male db/db Mice
Paul S. MacLean1
, Joseph F. Bower1
, Satyaprasad Vadlamudi1
, Jody
N. Osborne2
, John F. Bradfield2
, Hubert W. Burden3
, William H.
Bensch4
, Raymond F. Kauffman4
, Hisham A. Barakat1
Departments of Biochemistry1
, Comparative Medicine2
, and Anatomy and Cell Biology3
, in the Brody
School of Medicine, East Carolina University, Greenville, NC, 27858; Lilly Research Laboratories4
, a
Division of Eli Lilly and Company, Indiannapolis, IN, 46285
2. Cholesterol Ester Transfer Protein
• Structural Information
– Plasma glycoprotein, 4 glycosylation sites
– 2.2 kb mRNA, alternative splicing
• Functional Information
– Catalyzes the heteroexchange of neutral
lipids between the plasma lipoproteins
• Can modulate the composition and concentration
of the lipoproteins.
• Important role in Reverse Cholesterol Transport.
4. CETP and Vascular Health?
• Anti-Atherogenic
–Role in RCT,
clearing peripheral
cholesterol.
– Foger et al, 1999; Zhong et al, 1996;
Hayek et al, 1995; Breslow, 1995;
Sakai et al, 1995; Hirano et al, 1995;
Yamashita et al, 1995; Hennessy et al,
1993; Kinoshita et al, 1993; Stein et
al, 1985; Morton, 1988.
• Pro-Atherogenic
–Lowers HDL-C,
increases VLDL-
C and LDL-C.
– Rittershaus et al, 2000; Okamoto et
al, 2000; Mabuchi et al, 1995;
Inazu et al, 1994; Marotti et al,
1993; Bhatnagar et al, 1993;
Kinoshita et al, 1993; Agellon et
al, 1992; Quinet et al, 1991.
5. Why Study CETP?
• Perturbations in CETP expression could
lead to abnormal lipid/lipoprotein profiles
and contribute to vascular disease.
– Excess or deficiency in CETP expression
• CETP is viewed as a potential target in the
treatment of vascular disease, a tool to
modify atherogenic lipoproteins.
– CETP inhibitors, activators
6. CETP and the
Insulin Resistance Syndrome
• IRS
– dyslipidemia, obesity, diabetes, insulin
resistance, vascular disease, hypertension, etc.
• Alterations in CETP expression in IRS
– elevated in obesity
– type 2 diabetes?
7. Plasma CETP Activity and Mass
50
55
60
65
70
75
80
85
90
Activity
nmol/mL/hr
♣
♦
Values are given as means ± SEM (n).
♣ significantly different from the non-obese, p<0.001.
♦ significantly different from the obese, p<0.001.
1.5
1.75
2
2.25
2.5
Mass
ug/mL
♦
♣
(129) (95) (25) (20)(42)(57)
Values are given as means ± SEM (n).
♣ significantly different from the non-obese, p<0.05.
♦ significantly different from the obese, p<0.05.
Non-Obese Obese Obese NIDDM
8. How does Type 2 Diabetes influence the
characteristics of Obese Patients?
CVD
Risk ↑ over twice the risk
(Manson et al, 1990; Abbott et al, 1988)
Lipid
Abnormalities ↑ ↑TG, ↑LDL-C, ↓HDL-C
(Barakat et al, 1990; Barakat et al, 1992)
Lipoprotein
Abnormalities ↑ ↓LDLs, ↓HDLs, ↑VLDLs
(MacLean et al, 2000; Barakat et al, 1990-2)
Plasma
CETP Levels ↓ ~20%lower
(MacLean et al, 2000; Kahri et al, 1994)
9. Purpose
• To examine the effects of CETP
overexpression on vascular
health in a murine model of
diabetic obesity (db/db).
16. Summary
• The overexpression of CETP in db/db
mice:
–lowers total cholesterol concentrations
–lowers the amount of cholesterol in
VLDL and IDL/LDL subfractions
–prevented the formation of diet-induced
atherosclerotic plaques
17. Conclusions
• In this murine model of diabetic obesity, CETP
is clearly anti-atherogenic.
• In the metabolic context of diabetic obesity in
humans, CETP may play an important role in
maintaining vascular health.
• The suppressive effect of diabetes on CETP
expression in obese humans may contribute to
the higher risk of atherosclerosis.
Editor's Notes
CETP is a plasma glycoprotein that appears as a broad band between 61 and 74 kDa on an SDS-PAGE gel.
It is made up of 476 amino acids with four known glycosylation sites.
The 25 kb gene transcribes a 2.2 kb mRNA that appears to undergoes alternative splicing.
Once in the plasma, CETP catalyzes the heteroexchange of neutral lipids between the lipoproteins.
In this respect, it plays an important role in reverse cholesterol transport.
Reverse cholesterol tranport is the process by which cholesterol is cleared from the periphery and returned to the liver.
Cholesterol is collected from the tissues, esterified, and placed into HDL by the action of lecithin:cholesterol acyltransferase.
From the action of CETP, there is a net transfer of CE from HDL to VLDL and a net transfer of TG from VLDL to HDL.
VLDL is delipidated via the action of lipoprotein lipase and gradually becomes cholesterol-rich, triglyceride-poor LDL.
LDL is taken in by the liver where the CE are broken down and disposed of.
The TG-rich HDL is recycled by the delipiadting action of hepatic triglyceride lipase.
Thus, CETP is a pivotal step in RCT.
Two important points to mention here.
1. CET in the plasma is dependent upon both the concentration of CETP, as well as the composition and concentrations of the substrate lipoproteins.
2. The composition and concentration of the substrate lipoproteins is dependent upon a number of factors, only one of which is CETP.
Even so, it has been shown that CET from HDL to VLDL and LDLD critically depends upon CETP concentrations (Lottenberg 96, Bruce 98, Tg studies, 30% deficiency)
Several researchers have addressed this issue in studies involving humans, transgenic mice, and other mammalian models, with apparently conflicting results.
Some findings suggest that CETP is anti-atherogenic, linking its putative effects to it’s role in the reverse cholesteryl transport proccess.
In other words, CETP has been shown to facilitate the process of gathering and returning peripheral cholesterol to the liver.
On the other hand, other studies suggest that CETP is pro-atherogenic, linking its putative effects to atherogenic lipoprotein formation.
In other words, CETP has been linked to lowering HDL cholesterol and elevating VLDL and LDL cholesterol.
I observed that both CETP activity and mass were elevated in the plasma of obese patients when compared to lean, but that obese NIDDM patients had lower levels of both activity and mass when compared to obese non-diabetics.
The elevatedlevels in obese patients has been consistently reported in literature, but the effects of diabetes has been more controversial.
These data are consistent with all the reports in the literature in that only when the effect of diabetes was examined in obese subjects was a suppressive effect observed.
This raisesthe question, “What complicating factor of diabetes in obese subjects leadsto depressed CETP activity in the plasma.
To examine this question, I examined CETP expression in the liver and subcutaneous and omental adipose tissue of in a subset of obese and obese NIDDM subjects whose characteristics reflected that of all patients in which plasma CETP activity was measured.
In that the only differences I observed were in the liver, this is what I will present to you today.
The increased risk for cardiovascular disease, more atherogenic lipid concentrations, and more atherogenic lipoprotein profiles as exhibited by LDL and HDL size, and larger VLDL size.
In addition, we and others have reported that the well-documented elavation in plasma CETP activity that accompaniesobesity is suppressed by type 2 Diabetes.
Thus, we began to examine if this suppressive effect of type 2 diabetes on plasma CETP activity contributes to the formmation of these atherogenic lipid and lipoprotein profiles and the increased risk for cardiovascular disease.
Therefore, the purpose of this study was to examine the effects of CETP overexpression on vascular health in a murine model of diabetic obesity, or the dbdb mouse.
While the etiology of diabetes in this model is based upon the leptin receptor defect, it reflects the metabolic context with respect to insulin resistance, hyperinsulinemia, hyperglycemia, dyslipidemia, and, for male mice, susceptibility to atherosclerosis when fed an atherogenic diet.
We crossed genetically derived animals that were heterozygote for the db mutation and the human CETP trangene with its natural flanking regions in order acquire three groups of animals. Normal mice that expressed CETP, diabetic, obese mice without CETP expression, and diabetic obese mice that expressed CETP.
Plasma CETP activity in the lines expressing CETP was quite high, reflecting values 10x higher than what we normally see in humans. Both diabetic strains were hyperglycemic with elevated cholesterol and triglyceride levels.
After 16 weeks on an atherogenic diet, searched for lesion development in the proximal aorta and examined the cholesterol distribution among the lipoproteins.
Yet, the most striking differences between the strains of mice was observed when we excised and sectioned the proximal aorta for the analysis of atheroscleric lesion development.
5 cross sections in each animal, 80 um apart, were evaluated for the presence or absence of oil red O positive lesions.
We did not observe any lesions in CETP mice, as has been shown by others in the background strain when placed on a similar diet.
Out of 17 db mice, 15 developed lesions in response to the diet. In these 15 mice, 1 to 3 lesions were found per animal with mean area of 26,000 um2.
However, the introduction of CETP into this dibetic obese model led to no lesion development in the 22 mice we examined.
While db were characterized by higher total cholesterol levels, particularly during the diet, db/CETP mice had levels similar to lean, non-diabetic CETP mice.
FPLC analysis of the lipoproteins from pooled plasma samples collected at the end of the diet indicated that the majority of this cholesterol difference was due to higher amounts in the VLDL and IDL/LDL subfractions, without a substantial difference in HDL-C.
We subsequently looked at the timecourse of these differences in another set of mice. VLDL-C was elevated during the diet in all but the last time point, in db mice, but not in db/CETP mice. Ccholesterol in the IDL/LDL subfraction was elevated in the db mice, but not the CETP mice, before, during, and after the dietary regimen.
In summary, the overexpression of CETP in this murine model of diabetic obesity
1. Lowers total plasma cholesterol concentrations
2. Lowers the amount of cholesterol in VLDL and IDL/LDL subfractions
3. And most strikingly, prevented the formation of diet-induced atherosclerotic plaques.
So in this particular model, CETP is clearly atherogenic.
And these findings support the hypothesis that:
1. in the metabolic context of diabetic obesity in humans, CETP may play an important role in maintaining vascular health.
2. The suppressive effect of diabetes on CETP expression in obese humans may contribute to the higher risk of atherosclerosis.
