| Background:Type 2 diabetes patients tend to have lipid disorder.It is well accepted that reverse cholesterol transport(RCT) is abnormal in type 2 diabetes.This study will focus on change of cholesterol efflux among this group of patients,which is regarded as the initial important step of reverse cholesterol transport.Previous research shows that,efflux of cholesterol from cultured fibroblast to high-glucose plasma decreased compared to controls[1],and the ability of efflux from Fu5AH to plasma of diabetes was also injuried[2].However,it was recently reported that the ability of plasma of type 1 diabetes to induce cholesterol efflux from Fu5AH and HSF overexpressed ABCA1 was enhanced[3].Actually,the plasma of diabetes contains a lot of components,and some of these components would influence the cholesterol efflux,such as the levels of insulin[4],glucose,cytokine[5],glucosylated lipoprotein[7],et al.,so the efflux of cholesterol not only depends on HDL,but other factors.It is not clear whether the disorder of non-lipoprotein or the "quality" or "quantity" of HDL plays an important role in the cholesterol efflux.Among the studies of efflux of cholesterol,ABCA1,SR-BI are the most often studied membrane proteins[8].Recently,ABCG1 was reported to be an important half-tranportor in cholesterol efflux.Besides that,cholesterol can also efflux via ABCG4,ABCG8 and diffusion.Some cells,for example HepG2,HSF,can express membrane proteins exclusively,so that they can be used to study the function of some membrane protein.It was proved[7]that the efflux of cholesterol in vitro could predict the atherosclerosis levels in diabetes and non-diabetes.We are going to find out the characteristics of the cholesterol efflux in peripheral cells of type 2 diabetes through the study in vitro.Objectives:1.To determine the expression ofSR-BI,ABCA1,and ABCG1 in HepG2,HSF,and HUVEC cells.2.In order to find out of effects of plasma and HDL of type 2 diabetes on cholesterol efflux from peripheral cells,we investigate the different effect of HDL,LPDS and serum of type 2 diabetes patients on the cholesterol efflux via ABCA1 and SR-BI exclusivelyMethods:1.Isolate and culture the HSF and HUVEC cells with tissue adherence and collagenase perfusion.2.Test the expression of SR-BI,ABCA1,ABCG1 in HepG2,HSF,HUVEC with RT-PCR and Western Blot.3.Isolate HDL and LPDS from plasma with ultracentrifugation.4.Compare the ability of cholesterol efflux between type 2 diabetes and non-diabetes patients with H3 isotope labeled HDL.5.Determine the cholesterol efflux via ABCA1 with the difference between basic and post-stimulated with 22-OH cholesterol in HSF to determine the the cholesterol efflux via SR-BI with HepG2.6.Test the activity of PLTP and CETP with fluorescent self-quenched method.7.Determine the relationship between the ability of cholesterol efflux and basic data, activity of PLTP,CETP of patients. 8.Data analysis by SPSS 13.0.Results:1.HSF could be isolated,cultured with tissue adhesive method.2.HUVEC could be isolated,cultured successfully with collagenase perfusion method.3.RT-PCR and Western Blot proved that HepG2 could highly express SR-BI,and the ABCA1 and ABCG1 could be highly expressed in HSF and HUVEC respectively after stimulated with 22-OH cholesterol.4.HDL and LPDS could be isolated successfully from plasma with ultracentrifugation method.5.The HDL of type 2 diabetes patients has a reduced ability to induce cholesterol efflux from HepG2 cells than non-diabetes(P=0.0015).6.There was no significant difference in the ability of serum to induce cholesterol efflux from HepG2 between the diabetes and control group(P=0.19).7.The difference of the ability of HDL to induce cholesterol efflux from HSF between basic and post-stimulated with 22-OH cholesterol had no difference in both groups.8.The difference of the ability of serum to induce cholesterol efflux from HSF between basic and post-stimulated with 22-OH cholesterol had no significant difference in both groups.(control VS type 2 diabetes,5.7±4.4%VS 8.8±5.9%,P=0.33).9.The difference of the ability of LPDS to induce cholesterol efflux from HSF between basic and post-stimulated with 22-OH cholesterol was higher in controls (control VS type 2 diabetes,11.0±7.7%VS 6.6±4.7%,P=0.02). 10.The activity of PLTP in type 2 diabetes is significantly higher than in controls, and positively related with the ability of cholesterol efflux via ABCA1 in controls and in all patients.The difference of the activity of CETP in both groups was not observed.Conclusions:1.HepG2 strongly expresses SR-BI,while HSF and HUVEC highly express ABCA1 and ABCG1 exclusively after stimulating by 22-OH cholesterol.These cell strains can be used to study cholesterol efflux via SR-BI,ABCA1,ABCG1.2.HDL and LPDS(contains apoA1 and pre-βHDL)of type 2 diabetes patients is deficient in the ability to induce cholesterol efflux from HepG2,which may contribute to cholesterol deposit in peripheral cells of diabetes patients.3.The activity of PLTP is higher in type 2 diabetes compared with controls,which may contribute to the enhanced ability of cholesterol efflux of serum via ABCA1 in type 2 diabetes. Background:Observational studies as well as animal experiments have indicated that estrogen attenuates cardiac hypertrophy,indicating a cardioprotective role for estrogen. However,conflicting results have been reported.The controversies regarding the effect of estrogen on the heart could be related in part to the dose used.Our previous study using a mouse model of myocardial infarction showed that low dose of estrogen is cardioprotective,whereas at moderate and higher dose,it become detrimental to the heart and is associated with renal damage.This indicates that the dose-dependent effect of estrogen may play an important role in the outcomes of estrogen replacement therapy.Objectives:1.To determine the dose effect of estrogen replacement on cardiac hemodynamics, morphology,mass and cardiac function in ovariectomized mice.This includes measurement of blood pressure and heart rate,myocyte size,interstitial collage deposition,capillary density,left ventricular mass,dimensions and function,as well as cardiac reserves.2.To determine the effect of estrogen on renal function,including kidney mass, albuminuria and creatinine clearance;3.To study whether the dose dependent effects of estrogen are due to activation of different signaling pathways,including PKC,PI3K,ERK.Methods:1.Mice were subject ovariectomy(ovx) or sham ovx.Estrogen replacement was initiated after ovx at dose of 0.025(very low),0.24(low),4.2(moderate) and 28.3 μg/day(high) via subcutaneous implantation of estrogen-containing pellets.2.Using echocardiography and intra-cardiac catheterization,we evaluated the dose effects of estrogen on left ventricular(LV) mass,dimensions and function,as well as LV functional response to isoproterenol,a measure of functional preservation;3.Immunohistochemistry was used to study the dose effects of estrogen on myocardial interstitial collagen deposition and myocyte cross-sectional area capillary density;4.Western blot was used to determine the dose effect of estrogen on protein expression of PI3K,ERK,PKC.5.Metabolism cage was used to study the dose effect of estrogen on 24 hr albuminuria and creatinine clearance.Results:1.Low dose estrogen at(ovx+L) slightly increased heart weight,whereas at higher doses(4.2ug/d and 28.3μg/d),estrogen decreased heart weight(p＜0.05 ovx+H vs ovx+L);2.No dose-effect was observed in collagen deposition and capillary density in the heart,but the MCSA in ovx+H tends to deline.3.Estrogen had no effect on blood pressure in all groups;however,heart rate was gradually decreased with increased dosage(p＜0.05 ovx+H vs sham,ovx,ovx+VL and ovx+L);4.LVEF was decreased in the ovx+M,ovx+H compared to ovx+placebo(LVEF in ovx+placebo,ovx+M,ovx+H is 80.89±0.46,77.48±1.09,78.87±0.63,P＜0.05); however,with increased dose of estrogen,estrogen tended to increase LVDs, LVDd(P＜0.05). 5.Functional preservation was not changed by the different doses of estrogen;6.With increasing doses of estrogen,kidney mass and 24 hr albumin excretion increased significantly(P＜0.05 VS sham,P＜0.01 VS ovx+placebo,P＜0.05 VS sham+VL),whereas creatinine clearance tended to decline(P=0.056).7.Moderate and high doses of estrogen activated PCK and PI3K,whereas ERK and P70S6K tended to decline with increasing dose of estrogen.Conclusions:1.Low dose of estrogen may effect the structure and function of heart via ERK signaling pathway,while high dose of estrogen does via PKC and PI3K;then, Low and high doses of estrogen may influence the growth of myocytes through different signaling pathway.2.There is no significant effect on the collagen deposit and capillary density with the doses of estrogen which is used in this experiment,but the MCSA in ovx+H tends to deline.3.But high dose of estrogen may influence the structure and function to a certain extent:it resulted in the ventricular chamber enlargement,heart mass/body weight decline,and left ventricular ejection fraction fall a little;it also increase the mortality in the post-MI mice,decrease the left ventricular ejection fraction.It indicated that high dose of estrogen might impair the heart function both in basic and post-MI mice.4.Estrogen had a detrimental effect on renal function,including increasing albuminuria and enlarges kidney mass,which may result from the vascular endothelium injuries due to the change in blood lipid.
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