Functional Changes And Mechanisms Of Cholesterol Efflux In Patients With Type 2 Diabetes

Background:Type 2 diabetes mellitus patients have increased cardiovascular risks which arecommonly associated with multiple atherogenic mechanism involving low levels ofhigh-density lipoprotein cholesterol (HDL-C) and abnormalities in the reversecholesterol transport (RCT) system. The RCT pathway consists of multiple differentsteps. Efflux of free cholesterol from peripheral cells to extracellular acceptors like HDLis considered to represent the first step of RCT. This article focused on abnormalities incholesterol efflux and its regulation in type 2 diabetes mellitus patients.Objectives:1. To investigate the expression of ABCA1 and ABCG1 in MDM in type 2 diabetespatients and healthy controls.2. We examined the cellular cholesterol efflux from peripheral MDM to HDL andapoA1 from same commercial sources and autoserum and sdandard serum from type 2diabetes and healthy controls.3. The mRNA expression levels of LXRαand LXRβwere measured through RealTimePCR system. DNA-protein complex of LXR and LXR element (LXRE) located inABCG1 promoter were dectected by Electrophery Mobility Supershift Assay (EMSA).Methods1. Patients with type 2 diabetes mellitus and age and sex matched healthy controls wererecruited and the patients with diabetes were regrouped into with and withoutcoronary heart disease. 40ml peripheral blood was collected from all participants forthe study and routine clinical laboratory assays.2. Cell preparation-Human peripheral blood mononuclear cells (PBMC) were isolatedusing Ficoll-Paque density gradient centrifugation and monocytes were isolatedusing EasySep human CD14 selection Kit according manufacturer's instructions.Monocytes were allowed to differentiate for 3 days with human macrophages colonystimulant factor (hM-CSF).3. Quantitative Real-time PCR was used to measure the mRNA expression of ABCA1and ABCG1.4. Western blot was used to detect the protein expression of ABCA1 and ABCG1. 5. Cholesterol assays-Cells were radio labeled with 1μCi/ml of [3H] cholesterol for24 hours in the presence of 10% FBS medium and then equilibrated for 2 hours inthe presence of serum free medium containing 0.2% fatty acid free bovine serumalbumin (BSA). BLT-1, a SR-B1 receptor inhibitor, was used to block thecholesterol efflux mediated by SR-B1 during equilibration. HDL, apoA1 and 5%autoserum and standard serum were then used to induce cholesterol efflux from thelabeled cells for 4 hours.6. ELISA-Serum free fatty acids (FFA) was measured by a human FFA ELISA Kitaccording to the manufacturer's instruction.7. The mRNA expression of LXRαand LXRβwas detected through QuantitativeReal-time PCR.8. The DNA-protein complex of LXR and LXRE located in promoter of ABCG1 wasdetermined by Electrophoresis Mobility Supershift Assay.9. Statistic analysis-Data for all experiments were analyzed using the SPSS 13.0software program. Comparisons between groups were performed using ANOVAmethods. Data are graphically represented as mean±S.E. Pearson's correlations wereused to test the relationship between variables. and multiple linear regression modelwas used to assess the relationships between ABCG1 expression and variousvariables simultaneously.Results1. The study population included 15 control subjects and 15 patients with both type 2diabetes and CAD and 15 patients with diabetes but no CAD. The patients with type2 diabetes mellitus have laboratory profiles, with high levels of hemoglobin A1c.elevated fasting glucose, hs-CRP and low HDL cholesterol. Other factors among 3groups were similar.2. The yield of PBMC isolated by Ficoll-Paque was 1×10~6/mL mononuclear cells/ml ofblood and the yield of monocytes isolated by EasySep human CD14 selection Kitwas 0.10-0.12×10~6/mL.3. The mRNA expression of ABCG1 measured by Real-time PCR was significantlydecreased in diabetic macrophages while ABCA1 mRNA revealed no changesamong the study groups.4. The protein expression of ABCG1 detected by western blot was significantlyreduced in diabetes macrophages and ABCA1 protein levels have no changes amonggroups. 5. Cholesterol effluxes to HDL, autoserum and pool serum were decreased inmacrophages from type 2 diabetic subjects compared with healthy controls.Cholesterol efflux to lipid-poor ApoA1 was unchanged between diabetes withoutCHD and controls and. However, the significant difference occurred between thediabetes subjects with CHD and healthy controls.6. There is no significant difference of Serum levels of FFA among 3 groups.7. On univariant analysis of all subjects, there was a positive correlation between theexpression of ABCG1 and HDL or serum mediated cholesterol efflux, as well as theexpression of ABCA1 and ApoA-1 mediated cholesterol efflux.8. The results of multiple linear regression analysis showed that HbA1c was theindependent determinants of ABCG1 mRNA expressions.9. The expressions of LXRαand LXRβmRNA were unchanged between diabetessubjects and healthy controls.10. There is no significant difference on DNA-protein complex of LXR and LXRElocated in the promoter of ABCG1 gene detected by EMSA between diabetessubjects and healthy controls.Conclusion1. The expression of ABCG1 was significantly derceased in type 2 diabetes subjectswhereas the levels of ABCA1were comparable with healthy controls.2. Cholesterol efflux from macrophages to HDL or serum was significantly impaired indiabetic patients whereas the ApoA-1 mediated cholesterol efflux was impaired indiabetic subjects with CHD rather than those without CHD.3. HbA1C was the independent determinant of the expression of ABCG1.4. The expressions of ABCG1 in diabetes patients might be down-regulated in aLXR-independent manner.