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Insulin has been reported to influence cholesterol removal from different cells, but the results have been controversial. Based on our previous study, which we found that insulin promote cholesterol efflux from HepG2 cells and can reverse the decreased cholesterol efflux to HDL though PI3K-Akt pathway, we further investigate the effects of insulin on damaged cholesterol efflux by MCP-1 in this report.
3T3-L1 preadipocytes were obtained from the Cell Bank of the Chinese Academy of Sciences and differentiated into adipocytes as described previously. Fully differentiated adipocytes (day 13) seeded in collagen-coated 24-well plates were starved for 6 h and labeled with 3H-cholesterol (1 Ci/ml) (Perkin-Elmer Analytic Sciences, Boston, MA) for 24 h. Cellular cholesterol efflux was initiated by the addition of DMEM containing 0.2% BSA with 20μg/ml human apoA1 or 50 μg/ml HDL3 with the indicated dose of MCP-1 for the indicated period of time in the presence or absence of insulin. After incubation, the radioactivity of the medium and cells was measured using a liquid scintillation counter. Cholesterol efflux was calculated as the percentage of total [3H]-cholesterol released into the medium after subtraction of the values obtained in the absence of a cholesterol acceptor. 3T3-L1 adipocytes were harvested for Real-time PCR, western blotting, cell-surface protein assays and Confocal microscopy. The data were statistically analyzed using SPSS 13.0, and the results are expressed as the means ± SD.
1.MCP-1 reduced cholesterol efflux to HDL3 and apoA1 in differentiated 3T3-L1 adipocytes. 2. In differentiated 3T3-L1 adipocytes, MCP-1 reduced cholesterol efflux to HDL3 mainly by inhibiting SR-BI and ABCG1 and to apoA1 mainly by inhibiting ABCA1. 3. MCP-1 decreased ABCA1 and SR-BI mRNA expression but not ABCG1. 4. MCP-1 decreased total and cell surface ABCA1, ABCG1, and SR-BI protein expression as shown by Western blotting and confocal microscopy in differentiated 3T3-L1 adipocytes. 5. Insulin increased MCP-1 suppressed cholesterol efflux to HDL3 and apoA1 depending on Akt phosphorylation. 6. Insulin reversed MCP-1 suppressed ABCA1 and SR-BI mRNA expression, and ABCA1, ABCG1 and SR-BI protein expression via PI3K/Akt pathway.
Insulin reverses the suppressed cholesterol efflux to HDL3 and apoA1 by MCP-1 through up-regulation of ABCA1, ABCG1 and SR-BI through PI3K/Akt pathway in 3T3-L1 adipocytes, which provides evidences that insulin may improve the MCP-1-induced adipocyte cholesterol imbalance to exert the anti-inflammatory effect.