Adipophilin Accumlated Cellular Cholesteryl Ester By ACAT1 In THP-1 Macrophages

The accumulation of neutral lipid,such as cholesterol, plays a key role in the foam-cell formation and in the development of atherosclerotic lesion. In eukaryocytes, the deposited lipid is in the form of lipid droplets. The content of adipophilin is highest among some 40 lipid droplet-related proteins.Its main function is to promote lipid accumulation and inhibit cholesterol efflux . ACAT(acyl-coenzyme A:cholesterol acyltransferase, ACAT) is the unique intracellular cholesterol ester synthetase .It catalyzes free cholesterol and long-chain fatty acid to synthesize cholesterol ester so that it can influences the development of atherosclerosis. The concordance of adipophilin and ACAT in function and location suggests that there is close correlation between them. To have a further study for the regulation factors in lipid-accumulation mediated by adipophilin,our study , firstly, observed the expression changes of adipophilin and ACAT1 and also the lipid accumulation condition in THP-1 macrophages loaded with lipid; then overviewed the expression of ACAT1 and the lipid accumulation condition in THP-1 macrophages incubated with ACAT1 inhibitor S-58035;finally, measured the expression of ACAT1 and the lipid content in THP-1 macrophages which overexpressed adipophilin.Part 1 Effects of oxLDL or ACAT inhibitor S-58035 on Adipophilin and ACAT1 Expressions and lipid accumulation in THP-1 MacrophagesObjective: To investigate the effects of oxLDL or ACAT inhibitor S-58035 on adipophilin or ACAT1 expression and lipid accumulation in THP-1 macrophages.Methods:①THP-1 macrophages were treated with 0,10,20,40,80μg/ml oxLDL for 24hr, respectively; or 80μg/ml oxLDL for 0,3,6,12,24hr,respectively.The mRNA level of adipophilin or ACAT1 was determined by reverse transcription-polymerase chain reaction(RT-PCR) ;Cellular lipid accumulation was determined by Oil Red O staining.②After incubated with 80μg /ml oxLDL for 24hr,THP-1 macrophages were treated with 0,2,5,10,20μg/ml S-58035 for 24hr, respectively; or 10μg/ml S-58035 for 0,3,6,12,24hr.The mRNA level of ACAT1 was determined by reverse transcription-polymerase chain reaction(RT-PCR) ;Cellular lipid accumulation was determined by Oil Red O staining.Results: With the increasing incubated concentration or time of oxLDL , the mRNA levels of adipophilin and ACAT1 both up-regulated obviously.Intracellar lipid droplets also increased.Reversely, the upregulated expression of ACAT1 induced by oxLDL was inhibited with the increasing incubated concentration or time of S-58035, and intracellar lipid droplets also decreased.Conclusion: When THP-1 macrophages incubated with oxLDL, both adipophilin and ACAT1 were high expression, and cellular cholesteryl ester also accumulated. ACAT inhibitor S-58035 inhibited ACAT1 high expression induced by oxLDL with dose-dependent and time-dependent manner, and decreased the cellular cholesteryl ester.Part 2 Effects of overexpressed adipophilin on ACAT1 Expression and lipid accumulation in THP-1 MacrophagesObjective: To investigate the effects of overexpressed adipophilin on ACAT1 expression and lipid accumulation in THP-1 macrophages.Methods: The isolated plamids were tested with the methods of enzyme-shearing and RT-PCR and then used to transfect THP-1 macrophages.The cells devided into 8 groups as follows: 1.control; 2.oxLDL; 3. pcDNA3.1 transfection; 4.oxLDL+ pcDNA3.1 transfection; 5. pcDNA3.1-HA-adi transfection; 6. oxLDL+pcDNA3.1-HA-adi transfection; 7. pcDNA3.1-HA-adi transfection +S-58035 8.oxLDL+pcDNA3.1-HA-ad transfection+S-58035.The doses of oxLDL and S-58035 are 80μg/ml and 10μg/ml,respectively. The mRNA level of ACAT1 was determined by reverse transcription-polymerase chain reaction(RT-PCR) ;Cellular lipid content was determined by High Performance Liquid Chromatography(HPLC). Results: The results of enzyme-shearing and RT-PCR confirmed the plasmids were as expected.RT-PCR and Western blot results showed that pcDNA3.1-HA-adi transfection upregulated mRNA and protein levels of adipophilin in THP-1 macrophages. Acccording to RT-PCR and HPLC results, pcDNA3.1 transfection had no effects on ACAT1 expression and intraceller lipid accumulation , but pcDNA3.1-HA-adi transfection significantly upregulated ACAT1 expression and promoted intraceller lipid accumulation.These effects had synergism with oxLDL。Notably,S-58035 inhibited these effects of pcDNA3.1-HA-adi transfection.Conclusion: We successful constructed the pcDNA3.1-HA-adi plasmid. Adipophilin mRNA and protein were highly expressed in THP-1 macrophages thransfected with pcDNA3.1-HA-adi. ACAT1 also highly expressed in THP-1 macrophages thransfected with pcDNA3.1-HA-adi and the effect was enhanced by oxLDL, at the same time, cellular cholesteryl ester accumulated, but all the effects were inhibited by S-58035.From the two parts of expreriments, we concluded:①Both adipophilin and ACAT1 were highly expressed by induced with oxLDL in THP-1 macrophages, companyed with cellular cholesteryl ester accumulation. S-58035 inhibited the ACAT1 high expression and cellular cholesteryl ester accumulation induced by oxLDL in THP-1 macrophages.②Overexpressed adipophilin upregulated ACAT1 expression and induced intraceller lipid accumulation in THP-1 macrophages, but the effect were attenuated by S-58035.③It suggested that adipophilin accumlated cellular cholesteryl ester by ACAT1 in THP-1 macrophages.