Abstract
Purpose: :
Acyl-coenzyme A: cholesterol acyltransferase (ACAT1/SOAT1) is an endoplasmic reticulum enzyme that generally catalyzes the formation of cholesteryl esters from cholesterol (Ch) and long-chain fatty acids. The purpose of this study is to investigate the involvement of ACAT1 in the metabolism of 7-ketocholesterol (7KCh) in ARPE-19 cells.
Methods: :
ACAT1 mRNA was measured in human tissues and ARPE-19 cells by real time qRT-PCR. A rabbit polyclonal affinity purified anti-ACAT1 antibody was used for immnublot and immunohistochemistry. Immunolocalization of ACAT1 was performed on monkey retina vibrotome sections (100 μm). ARPE-19 cells were preincubated with either ACAT1 inhibitor or cytosolic phospholipase A2a (cPLA2a) inhibitor prior to 7KCh treatment. Cells were collected and analyzed for 7KCh, Ch and 7KCh-fatty acid esters (7KFAEs) 24 h after 7KCh treatment. Cell viability was determined by celluar dehydrogenase activity. Identification and quantification of 7KCh, Ch and 7KFAEs was performed by HPLC-UV and LCMS.
Results: :
qRT-PCR analyses and immunoblots detected a significant level of ACAT1 mRNA and protein in human retina and ARPE-19 cells. In monkey retina ACAT1 localized mainly to the photoreceptor outer segments, outer plexiform and ganglion cell layers. A lower level of expression was observed in the RPE and choroid. HPLC-UV analyses of extracts from 7KCh-treated ARPE-19 cells detected 4 main 7KFAEs. Preliminary LCMS results indicate these esters are to 16:1 (palmitoleic), 18:3 (linolenic), 20:4 (arachidonic), and 20:3 (eicosatrienoic). The 7KFAEs accounted for 15-25% of the internalized 7KCh. No hydroxylated or sulfated forms of 7KCh were detected by LCMS. Inhibition of ACAT1 ablated the 7KFAEs synthesis but did not prevent cytotoxicity. By contrast, inhibition of cPLA2a while also ablating the 7KFAE synthesis provided some protection from 7KCh cytotoxicity.
Conclusions: :
Our results indicate that the only significant metabolism of 7KCh in these cultured cells is via fatty acid esterification catalyzed by ACAT1. Inhibition of ACAT1 provided no protection from cytotoxicity suggesting the 7KFAEs are not involved in the 7KCh-mediated inflammatory pathways. Inhibition of cPLA2a did provide some protection to 7KCh-mediated cytotoxicity suggesting that the fatty acid released by cPLA2 while serving as substrates for ACAT1 may also be involved in the 7KCh-mediated inflammation.
Keywords: lipids • metabolism • retina