AcLDL-stimulated CO synthesis in Natural 264

AcLDL-stimulated CO synthesis in Natural 264.7 macrophages (Fig. 1.2 M and 2.2 1.1 M for CHO-ACAT1 and CHO-ACAT2, respectively). Consistent with ACAT inhibition, Rimonabant treatment clogged ACAT-dependent processes in macrophages, oxysterol-induced apoptosis and acetylated-LDL induced foam cell formation. From these results we conclude that Rimonabant is an ACAT1/2 dual inhibitor and suggest that some of the atherosclerotic beneficial effects of Rimonabant Atractylenolide III are, at least partly, due to inhibition of ACAT. and 0.05. We also examined the ability of Rimonabant to inhibit CO synthesis in macrophages in which ACAT activity was stimulated by the addition of an oxysterol, 7-ketocholesterol (7KC), or acetylated low denseness lipoproteins (AcLDL) (Fig. 2). 7KC-stimulated CO synthesis in Natural 264.7 macrophages was significantly reduced by Rimonabant at concentrations 1 M (Fig 2A). AcLDL-stimulated CO synthesis in Natural 264.7 macrophages (Fig. 2B) and mouse peritoneal macrophages (Fig. 2C) was also significantly inhibited by Rimonabant. Open in a separate window Fig. 2 Rimonabant inhibits oxysterol-stimulated and AcLDL-stimulated ACAT activity in macrophages. (A) Natural 264.7 macrophages were cultured in the presence of increasing concentrations of Rimonabant as indicated for 1h prior to activation of ACAT activity with 7-ketocholesterol (7KC, 20 g/ml). The Atractylenolide III formation of [3H] CO from [3H] oleate was then identified as explained in fig 1. Natural 264.7 macrophages (B) and isolated peritoneal macrophages (C) were incubated in the presence of increasing amounts of Rimonabant as indicated for 1h prior to activation of ACAT activity with AcLDL (100 g/ml) and [3H] CO synthesis was determined. ACAT activity is definitely indicated as the percentage of the mean dpm/mg protein SD identified for untreated settings for triplicate samples. Graphs are representative of duplicate experiments performed individually. * = 0.05. Rimonabant is definitely a nonselective dual inhibitor of ACAT isoforms We next ITSN2 monitored the effect of Rimonabant on sterol esterification in AC29 cells, a mutant Chinese hamster ovary (CHO) cell collection lacking endogenous ACAT, stably expressing human being ACAT1 or ACAT2 (Fig. 3). Rimonabant at concentrations 1M significantly inhibited CO synthesis in CHO-ACAT1 and CHO-ACAT2 cells inside a concentration-dependent manner with similar effectiveness (IC50 values of 1 1.5 1.2 M and 2.2 1.1 M, respectively, Fig. 3B). Rimonabant also dose-dependently inhibited ACAT activity 0.05. Rimonabant inhibits 7KC-induced apoptosis and AcLDL-induced cytosolic lipid Atractylenolide III droplet build up in macrophages We next investigated the effects of Rimonabant on ACAT-dependent processes in macrophages relevant to atherosclerosis, including macrophage-derived foam cell formation and oxysterol-induced apoptosis of macrophages [4; 6]. Earlier work Atractylenolide III shown that 7-ketocholesterol (7KC)-induced apoptosis in macrophages is definitely a caspase-3 dependent process that is reliably recognized by assaying for caspase-3 activity and immunoblotting for cleavage of procaspase-3 and poly (ADP-ribose) polymerase (PARP) [4]. Rimonabant significantly reduced caspase-3 activity in Natural 264.7 macrophages induced by 7KC treatment (Fig. 4A). Consistent with inhibition of 7KC-induced apoptosis, Natural 264.7 macrophages treated with Rimonabant also contained reduced levels of cleaved (active) caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) compared to macrophages treated with 7KC in the absence of Rimonabant (Fig 4B). Rimonabant experienced no effect on capase-3 activity in macrophages cultured in the absence of 7KC (Fig. 4A) or in macrophages undergoing apoptosis induced by staurosporine (Fig 4C). At these concentrations of Rimonabant, no effect on cell viability as evaluated by altered MTT assays was observed (Fig. 4D). Open in a separate windows Fig. 4 Rimonabant inhibits oxysterol-induced macrophage apoptosis and AcLDL-induced foam cell formation. Natural 264.7 macrophages were treated with varying amounts of Rimonabant 1h prior to the addition of 7KC (20 g/ml) as indicated in the figure. After 16h, the induction of apoptosis was evaluated by (A) caspase-3 activity assays and (B) immunoblotting for cleavage of caspase 3 and PARP. (C) Effect of Rimonabant on induction of apoptosis by staurosporine (1 M) in Natural 264.7 macrophages as determined by caspase-3 activity. (D) Rimonabant does not impact cell viability as determined by altered MTT assay. (E) Representative photomicrographs of Natural 264.7 macrophages cultured in the absence or presence of Rimonabant (8 M) for 1hr prior to addition of AcLDL (100 g/ml) for 16h prior to staining with oil red-O to visualize cytosolic accumulation of neutral lipid droplets. Initial magnification = 40. Pub, 10 m. * = 0.05. Macrophage foam cell formation depends upon ACAT-mediated esterification of LDL-derived cholesterol resulting in the build up of CE in.