AM-251 and SR144528 inhibit sterol esterification in vivo and in vitro. The affect of (A) AM-251 and (B) SR144528 on the incorporation of [³H]oleate into neutral lipids in Raw 264.7 macrophages. Cells were incubated in medium supplemented with increasing concentrations of AM-251 and SR144528 as indicated for 1 h prior to the addition of [³H]oleate (1 μCi/ml). After a 6 h incubation, the total lipids were extracted and the amount of cholesteryl [³H]oleate, [³H]triacylglycerol and [³H]diacylglycerol formed was determined. (C) AM-251 and SR144528 prevent esterification of 7-ketocholesterol. Raw 264.7 macrophages were pretreated for 1 h with the antagonists, as indicated, prior to the addition of [³H]oleate (1 μCi/ml) and 7KC (10 μg/ml). After 6 h, the formation of 7-ketocholesteryl [³H]oleate was determined. Values are the percentage of the mean dpm/mg protein ± SD determined for control cells (vehicle only) for duplicate experiments each performed in triplicate. * p<0.05.

Lipid droplet accumulation in Raw 264.7 macrophages is inhibited by AM-251 and SR144528. (A) Cells were cultured for 16 h in medium alone (-acLDL), or medium supplemented with (B) 100 μg/ml acLDL, (C) 100 μg/ml acLDL and 8 μM AM-251 or (D) 100 μg/ml acLDL and 8 μM SR144528. The cells were fixed and the neutral lipids stained with oil red O. Original magnification = 40X. Bar, 10 μm.

AM-251 and SR144528 inhibit 7-ketocholesterol induced apoptosis. Raw 264.7 macrophages were pretreated for 1 h with AM-251 or SR144528, prior to treatment with 7KC (10 μg/ml) as indicated. After 16 h, apoptosis was evaluated by (A) caspase-3 activity assay and (B) APOPercentage assay. (C) Caspase-3 activity induced in Raw 264.7 macrophages after a 6h treatment with staurosporine (1 μM) in the absence and presence of AM-251 or SR144528 as indicated. (D) Cell viability of Raw 264.7 macrophages incubated for 24 h in medium supplemented with AM-251 or SR144528, as indicated, was determined by a modified MTT assay and presented as the percentage ± SD of the mean of the control cells treated with vehicle only. * p<0.05

AM-251 and SR144528 inhibit the stimulation of cholesterol esterification by acLDL independent of CB2 expression. (A) Raw 264.7 macrophages were pretreated with increasing concentrations of AM-251, SR144528 or 58-035 for 1h prior to supplementation with acLDL (100 μg/ml) and [³H]oleate (1 μCi/ml) as indicated. Following a 6 h incubation, the amount of cholesteryl [³H]oleate formed was determined and the ACAT activity expressed as percentage of the mean cholesteryl [³H]oleate dpm/mg realized for controls (vehicle only). Each experiment was performed independently twice, each with triplicate samples. (B) AM-251 and SR144528 inhibit acLDL-stimulated cholesterol esterification in resident peritoneal macrophages from wild type and CB2 null mice. Wild type (CB2+/+) and CB2 null (CB2−/−) resident macrophages were pretreated with AM-251 or SR144528 for 1h prior to the addition of acLDL (100 μg/ml) and [³H]oleate (1 μCi/ml). After 6 h, total lipids were extracted and the amount of cholesteryl [³H]oleate formed was determined. ACAT activity is expressed as the percentage of the mean cholesteryl [³H]oleate dpm/mg realized for controls (vehicle only). Each experiment was performed independently twice, each with triplicate samples. (C) Mouse liver microsomes were preincubated for 30 min with various concentrations (0–20 μM) of the inhibitors as indicated. [¹⁴C]Oleoyl-CoA (0.5 μCi) was added and the amount of cholesteryl [¹⁴C]oleate formed after a 5 min incubation at 37°C was determined. ACAT activity is expressed as the percentage of cholesteryl [¹⁴C]oleate formed in the absence of inhibitors ± SD for triplicate samples. In some cases the error bars are smaller than the symbol and are not visible.