Ezetimibe (SCH 58235) is a dietary cholesterol absorption inhibitor that exerts its physiologic effect by decreasing cholesterol absorption.

Ezetimibe effectively inhibits the transport of cholesterol across the intestinal wall, thereby reducing plasma cholesterol in preclinical animal models of hypercholesterolemia. It eliminates exocrine pancreatic function in rats while maintaining bile flow. In hamsters fed with cholesterol, ezetimibe reduces plasma cholesterol and hepatic cholesterol accumulation, with an effective dose (ED50) of 0.04 mg/kg. It also decreases the surface area of aortic atherosclerotic lesions, from 20.2% in the control group to 4.1% in the group on a Western diet and 7.0% in mice on a low-fat cholesterol diet. Furthermore, ezetimibe reduces the cross-sectional area of carotid atherosclerotic lesions by 97% in the Western low-fat cholesterol diet group and by 91% in cholesterol-free mice.

Ezetimibe significantly reduces the expression of mRNA for scavenger receptor class B type I (SR-BI), Niemann-Pick C1-like 1 protein (NPC1L1), ATP-binding cassette transporters, sub-family A member (ABCA), the β subunit of liver X receptor (LXRβ), retinoid X receptor γ (RXRγ), and steroid regulatory element-binding proteins 1 and 2 (SREBP-1 and -2). Ezetimibe notably decreases total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides, while moderately increasing high-density lipoprotein (HDL) cholesterol levels. In Caco-2 cells, Ezetimibe reduces cholesterol transport by 31% without affecting retinol transport.

GST-p62 is prepared from Escherichia coli and 0.5 μg of the purified GST-p62 protein is used for in vitro AMPK phosphorylation assay. Phosphorylation of p62 protein by AMPK is determined by non-radioisotope method using γS-ATP. AMPK complex is immuno-purified from the HEK293 cells, to which either myc-AMPKα1 wild-type (WT) or myc-AMPKα1 kinase-dead mutant (KD, D157A) is transfected with Flag-AMPKβ1 and HA-AMPKγ1. AMPK complex is added into the reaction mixture containing 20 mM HEPES, pH7.4, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 0.5 μg GST-p62, 0.2 mM AMP, and 1 mM ATPγS. Reaction is carried out at 37°C for 30 min, and then terminated by adding 20 mM EDTA. To detect γS-labeled p62 protein, the reaction product is alkylated with 2.5 mM PNBM for 2 h at room temperature and analyzed the products by western blotting using anti-thiophosphate antibody[1].

Ezetimibe is dissolved in DMSO and stored, and then diluted with appropriate medium before use[2]. Huh7 human hepatocytes are cultured in high glucose DMEM containing 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin at 37°C in a 95% air/5% CO2 atmosphere. Hepatocytes are treated with or without Ezetimibe (10 μM, 1 h) and incubated with palmitic acid (PA, 0.5 mM, 24 h)[2].