To assess the underlying mechanism of altered ubiquitination of hepatic MTP in FLS mice treated with ezetimibe, we investigated liver protein levels of Skp2 and CDC20, which are two major molecules involved in ubiquitin–protein check details ligation. Hepatic protein levels of Skp2 and CDC20 were significantly lower in EZ than in CT (Fig. 4b,c) (P < 0.01 and P < 0.05,
respectively). These findings suggest that ezetimibe may lead to suppression of ubiquitination of MTP protein via reduced expression of hepatic Skp2 and CDC20. Ezetimibe administration significantly decreased liver ROS level, and a previous study demonstrated that CCL4 induced post-translational degradation of MTP in MCA-RH7777 cells;[19] thus, we next examined the effects of ezetimibe on CCL4-induced post-translational degradation of MTP using MCA-RH7777 cells. A higher ROS level using
fluorescence microscopic imaging was observed in CCL4-treated cells, and ezetimibe significantly inhibited CCL4-induced ROS production (Fig. 5a). Increased intracellular ROS level determined by DCFDA was higher in CCL4-treated cells compared with DMSO-treated cells (control) and ezetimibe-treated cells (P < 0.01 and P < 0.01, respectively), and ezetimibe significantly decreased CCL4-induced ROS production (P < 0.01) (Fig. 5a). We examined the effect of ezetimibe on ubiquitination http://www.selleckchem.com/products/gdc-0068.html of MTP in MCA-RH7777 cells. CCL4 treatment enhanced ubiquitination of MTP compared with that in DMSO- and ezetimibe-treated Adenosine cells, and ezetimibe significantly decreased ubiquitination of MTP in CCL4-treated cells (Fig. 5c). To assess whether ezetimibe has a direct effect on post-translational degradation of MTP in MCA-RH7777 cells, we examined the protein expression of MTP, Skp2 and CDC20. Ezetimibe-treated
cells did not show enhanced protein expression of MTP or decreased ubiquitination of MTP compared with DMSO-treated cells; however, the protein levels of Skp2 and CDC20 were decreased in ezetimibe-treated cells compared with DMSO-treated cells (P < 0.05 and P < 0.01, respectively) (Fig. 5b–e). On the other hand, ezetimibe significantly increased the protein expression of MTP in CCL4-treated cells, and decreased the protein expression of Skp2 compared with that in CCL4-treated cells (P < 0.01 and P < 0.05, respectively) (Fig. 5b,d). IN THE PRESENT study, we investigated the in vivo and in vitro effects of ezetimibe on NAFLD using FLS mice and rat hepatoma cells. In the spontaneous model, FLS mice, ezetimibe was associated with improvement of hepatic steatosis and fibrosis and a reduction of ROS. Ezetimibe also induced upregulation of hepatic MTP expression via suppression of ubiquitination and degradation of the MTP protein. Thus far, several groups have investigated the effect of ezetimibe on NAFLD in diet-induced models, and demonstrated that it attenuates liver steatosis in high-fat models by inhibiting intestinal cholesterol absorption, thus suppressing the inflow of lipids to the liver.