Abstract
We have reported previously that NB-598 competitively inhibits human squalene epoxidase and strongly inhibits cholesterol synthesis from [14C]acetate in cultured cells. Furthermore, multiple oral administration of NB-598 decreased serum cholesterol levels in dogs (Horie, M., Tsuchiya, Y., Hayashi, M., Iida, Y., Iwasawa, Y., Nagata, Y., Sawasaki, Y., Fukuzumi, H., Kitani, K., and Kamei, T. (1990) J. Biol. Chem. 265, 18075-18078). In the present study, the effects of NB-598 on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and low-density-lipoprotein (LDL) receptor were examined using a human hepatoma cell line Hep G2. Incubation of Hep G2 cells with NB-598 for 18 h increased HMG-CoA reductase activity in a dose-dependent manner. However, the increase in activity induced by NB-598 was lower than that induced by L-654,969 (a potent HMG-CoA reductase inhibitor), although NB-598 inhibited cholesterol synthesis more potently than L-654,969. On the other hand, HMG-CoA reductase mRNA was increased to the same extent by both inhibitors. These results demonstrate that NB-598 does not inhibit the synthesis of non-sterol derivative(s) of mevalonate, which regulate HMG-CoA reductase activity at the post-transcriptional level. NB-598 increased the binding of 125I-LDL to Hep G2 cells. LDL receptor mRNA was also induced by NB-598. In the presence of LDL or cycloheximide, NB-598 did not increase LDL receptor activity. These results demonstrate that the induction of LDL receptor activity by NB-598 is due to increases in mRNA and protein through the inhibition of cholesterol synthesis at the squalene epoxidase step. From these observations, squalene epoxidase inhibitor is expected to be highly effective in the treatment of hypercholesterolemia and also is very useful as a research tool for studying the regulation of cholesterol metabolism.
Highlights
Hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase and low-density-lipoprotein(LDL) receptor were examined using a human hepatoma cell line Hep G2
In thepresence of low density lipoprotein (LDL) or cycloheximide, NB-598 did not increase LDL receptor activity. These results demonstrate that the inductionof LDL receptor activity by NB-598 is due to increases minRNA and protein through the inhibition of cholesterol synthesis at the squalene epoxidasestep
At 18 h before the experiment, the cell culture medium (200-300 metabolism, we examined the effects of NB-598 on HMG- pgof cell protein/dish) grown in multiwell dishes (22 mm diameter, CoA reductase activity, LDL receptor activity, and squalene Corning, NY) was replaced with medium A containing 10% LPDS, epoxidase activity usinga human hepatoma cell line, HepG2
Summary
NB-598 sults i n a biphasicreductaseactivityresponse [19, 20]. h in medium A containing 10% LPDS or 10% fetal bovine serum. After centrifugation (1000 X g, 5 min sulfoxidedid not exceed 0.4% (v/v) Under these conditions, dimethyl at 4 "C), thecell pellet (approximately 2 X 10' cells) was suspended sulfoxide had no significant effect on HMG-CoA reductase, squalene in 3 ml of 0.1 M Tris-HC1,pH 7.5, 1 mM EDTA (buffer A) and was epoxidase, and LDL receptor activities. The reaction mixture was incubated for 90 min at 37 "C in a clone, HMG-CoA reductase (pHRed-102) cDNA clone, and Hep G2 final volume of0.3ml containing 0.1 M Tris-HCI, pH 7.5, 1 mM cells were obtained from American Type Culture Collection (Rock- EDTA, 1mM NADPH, 0.1 mM FAD, 0.4 mM AM0 1618(an inhibitor ville, MD).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
