Squalene synthase inhibition alters metabolism of nonsterols in rat liver

Abstract

We have used the potent squalene synthase inhibitor squalestatin I to investigate the regulation of isoprenoid metabolism in rat liver. Fresh-frozen liver pieces from normal rats and rats infused with squalestatin I at 16 μg h −1 for 16 h were assayed for farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) by HPLC after dephosphorylation. Levels of FPP and GGPP were 5.4 ± 1.6 nmol g −1 and 1.6 ± 0.7 nmol g −1 ( n = 13) wet wt., respectively, in control livers and 110 + 41 nmol g −1 and 3.0 ± 2.2 nmol g −1 ( n = 13) in livers from squalestatin I infused rats. In order to determine the relative level of isopentenyl pyrophosphate, liver slices from normal and squalestatin I infused rats were labeled to steady-state with [ 3H]acetate. Analysis of isoprenoid pyrophosphate intermediates by radio-HPLC after dephosphorylation indicated that squalestatin I brought about a 20-fold increase in the relative level of FPP (confirming direct analysis) and a 5-fold increase in the relative level of IPP. No change in either of these compounds was observed in livers from cholesterol-fed rats. To determine if squalestatin I altered the synthesis of nonsterol products, rats were subjected to long term subcutaneous infusion. After 14 days of infusion of 15 μg h −1, the median chain length of hepatic dolichol and dolichyl phosphate increased from C95 to C115 and the levels of these lipids increased ≈ 3-fold. In addition, dolichyl phosphate mannose synthase activity in microsomes from squalestatin I treated rats was increased relative to controls when assayed in the absence of dolichyl phosphate. Squalestatin I affected ubiquinone metabolism to a lesser extent: chain lengths shifted from a Q 10/Q 9 ratio of 0.118 ± 0.021 in the normal rat to 0.185 ± 0.016 in the squalestatin I treated animals, and levels rose by ≈ 90%. These results suggest that the isoprenoid pyrophosphate intermediates are shared by the cholesterol, dolichol and ubiquinone pathways and further show that the dolichol and ubiquinone pathways are not saturated. Apparently, under normal conditions, the levels of these intermediates are maintained relatively constant by coordinate enzyme regulation, thereby ensuring a constant rate of synthesis of nonsterols.