We recently demonstrated that squalene synthase (SQS) inhibitors reduce plasma triglyceride through an LDL receptor-independent mechanism in Watanabe heritable hyperlipidemic rabbits (Hiyoshi et al. 2001. Eur. J Pharmacol. 431: 345-352). The present study deals with the mechanism of the inhibition of triglyceride biosynthesis by the SQS inhibitors ER-27856 and RPR-107393 in rat primary cultured hepatocytes. Atorvastatin, an HMG-CoA reductase inhibitor, had no effect on triglyceride biosynthesis, but reversed the inhibitory effect of the SQS inhibitors. A squalene epoxidase inhibitor, NB-598, affected neither triglyceride biosynthesis nor its inhibition by ER-27856 and RPR-107393. The reduction of triglyceride biosynthesis by ER-27856 and RPR-107393 was potentiated by mevalonolactone supplementation. Treatment of hepatocytes with farnesol and its derivatives reduced triglyceride biosynthesis. In addition, we found that ER-27856 and RPR-107393 significantly reduced the incorporation of [1-C-14]acetic acid into oleic acid, but not the incorporation of [1-C-14]oleic acid into triglyceride. Though ER-27856 and RPR-107393 increased mitochondrial fatty acid beta-oxidation, the inhibition of beta-oxidation by RS-etomoxir had little effect on their inhibition of triglyceride biosynthesis. These results suggest that SQS inhibitors reduce triglyceride biosynthesis by suppressing fatty acid biosynthesis via an increase in intracellular farnesol and its derivatives.

Subfractions of VLDL (VLDL1, Sf 100–400; VLDL2V Sf 60–100; VLDL3, Sf 20–60) and LDL (LDL1, Sf 12–20; LDL2, Sf 6–12; LDL3, Sf 3–6) were isolated from the plasma of three normal, three type in and four type IV hyperlipoproteinemic subjects. In the type IV group, all VLDL subspecies Were of normal composition but were increased in concentration in the order VLDL1 >VLDL2 >VLDL3. In the same subjects, although LDL1 was lowered and LDL3 increased, the total plasma LDL concentration Was normal. All VLDL subfractions were elevated in the type III group, but in this ease VLDL3 predominated. These subfractions were enriched in cholesteryl esters and depleted in triglyceride. In the LDL density range there was a shift of mass towards the least dense fraction, LDL1, which was of normal composition. EPR studies of the VLDL and LDL subfractions in a type IV subject demonstrated a decrease in fluidity with increasing density. The major change occurred between VLDL3 and LDL, and was attributed to a substantial alteration in the cholesteryl ester: triglyceride ratio in the particle. A similar argument was used to explain the reduced fluidity of type III VLDL3 with respect to that of the same subfraction in normal or type IV subjects. Particle diameters, determined by laser light-scattering speetroscopy were-in good agreement with the values obtained by electron microscopy. This study provides a baseline for the examination of the relationship' between the physical and metabolic properties of VLDL and LDL subtractions in type in and IV hyperlipoproteinemia.

Inhibitors of squalene synthase are considered to be candidate drugs to reduce both plasma cholesterol and triglyceride. However, little is known about the mechanism of squalene synthase inhibitor-specific effect on plasma triglyceride. In this study, we confirmed the triglyceride-lowering effect of ER-27856, a potent squalene synthase inhibitor prodrug, in rhesus monkeys. To determine the role of low-density lipoprotein (LDL) receptor in the triglyceride-lowering effect of squalene synthase inhibitors, we intravenously administered ER-28448, the active form of ER-27856, to Watanabe heritable hyperlipidemic (WHHL) rabbits for 4 days. In heterozygotes, ER-28448 reduced plasma cholesterol and triglyceride by 52% and 37%, respectively. In homozygous rabbits, in contrast, ER-28448 lowered plasma triglyceride by 40% but did not lower plasma cholesterol. Orally administered ER-27856 reduced plasma triglyceride in homozygous animals but atorvastatin and bezafibrate did not. In hepatocytes isolated from homozygous WHHL rabbits, squalene synthase inhibitors but not atorvastatin reduced triglyceride biosynthesis. These data demonstrate that squalene synthase inhibitors reduced plasma triglyceride through an LDL receptor-independent mechanism, which was distinct from that of the triglyceride-lowering action of atorvastatin or bezafibrate. The reduction of hepatic triglyceride biosynthesis may play an important role in the hypotrigyceridemic action of squalene synthase inhibitors.