Squalene synthase inhibitors suppress triglyceride biosynthesis through the farnesol pathway in rat hepatocytes
2003; Elsevier BV; Volume: 44; Issue: 1 Linguagem: Inglês
10.1194/jlr.m200316-jlr200
ISSN1539-7262
AutoresHironobu Hiyoshi, Mamoru Yanagimachi, Masashi Ito, Nobuyuki Yasuda, Toshimi Okada, Hironori Ikuta, Daisuke Shinmyo, Keigo Tanaka, Nobuyuki Kurusu, Ichiro Yoshida, Shinya Abe, T. Saeki, Hiroshi Tanaka,
Tópico(s)Protein Structure and Dynamics
ResumoWe recently demonstrated that squalene synthase (S111111148) 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 [1-14C]acetic acid into oleic acid, but not the incorporation of [1-14C]oleic acid into triglyceride. Though ER-27856 and RPR-107393 increased mitochondrial fatty acid β-oxidation, the inhibition of β-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. We recently demonstrated that squalene synthase (S111111148) 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 [1-14C]acetic acid into oleic acid, but not the incorporation of [1-14C]oleic acid into triglyceride. Though ER-27856 and RPR-107393 increased mitochondrial fatty acid β-oxidation, the inhibition of β-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. The isoprenoid metabolic pathway is involved in the synthesis of a wide range of cellular products (1Goldstein J.L. Brown M.S. Regulation of the mevalonate pathway.Nature. 1990; 343: 425-430Google Scholar). Farnesyl pyrophosphate (FPP) is at the key branch point of the pathway with the potential to be incorporated into either sterols or other non-sterol products, such as ubiquinone, dolichol, heme A, prenylated proteins, and farnesol. Squalene synthase (SQS, EC 2.5.1.21) reductively dimerizes FPP to form squalene, which is the first committed intermediate in the pathway to cholesterol. Selective inhibitors of SQS are thus of interest, because they should inhibit cholesterol biosynthesis without any deleterious effect on the branching pathways of isoprenoid metabolism (2Biller S.A. Neuenschwander K. Ponpipom M.M. Poulter C.D. Squalene synthase inhibitors.Curr. Pharm. Des. 1996; 2: 1-40Google Scholar, 3Fung A.K. Baker W.R. Fakhoury S. Stein H.H. Cohen J. Donner B.G. Garvey D.S. Spina K.P. Rosenberg S.H. (1 alpha, 2 beta, 3 beta, 4 alpha)-1,2-bis[N-propyl-N-(4-phenoxybenzyl)amino] carbonyl]cyclobutane-3,4-dicarboxylic acid (A-87049): a novel potent squalene synthase inhibitor.J. Med. Chem. 1997; 40: 2123-2125Google Scholar, 4Amin D. Rutledge R.Z. Needle S.N. Galczenski H.F. Neuenschwander K. Scotese A.C. Maguire M.P. Bush R.C. Hele D.J. Bilder G.E. Perrone M.H. RPR 107393, a potent squalene synthase inhibitor and orally effective cholesterol-lowering agent: comparison with inhibitors of HMG-CoA reductase.J. Pharmacol. Exp. Ther. 1997; 281: 746-752Google Scholar, 5Ugawa T. Kakuta H. Moritani H. Matsuda K. Ishihara T. Yamaguchi M. Naganuma S. Iizumi Y. Shikama H. YM-53601, a novel squalene synthase inhibitor, reduces plasma cholesterol and triglyceride levels in several animal species.Br. J. Pharmacol. 2000; 131: 63-70Google Scholar, 6Hiyoshi H. Yanagimachi M. Ito M. Ohtsuka I. Yoshida I. Saeki T. Tanaka H. Effect of ER-27856, a novel squalene synthase inhibitor, on plasma cholesterol in rhesus monkeys: comparison with 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors.J. Lipid Res. 2000; 41: 1136-1144Google Scholar). We previously reported that ER-27856, a potent SQS inhibitor, lowered plasma cholesterol more potently, and with less adverse effects, than did HMG-CoA reductase inhibitors in rhesus monkeys (6Hiyoshi H. Yanagimachi M. Ito M. Ohtsuka I. Yoshida I. Saeki T. Tanaka H. Effect of ER-27856, a novel squalene synthase inhibitor, on plasma cholesterol in rhesus monkeys: comparison with 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors.J. Lipid Res. 2000; 41: 1136-1144Google Scholar). 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Because elevated plasma triglyceride has attracted increased attention as a risk factor for coronary heart disease (CHD) in recent years (8Jeppesen J. Hein H.O. Suadicani P. Gyntelberg F. Triglyceride concentration and ischemic heart disease: an eight-year follow-up in the Copenhagen Male Study.Circulation. 1998; 97: 1029-1036Google Scholar, 9Sprecher D.L. Triglycerides as a risk factor for coronary artery disease.Am. J. Cardiol. 1998; 82: 49U-56UGoogle Scholar, 10Austin M.A. Hokanson J.E. Edwards K.L. Hypertriglyceridemia as a cardiovascular risk factor.Am. J. Cardiol. 1998; 81: 7B-12BGoogle Scholar, 11Miller M. Seidler A. Moalemi A. Pearson T.A. Normal triglyceride levels and coronary artery disease events: the Baltimore Coronary Observational Long-Term Study.J. Am. Coll. 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Implications for regulation of apolipoprotein B synthesis.J. Clin. Invest. 1987; 80: 1692-1697Google Scholar, 15Arad Y. Ramakrishnan R. Ginsberg H.N. Lovastatin therapy reduces low density lipoprotein apoB levels in subjects with combined hyperlipidemia by reducing the production of apoB-containing lipoproteins: implications for the pathophysiology of apoB production.J. Lipid Res. 1990; 31: 567-582Google Scholar, 16Reihner E. Rudling M. Stahlberg D. Berglund L. Ewerth S. Bjorkhem I. Einarsson K. Angelin B. Influence of pravastatin, a specific inhibitor of HMG-CoA reductase, on hepatic metabolism of cholesterol.N. Engl. J. Med. 1990; 323: 224-228Google Scholar), it remains unknown how SQS inhibitors lower plasma triglyceride levels. Recently, we have reported that ER-27856, but not atorvastatin (an HMG-CoA reductase inhibitor), reduced plasma triglyceride through an LDL receptor-independent mechanism in Watanabe heritable hyperlipidemic (WHHL) rabbits (7Hiyoshi H. Yanagimachi M. Ito M. Saeki T. Yoshida I. Okada T. Ikuta H. Shinmyo D. Tanaka K. Kurusu N. Tanaka H. Squalene synthase inhibitors reduce plasma triglyceride through a low-density lipoprotein receptor-independent mechanism.Eur. J. Pharmacol. 2001; 431: 345-352Google Scholar). SQS inhibitors also reduced triglyceride biosynthesis in hepatocytes isolated from WHHL rabbits. SQS inhibitors have been reported to increase farnesol and farnesol-derived dicarboxylic acids owing to the increase of FPP utilization in other branching pathways of isoprenoid metabolism (4Amin D. Rutledge R.Z. Needle S.N. Galczenski H.F. Neuenschwander K. Scotese A.C. Maguire M.P. Bush R.C. Hele D.J. Bilder G.E. Perrone M.H. RPR 107393, a potent squalene synthase inhibitor and orally effective cholesterol-lowering agent: comparison with inhibitors of HMG-CoA reductase.J. Pharmacol. Exp. Ther. 1997; 281: 746-752Google Scholar, 17Bergstrom J.D. Kurtz M.M. Rew D.J. Amend A.M. Karkas J.D. Bostedor R.G. Bansal V.S. Dufresne C. 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Farnesol blocks the L-type Ca2+ channel by targeting the alpha 1C subunit.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 959-966Google Scholar). In the lipid metabolism, farnesol accelerates the degradation of HMG-CoA reductase (29Bradfute D.L. Simoni R.D. Non-sterol compounds that regulate cholesterogenesis. Analogues of farnesyl pyrophosphate reduce 3-hydroxy-3-methylglutaryl-coenzyme A reductase levels.J. Biol. Chem. 1994; 269: 6645-6650Google Scholar, 30Correll C.C. Ng L. Edwards P.A. Identification of farnesol as the non-sterol derivative of mevalonic acid required for the accelerated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.J. Biol. Chem. 1994; 269: 17390-17393Google Scholar, 31Meigs T.E. Roseman D.S. Simoni R.D. Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation by the nonsterol mevalonate metabolite farnesol in vivo.J. Biol. Chem. 1996; 271: 7916-7922Google Scholar, 32Meigs T.E. Simoni R.D. 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Here, we report that SQS inhibitors suppressed triglyceride biosynthesis in rat primary cultured hepatocytes, and that an increase in farnesol and its derivatives contributed to the effect by suppressing fatty acid biosynthesis. The role of farnesol and its derivatives in fatty acid metabolism is discussed. ER-27856 (4-[N-[(2E)-3-(2-Methoxyphenyl)-2-butenyl]-N-methylamino]-1,1-butylidenebisphosphonic acid tris (pivaloyloxymethyl) ester), RPR-107393, atorvastatin (35Brower P.L. Butler D.E. Deering C.F. Le T.V. Millar A. Nanninga T.N. Roth B.D. The synthesis of (4R-cis)-1,1-dimethylethyl-6-cyanomethyl-2,2-dimethyl-1,3-dioxane-4-acetate, a key intermediate for the preparation of CI-981, a highly potent, tissue selective inhibitor of HMG-CoA reductase.Tetrahedron Lett. 1992; 33: 2279-2282Google Scholar, 36Baumann K.L. Butler D.E. Deering C.F. Mennen K.E. Millar A. Nanninga T.N. Palmer C.W. Roth B.D. The convergent synthesis of CI-981, an optically active, highly potent, tissue selective inhibitor of HMG-CoA reductase.Tetrahedron Lett. 1992; 33: 2283-2284Google Scholar), NB-598, farnesyl acetate, 3,7,11-trimethyl-2,6,10-dodecatriene-1,12-dioic acid methylester, 3,7-dimethyl-2,6-decadiene-1,10-dioic acid methylester, 3,7-dimethyl-2,6-octadiene-1,8-dioic acid methylester, and 3-methyl-2-hexamonoene-1,6-dioic acid methylester were synthesized in our laboratories. RS-Etomoxir was a gift from L. Agius (Department of Diabetes and Metabolism, University of Newcastle upon Tyne, UK). Insulin, dexamethasone, farnesol, squalene, and mevalonolactone (MVL) were purchased from Sigma (St. Louis, MO). Suberic acid was purchased from Nacalai tesque (Kyoto, Japan). Penicillin and streptomycin were from Life Technologies (Rockville, MD). Five-week-old male Sprague-Dawley (SD) rats were obtained from Japan SLC (Shizuoka, Japan). Animals were housed in a well-ventilated (10–15 changes/h), temperature-controlled (23°C ± 3°C) room with constant humidity (55% ± 15%) under a reversed 12 h light/dark (22:00/10:00) cycle. Animals were fed with normal diet. Primary hepatocytes were prepared from male SD rats (200–300 g) grown under an inverse light/dark cycle, as previously described (37Moldeus P. Hogberg J. Orrenius S. Isolation and use of liver cells.Methods Enzymol. 1978; 52: 60-71Google Scholar). In the night phase, rats were anesthetized and heparinized intravenously. The liver was perfused with Liver Perfusion Medium (Life Technologies Inc., MD) at 37°C for 15 min at 20 ml/min., then with Liver Digest Medium (Life Technologies Inc.) for another 15 min. Liver cells were dispersed in Williams' E medium (pH 7.4) supplemented with 10% FBS, 0.1 μM insulin, 1 μM dexamethasone, 100 U/ml penicillin, and 100 μg/ml streptomycin by dissection and gentle shaking. After filtration through 70 μm nylon mesh filter, hepatocytes were isolated by repeated centrifugation (3–5 times) at 50 g for 2 min. Hepatocytes with >90% viability were cultured in Type I collagen-coated 24-well plates (Iwaki) at a cell density of 1 × 105 cells/well. After a 2-h incubation at 37°C in a 5% CO2 atmosphere, non-attached cells were removed by washing with culture medium. Test compounds were added immediately after washing, and incubation was continued for 24 h. Hepatocytes were incubated overnight (18 h) before use in 6-h experiments. Inhibition of lipid biosynthesis was determined by measuring the conversion of [1-14C]acetic acid (185 kBq/ml, 1.85–2.29 GBq/mmol, Amersham Pharmacia Biotech, Little Chalfont, UK) or BSA-bound [1-14C]oleic acid (2.47 kBq/ml, 16.7 MBq/mmol, PerkinElmer Life Sciences, Boston, MA) into cellular lipids. For cholesterol and triglyceride biosynthesis, cells were incubated with or without compounds for the indicated time periods. [1-14C]acetic acid or [1-14C]oleic acid was added to the cells 2 h prior to harvest. Cells were washed twice and lipids were extracted by incubating the cells with 750 μl of hexane-2-propanol (3:2, v/v) for 30 min at room temperature. Aliquots were transferred to glass tubes and evaporated under a nitrogen stream. Samples were resuspended in 30 μl of chloroform, applied onto TLC plastic sheets (Silica gel 60 F254, Merck, Darmstadt, Germany), and developed twice, first with toluene-isopropyl ether (1:1, v/v) for 10 min and then with heptane for 15 min. The radioactivities in the cholesterol and triglyceride fractions were analyzed by means of a BAS 2000 imaging plate system (Fuji Film, Tokyo, Japan). To determine fatty acid biosynthesis, cells were incubated with a test compound for 4 h and with [1-14C]acetic acid for an additional 2 h. The cells were then dissolved in 500 μl of 4 N KOH. Ethanol (1 ml) was added, and an aliquot of the mixture was saponified at 80°C for 30 min. Non-saponifiable lipids were removed by extracting with 3 ml of petroleum ether. The aqueous phase was mixed with 1 ml of 6 N HCl, and acid-insoluble fatty acids were extracted with 3 ml of petroleum ether. The extract was evaporated, and the residue was resuspended in 25 μl of chloroform, applied onto high-performance TLC sheets (RP-8 F254S, Merck), and developed with a solvent (CH3CN-H2O-MeOH-HCOOH, 95:3:2:0.5, v/v/v/v). The radioactivities in the oleic acid fractions were analyzed by BAS 2000. β-Oxidation activity was determined by measuring 3H2O produced from [3H]palmitic acid (200 μM, 167 MBq/mmol, Amersham Pharmacia Biotech) as described previously (38Lee Y. Hirose H. Zhou Y.T. Esser V. McGarry J.D. Unger R.H. Increased lipogenic capacity of the islets of obese rats: a role in the pathogenesis of NIDDM.Diabetes. 1997; 46: 408-413Google Scholar), with some modifications. Cells were incubated with or without a test compound for 4 h. Bovine serum albumin-bound 9,10-[3H]palmitic acid was added, and the cells were incubated for an additional 2 h. An aliquot of the medium (300 μl) was taken and excess [3H]palmitic acid was removed by precipitation with an equal volume of 10% perchloric acid. The supernatant (500 μl) was transferred to a microtube, which was placed uncapped in a scintillation vial containing 1 ml of unlabeled water. The vial was tightly capped and incubated at 50°C for 18 h to equilibrate 3H2O in the aliquot with water in the vial. Radioactivity in the vial was then measured by liquid scintillation counting. The amount of cellular protein was determined using BCA Protein Assay Reagent Kit (Pierce, IL). Statistical analysis was conducted using the software package SAS 6.12 (SAS Institute Japan Ltd., Tokyo, Japan). The IC50 values were calculated by nonlinear regression analysis. Statistical evaluation was performed by means of a one-way ANOVA, followed by Dunnett's t-test for comparison with the control, unless otherwise specified. In order to study the effect of cholesterol biosynthesis inhibitors on lipid biosynthesis, rat hepatocytes were exposed to ER-27856 and PRR-107393 (SQS inhibitors), atorvastatin (an HMG-CoA reductase inhibitor), or NB-598 (a squalene epoxidase inhibitor), and the incorporations of [1-14C]acetic acid into cholesterol and triglyceride were determined (Fig. 1). In a 6-h experiment, all the test compounds concentration-dependently inhibited cholesterol biosynthesis (Fig. 1A). The IC50 values were 4.3 nM for ER-27856, 880 nM for RPR-107393, 20 nM for atorvastatin, and 89 nM for NB-598. In contrast, inhibition of triglyceride biosynthesis was specific to SQS inhibitors (Fig. 1B). ER-27856 and RPR-107393 decreased the incorporation of [1-14C]acetic acid into triglyceride in a concentration-dependent manner. The IC50 values for ER-27856 and RPR-107393 were 4.6 nM and 410 nM, respectively, which are comparable to the IC50 values for cholesterol biosynthesis. However, atorvastatin and NB-598 had no effect on the incorporation of [1-14C]acetic acid into triglyceride, even at 10 μM. In the time-course study, cells were treated with ER-27856 (1 μM), RPR-107393 (10 μM), atorvastatin (1 μM), or NB-598 (1 μM) for 2–24 h, and lipid biosynthesis during the last 2 h of the incubation was determined (Fig. 2). Again, all the test compounds inhibited cholesterol biosynthesis, but only the SQS inhibitors reduced triglyceride biosynthesis. The inhibition of cholesterol biosynthesis was maximal at 2 h, when [1-14C]acetic acid was added to the cells immediately after the addition of the test compounds (Fig. 2A). On the other hand, the reduction of triglyceride biosynthesis by the SQS inhibitors gradually increased as the incubation time was extended, becoming maximal after 12 h to 24 h (Fig. 2B). In contrast to the case of cholesterol biosynthesis, SQS inhibitors did not completely halt triglyceride biosynthesis, and more than 10% of the total triglyceride biosynthetic activity was retained. These findings indicated a possibility that the decrease of triglyceride biosynthesis by squalene synthase inhibitors was a secondary response to the enzyme inhibition. Since neither atorvastatin nor NB-598 affected triglyceride biosynthesis, some factor induced by SQS inhibitors, but not by atorvastatin or NB-598, may suppress triglyceride biosynthesis. As SQS inhibitors suppress one of the branching pathways of FPP metabolism, changes in the amounts of other non-sterol products may affect the triglyceride biosynthetic activity. Therefore, we examined the effect of SQS inhibitors on triglyceride biosynthesis in the presence of atorvastatin or NB-598. ER-27856 at 0.1 μM inhibited the biosynthesis of cholesterol and triglyceride by 98.2% and 71.1%, respectively (Fig. 3A). Although atorvastatin did not influence the cholesterol biosynthesis inhibition by ER-27856, it caused triglyceride biosynthesis to revert to the control level. NB-598 had no effect on the inhibition of either cholesterol or triglyceride biosynthesis by ER-27856. Similarly, 1 μM RPR-107393 inhibited cholesterol and triglyceride biosynthesis by 82.4% and 70.0%, respectively (Fig. 3B). Atorvastatin potentiated the inhibition of cholesterol biosynthesis by RPR-107393, but diminished the inhibition of triglyceride biosynthesis. NB-598 potentiated the inhibitory activity of RPR-107393 on cholesterol biosynthesis, but had no effect in the case of triglyceride biosynthesis. Next, cells were treated with ER-27856 and RPR-107393 in the presence of 2 mM or 10 mM MVL to increase the levels of FPP derivatives. We found that the reduction of triglyceride biosynthesis was potentiated by MVL supplementation (Fig. 4). In the absence of MVL, the decreases of triglyceride biosynthesis by 0.1 μM ER-27856 and 1 μM RPR-107393 amounted to 33.3% and 36.2% of the control, respectively. The decreases were further potentiated by 2 mM (25.0% and 28.0%, respectively) and 10 mM MVL (19.5% and 21.1%, respectively). MVL alone, at 2 or 10 mM, did not decrease triglyceride biosynthesis under these conditions (data not shown). These results suggest that the reduction of triglyceride biosynthesis was mediated by an increase of FPP derivatives. SQS inhibitors increase farnesol and farnesol-derived dicarboxylic acids in vitro (17Bergstrom J.D. Kurtz M.M. Rew D.J. Amend A.M. Karkas J.D. Bostedor R.G. Bansal V.S. Dufresne C. VanMiddlesworth F.L. Hensens O.D. Liesch J.M. Zink D.L. Wilson K.E. Onishi J. Milligan J.A. Bills G. Kaplan L. Nallin Omstead M. Jenkins R.G. Huang L. Meinz M.S. Quinn L. Burg R.W. Kong Y.L. Mochales S. Mojena M. Martin I. Pelaez F. Diez M.T. Alberts A.W. Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase.Proc. Natl. Acad. Sci. USA. 1993; 90: 80-84Google Scholar), and an increase was also reported in mice, rats, and beagle dogs (4Amin D. Rutledge R.Z. Needle S.N. Galczenski H.F. Neuenschwander K. Scotese A.C. Maguire M.P. Bush R.C. Hele D.J. Bilder G.E. Perrone M.H. RPR 107393, a potent squalene synthase inhibitor and orally effective cholesterol-lowering agent: comparison with inhibitors of HMG-CoA reductase.J. Pharmacol. Exp. Ther. 1997; 281: 746-752Google Scholar, 18Keller R.K. Squalene synthase inhibition alters metabolism of nonsterols in rat liver.Biochim. Biophys. Acta. 1996; 1303: 169-179Google Scholar, 19Vaidya S. Bostedor R. Kurtz M.M. Bergstrom J.D. Bansal V.S. Massive production of farnesol-derived dicarboxylic acids in mice treated with the squalene synthase inhibitor zaragozic acid A.Arch. Biochem. Biophys. 1998; 355: 84-92Google Scholar, 20Bostedor R.G. Karkas J.D. Arison B.H. Bansal V.S. Vaidya S. Germershausen J.I. Kurtz M.M. Bergstrom J.D. Farnesol-derived dicarboxylic acids in the urine of animals treated with zaragozic acid A or with farnesol.J. Biol. Chem. 1997; 272: 9197-9203Google Scholar). To study the effects of farnesol and its derivatives on triglyceride biosynthesis, we added 100 μM farnesol, squalene, farnesyl acetate, 3,7,11-trimethyl-2,6,10-dodecatriene-1,12-dioic acid methylester, 3,7-dimethyl-2,6-decadiene-1,10-dioic acid methylester, 3,7-dimethyl-2,6-octadiene-1,8-dioic acid methylester, 3-methyl-2-hexamonoene-1,6-dioic acid methylester, and suberic acid to hepatocytes, and evaluated their ability to inhibit triglyceride biosynthesis (Fig. 5). Although squalene and suberic acid did not affect triglyceride biosynthesis, farnesol and its derivatives significantly reduced it. Farnesol and its derivatives also decreased the rate of cholesterol biosynthesis, but to a lesser extent, though the effects were well correlated with those on triglyceride biosynthesis. Because farnesol and its derivatives did not affect the rate of protein biosynthesis (data not shown), the effects cannot be attributed to cytotoxicity. The results suggest that the SQS inhibitors decreased triglyceride biosynthesis via an increase of farnesol and its derivatives, and that a common mechanism may regulate triglyceride and cholesterol biosynthesis. Changes of fatty acid β-oxidation affect the rate of trigly-ceride biosynthesis (39Spurway T.D. Pogson C.I. Sherratt H.S. Agius L. Etomoxir, sodium 2-[6-(4-chlorophenoxy)hexyl] oxirane-2-carboxylate, inhibits triacylglycerol depletion in hepatocytes and lipolysis in adipocytes.FEBS Lett. 1997; 404: 11-14Google Scholar). In order to determine whether or not SQS inhibitors affected the fatty acid metabolic pathway, we first studied the effect of cholesterol biosynthesis inhibitors on fatty acid β-oxidation (Fig. 6A). In a 6-h incubation, ER-27856 increased the rate of overall β-oxidation by 23.6% at 10 nM, 25.2% at 100 nM, and 34.6% at 1 μM, and RPR-107393 did so by 26.5% at 1 μM, and 39.5% at 10 μM. However, atorvastatin and NB-598 had no effect at concentrations up to 1 μM. RS-Etomoxir, a carnitine palmitoyltransferase I (CPT I) inhibitor, reduced the overall β-oxidation activity by 70% at 1 μM. RS-Etomoxir suppressed the increase of β-oxidation by ER-27856 and RPR-107393, indicating that SQS inhibitors increase carnitine-dependent mitochondrial β-oxidation. However, RS-etomoxir had no effect on triglyceride biosynthesis or its reduction by ER-27856 and RPR-107393 (Fig. 6B). The data indicate that SQS inhibitors reduce overall triglyceride
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