2015/03/10 by Shuichi Nagashima, Hiroaki Yagyu, Ryuichi Tozawa +11 · 8 citations
Medicine · Biochemistry, Genetics and Molecular Biology · #Cholesterol and Lipid Metabolism #Plant biochemistry and biosynthesis #Lipid metabolism and biosynthesis
paper · pdf · doi:10.1194/jlr.m057406
Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality. Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality. Mammals have developed sophisticated and complex systems to maintain cellular content of cholesterol, an essential component of cellular membranes and a precursor of bile acids and steroid hormones (1.Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Protein sensors for membrane sterols.Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar). In addition to dietary intake, cholesterol is supplied by de novo synthesis from acetate. Squalene synthase (SS; farnesyl-diphosphate farnesyltransferase, EC2.5.1.21) catalyzes the reductive head-to-head condensation of two molecules of farnesyl diphosphate (FPP) to form squalene, the first committed intermediate in the cholesterol biosynthetic pathway (2.Tansey T.R. Shechter I. Structure and regulation of mammalian squalene synthase.Biochim. Biophys. Acta. 2000; 1529: 49-62Crossref PubMed Scopus (89) Google Scholar, 3.Do R. Kiss R.S. Gaudet D. Engert J.C. Squalene synthase: a critical enzyme in the cholesterol biosynthesis pathway.Clin. Genet. 2009; 75: 19-29Crossref PubMed Scopus (80) Google Scholar). SS contains ∼416 amino acids and is anchored to endoplasmic reticulum by a short C-terminal membrane-spanning domain, with its large N-terminal catalytic domain facing the cytosol, where water-soluble FPP and NADPH are present. Hepatic SS is highly regulated at the transcriptional level, not only by cellular cholesterol content (4.Jiang G. McKenzie T.L. Conrad D.G. Shechter I. Transcriptional regulation by lovastatin and 25-hydroxycholesterol in HepG2 cells and molecular cloning and expression of the cDNA for the human hepatic squalene synthase.J. Biol. Chem. 1993; 268: 12818-12824Abstract Full Text PDF PubMed Google Scholar) but also by proinflammatory cytokines: TNF-α and interleukin 1β (5.Memon R.A. Shechter I. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. Endotoxin, tumor necrosis factor, and interleukin-1 decrease hepatic squalene synthase activity, protein, and mRNA levels in Syrian hamsters.J. Lipid Res. 1997; 38: 1620-1629Abstract Full Text PDF PubMed Google Scholar). This enzyme has been an attractive target for cholesterol-lowering therapy because the inhibition of this step theoretically may not perturb the nonsterol pathway, which is a potential problem in the use of statins, inhibitors of HMG-CoA reductase (HMGCR). However, development of SS inhibitors such as zaragozic acid and lapaquistat acetate (TAK-475) has been halted because of safety concerns (6.Vaidya 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: 84-92Crossref PubMed Scopus (46) Google Scholar, 7.Stein E.A. Bays H. O'Brien D. Pedicano J. Piper E. Spezzi A. Lapaquistat acetate: development of a squalene synthase inhibitor for the treatment of hypercholesterolemia.Circulation. 2011; 123: 1974-1985Crossref PubMed Scopus (54) Google Scholar). To investigate the consequences of the systemic ablation of SS, we generated mice lacking SS in the whole body (8.Tozawa R. Ishibashi S. Osuga J. Yagyu H. Oka T. Chen Z. Ohashi K. Perrey S. Shionoiri F. Yahagi N. et al.Embryonic lethality and defective neural tube closure in mice lacking squalene synthase.J. Biol. Chem. 1999; 274: 30843-30848Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The SS−/− mice exhibited developmental defects and did not survive beyond E12.5. Supplementation of the dams' diet with squalene or cholesterol did not allow survival of SS−/− fetuses to term. In contrast, SS+/− mice were apparently normal and their plasma lipoprotein profiles were indistinguishable from those of wild-type mice, even though hepatic SS activity was reduced by 50%. These results suggest that fetal demands for cholesterol were not met by maternal supplies and/or that the accumulation of precursors of squalene such as FPP was toxic. In this context, it is noteworthy that naturally occurring inborn errors of cholesterol metabolism are frequently associated with severe developmental abnormalities particularly in the central nervous system (9.Porter F.D. Herman G.E. Malformation syndromes caused by disorders of cholesterol synthesis.J. Lipid Res. 2011; 52: 6-34Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Because deletion of SS theoretically does not block the nonsterol pathway, mice in which SS is specifically knocked out in the liver can be a viable model in which hepatic sterol pathway is selectively abrogated. To test this hypothesis and further examine the efficacy and safety of inhibition of SS, we used tissue-specific gene targeting with the Cre-loxP system to generate mice lacking SS in a liver-specific manner. The detailed procedures for the generation of liver-specific SS knockout (L-SSKO) mice and other assays are available in the supplementary Materials and Methods. The heterozygous floxed SS (SS+/f; f denotes flanked by loxP) carrying one copy of the Cre recombinase gene under the control of the albumin gene promoter (Alb-Cre) (10.Yakar S. Liu J.L. Stannard B. Butler A. Accili D. Sauer B. LeRoith D. Normal growth and development in the absence of hepatic insulin-like growth factor I.Proc. Natl. Acad. Sci. USA. 1999; 96: 7324-7329Crossref PubMed Scopus (1183) Google Scholar) were interbred with SS+/f littermates lacking Alb-Cre to generate L-SSKO mice. L-SSKO mice and littermate controls [SS+/+, SS+/+Alb-Cre, and SSf/f (fSS)] were generated. Because there were no differences in growth curves or metabolic parameters such as plasma lipid and glucose levels between the SS+/+, SS+/+Alb-Cre, and SSf/f (fSS) mice, we used fSS mice as a control. To determine whether SS expression was ablated in the liver, we performed a Southern blot analysis (supplementary Fig. 2A). At 4 weeks of age, the livers of L-SSKO mice contained both disrupted and floxed alleles with the former being more predominant (89% based on relative band density). Northern blot analysis (supplementary Fig. 2B) and real-time PCR (Fig. 1A) showed that the mRNA expression of SS in the livers of L-SSKO mice was reduced to 5% of that in the fSS mice. In contrast, the mRNA expressions of SS in small intestines, adrenal glands, and testes were not different between fSS and L-SSKO mice (Fig. 1A). In an immunoblot, SS protein was barely detectable in the livers of L-SSKO mice (Fig. 1B). Consistent with the profound reduction in SS at both the mRNA and protein levels, the SS activity in the livers of L-SSKO mice was <10% of that in fSS mice (Fig. 1C). L-SSKO mice were born at a rate in accordance with the rule of Mendelian inheritance and survived at a rate that was not significantly different from that of the control mice up to 24 weeks of age. Cholesterol synthesis was reduced by 71% in liver slices (Fig. 1D), while fatty acid synthesis was increased by 58% (Fig. 1E). In parallel, though less significantly, cholesterol synthesis from [3H]water injected intraperitoneally in vivo was decreased by 49% (Fig. 1F). Nevertheless, the hepatic levels of cholesterol were not decreased (Fig. 2H). A compensatory increase in the intestinal cholesterol absorption was not observed (supplementary Fig. 2C). The hepatic contents of triglycerides, phospholipids, and fatty acids were not altered either (Table 1). The hepatic contents of intermediary metabolites of cholesterol biosynthesis proximal to SS such as mevalonate and FPP were increased, while those of squalene, the end product of SS, were reduced by 86% (Fig. 2A–C; supplementary Fig. 5). By contrast, the hepatic contents of intermediary metabolites of cholesterol biosynthesis distal to SS such as lanosterol, zymosterol, lathosterol, desmosterol, and cholesterol were not altered (Fig. 2D–H; supplementary Fig. 5).TABLE 1Lipid contents in the plasma and liversAge 4 WeeksAge 12 WeeksfSS GenotypeL-SSKO GenotypefSS GenotypeL-SSKO GenotypePlasma total cholesterol (mg/dl)Fed80.8 ± 9.0 (5)58.8 ± 15.1 (5)aP < 0.05 versus fSS mice with the same age by Student's t-test.85.8 ± 12.2 (6)55.6 ± 11.5 (7)bP < 0.001 versus fSS mice with the same age by Student's t-test.16 h fasted86.3 ± 1.44 (4)86.5 ± 15.0 (10)95.9 ± 13.2 (7)98.3 ± 26.2 (7)Plasma triglyceride (mg/dl)Fed53.6 ± 22.3 (5)49.3 ± 17.0 (5)69.5 ± 33.2 (6)35.8 ± 13.2 (7)aP < 0.05 versus fSS mice with the same age by Student's t-test.16 h fasted103.8 ± 8.9 (4)81.1 ± 25.2 (10)106.2 ± 42.6 (7)102.5 ± 50.5 (7)Plasma free fatty acid (mM)Fed0.44 ± 0.17 (5)0.56 ± 0.27 (5)0.41 ± 0.09 (6)0.43 ± 0.11 (7)16 h fasted2.13 ± 0.28 (4)1.63 ± 0.50 (10)1.50 ± 0.4 (7)1.58 ± 0.24 (7)Hepatic cholesterol (mg/g)3.6 ± 0.1 (5)3.3 ± 0.5 (5)3.0 ± 0.5 (5)2.5 ± 0.3 (5)Hepatic triglyceride (mg/g)4.2 ± 1.1 (5)6.0 ± 2.7 (5)5.7 ± 1.9 (5)6.2 ± 1.7 (5)Hepatic phospholipid (mg/g)24.3 ± 2.1 (5)25.8 ± 2.1 (5)20.2 ± 1.8 (5)20.5 ± 1.8 (5)Hepatic free fatty acid (μmol/g)4.7 ± 0.6 (5)4.4 ± 0.5 (5)5.4 ± 1.9 (5)4.0 ± 0.7 (5)Blood samples were taken from male mice fed a normal chow diet ad libitum or fasted for 16 h before the study. Each value represents the mean ± SD. Sample sizes are provided in parentheses.a P < 0.05 versus fSS mice with the same age by Student's t-test.b P < 0.001 versus fSS mice with the same age by Student's t-test. Open table in a new tab Blood samples were taken from male mice fed a normal chow diet ad libitum or fasted for 16 h before the study. Each value represents the mean ± SD. Sample sizes are provided in parentheses. Next, we compared liver weight, liver functions, and plasma lipid levels between the control and L-SSKO mice (Fig. 3, Table 1, and supplementary Table 2). The livers of L-SSKO mice were those of the control mice (Fig. The of liver to body was in L-SSKO mice at 12 weeks of age and at 24 weeks of age (Fig. In to of the liver, plasma levels of in the L-SSKO were significantly compared with those in the control mice at 12 weeks of age (Fig. To determine the of the liver dysfunction associated with we performed a analysis of the and and cellular and necrosis of cells (Fig. showed no for accumulation of (Fig. The livers of the L-SSKO mice contained an increased of end cells indicating (Fig. liver activity in the L-SSKO mice was that in the fSS mice (Fig. In contrast, the of was significantly increased in the L-SSKO mice (Fig. indicating that the at an increased The accumulation of FPP may be to the increased and of of L-SSKO mice. To this we the farnesol biosynthesis The farnesol biosynthesis rate was significantly increased in L-SSKO mice by and at 12 and 24 weeks of age, (supplementary Fig. The of the of farnesol synthesis in with that of the of that the of farnesol and/or increasing of farnesol decreased the of the in (supplementary Fig. To examine whether the we fed the mice a and diet for 12 weeks to fatty was observed in the plasma levels between fSS and L-SSKO mice (supplementary Fig. In a fed state, plasma total cholesterol levels were decreased by at 4 and 12 weeks of age (Table 1). triglyceride levels were decreased by at 12 weeks of age. free fatty acid levels were not different between fSS and L-SSKO mice. a 16 h plasma levels of both total cholesterol and were increased, and their differences between the control and L-SSKO mice To determine which were by the of SS, we performed of plasma of the lipoprotein were by the of SS cholesterol contents were significantly decreased in the and (Fig. and triglyceride contents were decreased in the lipoprotein (Fig. Consistent with the in and plasma levels of and were decreased (Fig. The mice were injected with to the plasma of lipoprotein by with lipoprotein and the lipoprotein by the The rate of increase in plasma triglyceride levels the of was decreased by in the L-SSKO mice, indicating that hepatic VLDL production was decreased (Fig. was in receptor mice compared with fSS mice, while it was not significantly different between the fSS and L-SSKO mice (Fig. results that the hypolipidemia of L-SSKO mice primarily from of VLDL by the SS synthesis in the liver the mRNA expressions of genes in cholesterol and fatty acid metabolism (Fig. and supplementary Fig. 5). by a factor of 2 or more FPP synthase squalene and by a factor of 2 or more sterol-regulatory element binding protein protein 1, receptor factor liver X receptor cholesterol and The mRNA expression of was not The in protein expression of and were in with the in mRNA no and decrease and (Fig. activity, as the rate of biosynthesis of mevalonate from was in the L-SSKO mice in the control mice (Fig. Consistent with the increased activity, hepatic content of mevalonate was increased by in the L-SSKO as compared with the control mice (Fig. 2A). In to the severe liver dysfunction and from the of in the liver S. Yagyu H. Ohashi K. F. T. T. K. Osuga J. et deletion of a reductase hepatic and Biol. PubMed Scopus Google the liver dysfunction of L-SSKO mice was and transient and did not the almost absence of cholesterol synthesis hepatic cholesterol synthesis in vivo was only and hepatic contents of cholesterol and its precursors distal to squalene in L-SSKO mice were indistinguishable from those in fSS mice. the of cholesterol in L-SSKO mice, VLDL production was reduced, which the plasma of was the hepatic cholesterol synthesis in vivo only to et of sterol synthesis and in the of the in Lipid Res. Full Text PDF PubMed Google of cholesterol synthesis in small of and a significant of it is to liver where of total cholesterol synthesis a of intraperitoneally injected [3H]water is used for cholesterol synthesis in the and in the liver, the of the of cholesterol synthesis in the was the reduction of cholesterol synthesis in vivo less compared with that in the liver-specific protein knockout mice where of cholesterol synthesis was H. G. J.D. J.L. Brown M.S. increase in fatty acid synthesis in of mice with of in Full Text Full Text PDF PubMed Scopus Google In to L-SSKO mice, mice showed a significant decrease in the expression of centripetal of cholesterol in mice the of cholesterol synthesis in the extrahepatic particularly in the to the cholesterol synthesis in increasing the of the is also to that hepatic contents of cholesterol and its precursors distal to squalene were not altered in L-SSKO mice (Fig. 2). may be from the such as the to et R.S. Squalene and sterol synthesis in cells of the J. PubMed Scopus Google of which are cholesterol are out of the intestinal cells compared with and may not be in this because the centripetal of cholesterol was not altered in mice the absence of C. no in the centripetal of cholesterol from to the liver and in the Lipid Res. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). The in the gene expression in Fig. in the gene expressions of and and in the gene expressions of and also to the of cholesterol in the of may from of the pathway (Fig. owing to the reduced cholesterol or in the livers of L-SSKO mice J.D. J.L. Brown M.S. of the of cholesterol and fatty acid synthesis in the PubMed Scopus Google as for D. to inhibition of hepatic squalene Biochem. Biophys. 1998; PubMed Scopus Google Scholar, of hepatic expression by sterol biosynthesis inhibitors of squalene synthase are of expression in and 1998; PubMed Scopus Google Scholar) and D. of squalene synthase expression in Biochem. Biophys. PubMed Scopus Google Scholar) in hepatic cells or livers of treated with SS of may also for the in the expression of Transcriptional of metabolism by the and Biol. PubMed Scopus Google A. T. induces activity in cells and Biophys. Res. PubMed Scopus Google and H. A. C. K. hepatic and production of J. 2009; PubMed Scopus Google Scholar). In addition to the reduced expression of (Fig. of its may to while as for The a new of Genet. 1998; PubMed Scopus Google Scholar). such as which are from cholesterol, a with activity Cholesterol and bile acid metabolism are in mice lacking the receptor 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Brown control of cellular cholesterol Biol. Chem. Full Text Full Text PDF PubMed Scopus Google is from desmosterol, an precursor of cholesterol, has been to both the and C. A. F. J.C. et from cholesterol biosynthetic pathway as liver X receptor Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). However, contents were decreased in the livers of L-SSKO mice (supplementary Fig. 4 and Fig. it is that the of gene and protein expressions in L-SSKO mice were caused by of L-SSKO mice decreased plasma levels of both and in a fed (Table Fig. At the of this we that the lipid levels in L-SSKO mice were caused by of expression of in the livers and of as in the of J.L. Brown M.S. for hepatic lipoprotein in vivo in the Natl. Acad. Sci. USA. PubMed Scopus Google Scholar). However, expression was not significantly increased at either the mRNA (Fig. or the protein (Fig. plasma of was not (Fig. Because mevalonate was to mRNA in liver Z. of squalene synthase inhibition on the expression of hepatic cholesterol biosynthetic and cholesterol Biochem. Biophys. PubMed Scopus Google the increased hepatic contents (Fig. have the the of pathway in L-SSKO mice. The hypolipidemia was to the decrease in hepatic VLDL production. VLDL production and plasma lipid lowering have also been in T. R. E. H. of a squalene synthase in vivo and in J. PubMed Scopus Google Scholar) or T. R. E. of a squalene synthase in of J. PubMed Scopus Google Scholar) treated with an SS the mice SS in the liver showed an in of plasma lipoprotein were to an increase in hepatic VLDL production H. F. S. N. K. Yahagi N. Ohashi K. T. et cholesterol biosynthesis and in mice squalene synthase in the Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). does SS activity determine hepatic VLDL The decreased expression of and to the decreased hepatic VLDL production (supplementary Fig. because genes VLDL production H. N. T. J. F. M.M. receptor hepatic lipoprotein production in Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar, of the gene results in lethality in and in Natl. Acad. Sci. USA. PubMed Scopus Google Scholar, J. Liu T. Z. an and lipid protein, VLDL and by with 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). Although both expression of mRNA (Fig. and fatty acid synthesis (Fig. were increased, hepatic triglyceride synthesis be because of the decreased expression of the transcriptional factor J.D. J.L. Brown M.S. of the of cholesterol and fatty acid synthesis in the PubMed Scopus Google and accumulation of farnesol and its which triglyceride biosynthesis H. N. T. H. D. K. N. I. et synthase inhibitors triglyceride biosynthesis the farnesol pathway in Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). is also noteworthy that expressions of of target genes were decreased as Because activity is a of VLDL production H. A. C. K. hepatic and production of J. 2009; PubMed Scopus Google it is to that ablation of SS activity, VLDL production. Because we to in hepatic contents of the that can other may the of In this context, it is to that the nonsterol of diphosphate and its B. Chen J. The receptor is and regulated by of mevalonate Natl. Acad. Sci. USA. 1997; PubMed Scopus Google Scholar). This may a because a precursor of was increased in the livers of L-SSKO mice (Fig. The hypolipidemia observed in the L-SSKO mice in a fed in a fasted (Table 1). may the caused by inhibition of cholesterol by the biosynthesis of cholesterol in the liver Cholesterol synthesis by I. of and of Biol. Chem. Full Text PDF PubMed Google Scholar). the transient liver dysfunction has been to and in cells in PubMed Scopus Google Scholar). a on (supplementary Fig. liver dysfunction and increased synthesis of farnesol (Fig. supplementary Fig. it is highly that the transient of farnesol of in L-SSKO mice. The increased have as a compensatory (Fig. The of lapaquistat or was the for the of its development E.A. Bays H. O'Brien D. Pedicano J. Piper E. Spezzi A. Lapaquistat acetate: development of a squalene synthase inhibitor for the treatment of hypercholesterolemia.Circulation. 2011; 123: 1974-1985Crossref PubMed Scopus (54) Google Scholar). a of farnesol is a factor, of can the liver by the synthesis of A has been to nonsterol associated with treatment with A inhibitor of squalene synthase with a or a in Lipid Res. 2011; 52: Full Text Full Text PDF PubMed Scopus Google Scholar). from the liver of SS inhibition is and even though the inhibition is it be to up the development of SS inhibitors as a to and In conclusion, L-SSKO mice are hypolipidemic to a decrease in VLDL production. and transient liver This model can be used to further the of hepatic cholesterol synthesis in the regulation of plasma and to the of of SS The and for with Cre recombinase gene under the control of the albumin gene promoter cholesterol flanked by farnesyl diphosphate and HMG-CoA reductase receptor liver-specific protein knockout liver-specific SS knockout liver X receptor receptor sterol-regulatory element binding protein squalene synthase end