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Inhibitory effect of squalene synthase inhibitors on triglyceride synthesis
Thursday June 29, 2000: Poster Abstracts P: W32 lntracellular Lipid Metabolism
288
I Th P14:W32 [ Inhibitory effect of squalene synthase inhibitors on triglyceride synthesis i
M. Yanagimachi, H. Hiyoshi, T. Saeki, H. Tanaka. Tsukuba Res. Lab., CV
Research Unit, Eisai Co., Tsukuba, Japan
Objective: Squalene synthase (SQS), a key enzyme in cholesterol (Chol) synthetic pathway, has been reported to be a novel target to treat hyperlipidemia. We found that oral administration of ER-27856, a potent SQS inhibitor (SQSI), but not atorvastafin (ATOV) decreased plasma triglyceride (TG) in Watanabe heritable hyperlipidemic rabbits. To determine the mechanism how SQSIs decreased plasma TG, we focused on the effect of SQSIs (ER-27856 and RPR-107393) on de novo TG synthesis in rat hepatocytes. Methods: Cells were incubated with wiUiam's medium E containing 10% fetal bovine serum (FBS) or lipoprotein deficient FBS (LPDS). Cells were incubated with SQSIs for 2-24 hr in the presence or absence of 2 mM mevalonolactune (MVL) or l /zM ATOV prior to addition of [14C]acetate. After 2 hr incubation with [14C]acetate, cellular [14C]Chol and [14C]TG were measured. Results: In the time course study, SQSIs inhibited Chol synthesis in 2-24 hr incubation and suppressed TG synthesis in 6-24 hr incubation, but not in 2 hr incubation. ATOV potently inhibited Chol synthesis, but not TG synthesis. Importantly, MVL or LPDS potentiated the inhibitory activity of SQSIs to TG synthesis. In contrast, the effect was diminished in the presence of ATOV:
FCS 100 1040
ER-27856 RPR-107393
IC50of TG synthesis(nM) FCS,2 mMMVL LPDS 3.5 4.8 47 130
I ThP17:W32 I The proteasome ymediates the degradation of endocytosed iipoproteins U. Tistiar 1, H. Scharnagl ~, H. Wieland t , M. Htittinger2, W. Marz.
FCS, 1 //,MATOV > 1000 > 10.000
Contusions: We provided the first evidence that the continuous inhibition of SQS suppressed TG synthesis by increasing mevalonate-derived non-sterol product. SQSIs, which suppress both Chol and TG synthesis, may be a novel therapeutic agents to prevent atherosclerosis.
ThP15:W32 ]
lipoprotein (LDL) with autologous blood plasma-derived serum leads to loss of sialic acid by lipoprotein particles. Later we have established that this modification is produced by trans-sialidase of human blood plasma. The goal of this study is to investigate the properties of plasma trans-sialidase and to elucidate an affect of this enzyme on the intracellular LDL metabolism. Methods: Trans-sialidase was isolated from lipoprotein deficient serum by the affinity chromatography on polysialic acid-sepharose. Results: Isolated trans-sialidase removes sialic acid from apofipoproteins and ganglyosides of LDL, VLDL, IDL, and HDL particles (in order to decrease of rate) as well as from plasma glycoproteins (fetuin, transferrin, ferritin). Removed molecule of sialic acid can be bound to galactose or N-acetyl-galactosamine of acceptors by the alpha-2.3 and alpha-2.6 linkage. The lipoprotein particles, plasma proteins and glycosphingofipids may serve as the acceptors of transferred sialic acid. Trans-sialidase treatment of native LDL leads to lipoprotein aggregation. Aggregates of trans-sialidase treated LDL is taken up by human aortic intimal smooth muscle cells via phagocytosis that leads to cholesteryl ester accumulation. Conclusions: Trans-sialidase may be involved in earlier stages of atherogenesis.
Deglycosylation is an atherogenic modification of ApoB-containing lipoproteins
V.V. Tertov, A.N. Orekhov. Institute of Experimental Cardiology, Moscow;
Institute for Atherosclerosis Research, Moscow, Russia
Objective: Several years ago we have found and isolated a subfractiun of LDL with low level of sialic acid. In contrast to native sialylated LDL, desialylated LDL was capable to accumulate cholesteryl esters in human aortic intimal smooth muscle cells and macrophages. Moreover, after sialic acid remove by neuraminidase, native LDL increased intracellular lipid content. In present study we studied the effect of desialylation and deglycosylation on apoB-containing fipoprotein atherogenicity. Methods: Native and in vivo desialylated VLDL, IDL and LDL were isolated by lectin chromatography on RCA120-agarose. Native lipoproteins were deglycosylated with specific endoglycosidases. Lipoprutein aggregation was measured by laser scattering. Results: Like desialylated LDL, in vivo desialylated VLDL and IDL stimulated free and esterified cholesterol content in human intimal smooth muscle cells and macrophages. In vitro treatment of VLDL and IDL with 2.6-and 2.3-specific sialidases causes an appearance of lipoprotein atherogenicity. Moreover, it was observed that demannosylation of VLDL, IDL and LDL with alpha-mannosidase is accompanied with an increase of lipoprotein atherogenic potential. Strong atherogenic effect was produced by the remove of lipoprotein high mannose and biantennary carbohydrate chains using endoglycosidases FI and F2 and peptide-N-glycosidase F. In all cases, deglycosylation of VLDL, IDL and LDL was accompanied by aggregation of lipoprotein particles. The filtration of deglycosylated lipoprotein samples prevented intracellular lipid accumulation. Conclusions: Deglycosylation-mediated lipoprotein aggregation is the reason for lipid accumulation in human intimal smooth muscle cells and macrophages. ThP16:W32 I Human plasma traus-sialidase affects LDL metabolism in aortic intimai smooth muscle cells [
V.V. Tertov, V.V. Kaplun, K.A. Boytsova, N.V. Bovin 1. Institute of
Experimental Cardiology, Moscow;/Institute of Bioorganic Chemistry, Moscow, Russia
Objective: Earlier we have found that incubation of human low density
/Department of Clinical Chemistry, Medical University Clinic, Freiburg, Germany; 2Institute of Medical Chemistry, University of Vienna, Austria
Objective: Lipoprotein particles are receptor mediated endocytosed and then int~acellularly degraded in the lysosomes. We now present data which indicate that in addition to the lysosomal enzymes, the proteasome contributes to the degradation of apolipoproteins. Methods: In cultured human skin fibroblasts degradation of endocytosed 1251-VLDL was measured in the presence of proteinase inhibitors. In addition subcellular fractionation and gel filtration was included in the study. Results: E64d, an inhibitor of cystein proteinases that penetrates into cells caused 24% inhibition. In combination with the peptidyl aldehydes MG132 or PSI inhibition increase up to 36 and 43%, respectively. MG132 and PSI are good inhibitors of the proteasome but have also some effect on cystein proteinases. We therefore used them in combination with E64d. In contrast lactacystin is considered specific for the proteasome and suitable for the use in cell culture. We measured 16% inhibition with lactacystin. In subcellular fractionation we detected undegraded 125I-VLDL in the cytoplasm. Condnsion: It has previously been reported that de novo synthesized apoB and apoE are degraded by the proteasome. It is also known that virus are receptor mediated endocytosed and then released into the cytoplasm and there hydrolyzed by the proteasome. Up to our knowledge the results presented here indicate for the first time that the proteasome mediated the degradation of endocytosed lipoproteins. T h P 1 8 : W 3 2 ] Effect of atorvastatin on the metabolism of cholesterol !
and triacylglycerides in HepG2 cdis
H. Scharnagl, R. Schinker, H. Gierens, M. Nauck, H. Wieland, W. M~z.
Albert-Ludwigs-University, Dept. of Clinical Chemistry, Freiburg, Germany Atorvastatin is a synthetic hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor that effectively reduces plasma cholesterol and triacylglyceride levels. The effect of atorvastatin on plasma lipids in vivo is stronger compared to other HMG-CoA reductase inhibitors known so far. The aim of our study was to evaluate the effect of atorvastatin on the metabolism of cholesterol and triacylglycerides in vitro in comparison to lovastatin and simvastatin. In HepG2 cells all drugs were equally effective in inhibiting cholesterol synthesis. The IC50 values of the three drugs were only slightly different (atorvastatin 68 nM, lovastatin 31 nM, simvastatin 93 nM). All compounds increased triacylglyceride synthesis by about 50%. Atorvastatin increased the receptor mediated endocytosis of LDL in a dose-dependent manner. At a concentration of 10-6 M, binding, uptake and degradation of 12SI-labeled LDL was enhanced 1.6 to 2.1 fold. The effect of atorvastatin on LDL receptor expression was not significantly different compared to simvastatin and lovastatin. In transfection experiments using a plasmid coding for firefly-luciferase coupled to the LDL receptor promotor gene, atorvastatin increased the expression of the LDL receptor gene. As determined by reporter gene assays and Northern blot analysis all HMG-CoA reductase inhibitors significantly induced expression of fatty acid synthase (FAS). These data suggest that
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
288
I Th P14:W32 [ Inhibitory effect of squalene synthase inhibitors on triglyceride synthesis i
M. Yanagimachi, H. Hiyoshi, T. Saeki, H. Tanaka. Tsukuba Res. Lab., CV
Research Unit, Eisai Co., Tsukuba, Japan
Objective: Squalene synthase (SQS), a key enzyme in cholesterol (Chol) synthetic pathway, has been reported to be a novel target to treat hyperlipidemia. We found that oral administration of ER-27856, a potent SQS inhibitor (SQSI), but not atorvastafin (ATOV) decreased plasma triglyceride (TG) in Watanabe heritable hyperlipidemic rabbits. To determine the mechanism how SQSIs decreased plasma TG, we focused on the effect of SQSIs (ER-27856 and RPR-107393) on de novo TG synthesis in rat hepatocytes. Methods: Cells were incubated with wiUiam's medium E containing 10% fetal bovine serum (FBS) or lipoprotein deficient FBS (LPDS). Cells were incubated with SQSIs for 2-24 hr in the presence or absence of 2 mM mevalonolactune (MVL) or l /zM ATOV prior to addition of [14C]acetate. After 2 hr incubation with [14C]acetate, cellular [14C]Chol and [14C]TG were measured. Results: In the time course study, SQSIs inhibited Chol synthesis in 2-24 hr incubation and suppressed TG synthesis in 6-24 hr incubation, but not in 2 hr incubation. ATOV potently inhibited Chol synthesis, but not TG synthesis. Importantly, MVL or LPDS potentiated the inhibitory activity of SQSIs to TG synthesis. In contrast, the effect was diminished in the presence of ATOV:
FCS 100 1040
ER-27856 RPR-107393
IC50of TG synthesis(nM) FCS,2 mMMVL LPDS 3.5 4.8 47 130
I ThP17:W32 I The proteasome ymediates the degradation of endocytosed iipoproteins U. Tistiar 1, H. Scharnagl ~, H. Wieland t , M. Htittinger2, W. Marz.
FCS, 1 //,MATOV > 1000 > 10.000
Contusions: We provided the first evidence that the continuous inhibition of SQS suppressed TG synthesis by increasing mevalonate-derived non-sterol product. SQSIs, which suppress both Chol and TG synthesis, may be a novel therapeutic agents to prevent atherosclerosis.
ThP15:W32 ]
lipoprotein (LDL) with autologous blood plasma-derived serum leads to loss of sialic acid by lipoprotein particles. Later we have established that this modification is produced by trans-sialidase of human blood plasma. The goal of this study is to investigate the properties of plasma trans-sialidase and to elucidate an affect of this enzyme on the intracellular LDL metabolism. Methods: Trans-sialidase was isolated from lipoprotein deficient serum by the affinity chromatography on polysialic acid-sepharose. Results: Isolated trans-sialidase removes sialic acid from apofipoproteins and ganglyosides of LDL, VLDL, IDL, and HDL particles (in order to decrease of rate) as well as from plasma glycoproteins (fetuin, transferrin, ferritin). Removed molecule of sialic acid can be bound to galactose or N-acetyl-galactosamine of acceptors by the alpha-2.3 and alpha-2.6 linkage. The lipoprotein particles, plasma proteins and glycosphingofipids may serve as the acceptors of transferred sialic acid. Trans-sialidase treatment of native LDL leads to lipoprotein aggregation. Aggregates of trans-sialidase treated LDL is taken up by human aortic intimal smooth muscle cells via phagocytosis that leads to cholesteryl ester accumulation. Conclusions: Trans-sialidase may be involved in earlier stages of atherogenesis.
Deglycosylation is an atherogenic modification of ApoB-containing lipoproteins
V.V. Tertov, A.N. Orekhov. Institute of Experimental Cardiology, Moscow;
Institute for Atherosclerosis Research, Moscow, Russia
Objective: Several years ago we have found and isolated a subfractiun of LDL with low level of sialic acid. In contrast to native sialylated LDL, desialylated LDL was capable to accumulate cholesteryl esters in human aortic intimal smooth muscle cells and macrophages. Moreover, after sialic acid remove by neuraminidase, native LDL increased intracellular lipid content. In present study we studied the effect of desialylation and deglycosylation on apoB-containing fipoprotein atherogenicity. Methods: Native and in vivo desialylated VLDL, IDL and LDL were isolated by lectin chromatography on RCA120-agarose. Native lipoproteins were deglycosylated with specific endoglycosidases. Lipoprutein aggregation was measured by laser scattering. Results: Like desialylated LDL, in vivo desialylated VLDL and IDL stimulated free and esterified cholesterol content in human intimal smooth muscle cells and macrophages. In vitro treatment of VLDL and IDL with 2.6-and 2.3-specific sialidases causes an appearance of lipoprotein atherogenicity. Moreover, it was observed that demannosylation of VLDL, IDL and LDL with alpha-mannosidase is accompanied with an increase of lipoprotein atherogenic potential. Strong atherogenic effect was produced by the remove of lipoprotein high mannose and biantennary carbohydrate chains using endoglycosidases FI and F2 and peptide-N-glycosidase F. In all cases, deglycosylation of VLDL, IDL and LDL was accompanied by aggregation of lipoprotein particles. The filtration of deglycosylated lipoprotein samples prevented intracellular lipid accumulation. Conclusions: Deglycosylation-mediated lipoprotein aggregation is the reason for lipid accumulation in human intimal smooth muscle cells and macrophages. ThP16:W32 I Human plasma traus-sialidase affects LDL metabolism in aortic intimai smooth muscle cells [
V.V. Tertov, V.V. Kaplun, K.A. Boytsova, N.V. Bovin 1. Institute of
Experimental Cardiology, Moscow;/Institute of Bioorganic Chemistry, Moscow, Russia
Objective: Earlier we have found that incubation of human low density
/Department of Clinical Chemistry, Medical University Clinic, Freiburg, Germany; 2Institute of Medical Chemistry, University of Vienna, Austria
Objective: Lipoprotein particles are receptor mediated endocytosed and then int~acellularly degraded in the lysosomes. We now present data which indicate that in addition to the lysosomal enzymes, the proteasome contributes to the degradation of apolipoproteins. Methods: In cultured human skin fibroblasts degradation of endocytosed 1251-VLDL was measured in the presence of proteinase inhibitors. In addition subcellular fractionation and gel filtration was included in the study. Results: E64d, an inhibitor of cystein proteinases that penetrates into cells caused 24% inhibition. In combination with the peptidyl aldehydes MG132 or PSI inhibition increase up to 36 and 43%, respectively. MG132 and PSI are good inhibitors of the proteasome but have also some effect on cystein proteinases. We therefore used them in combination with E64d. In contrast lactacystin is considered specific for the proteasome and suitable for the use in cell culture. We measured 16% inhibition with lactacystin. In subcellular fractionation we detected undegraded 125I-VLDL in the cytoplasm. Condnsion: It has previously been reported that de novo synthesized apoB and apoE are degraded by the proteasome. It is also known that virus are receptor mediated endocytosed and then released into the cytoplasm and there hydrolyzed by the proteasome. Up to our knowledge the results presented here indicate for the first time that the proteasome mediated the degradation of endocytosed lipoproteins. T h P 1 8 : W 3 2 ] Effect of atorvastatin on the metabolism of cholesterol !
and triacylglycerides in HepG2 cdis
H. Scharnagl, R. Schinker, H. Gierens, M. Nauck, H. Wieland, W. M~z.
Albert-Ludwigs-University, Dept. of Clinical Chemistry, Freiburg, Germany Atorvastatin is a synthetic hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor that effectively reduces plasma cholesterol and triacylglyceride levels. The effect of atorvastatin on plasma lipids in vivo is stronger compared to other HMG-CoA reductase inhibitors known so far. The aim of our study was to evaluate the effect of atorvastatin on the metabolism of cholesterol and triacylglycerides in vitro in comparison to lovastatin and simvastatin. In HepG2 cells all drugs were equally effective in inhibiting cholesterol synthesis. The IC50 values of the three drugs were only slightly different (atorvastatin 68 nM, lovastatin 31 nM, simvastatin 93 nM). All compounds increased triacylglyceride synthesis by about 50%. Atorvastatin increased the receptor mediated endocytosis of LDL in a dose-dependent manner. At a concentration of 10-6 M, binding, uptake and degradation of 12SI-labeled LDL was enhanced 1.6 to 2.1 fold. The effect of atorvastatin on LDL receptor expression was not significantly different compared to simvastatin and lovastatin. In transfection experiments using a plasmid coding for firefly-luciferase coupled to the LDL receptor promotor gene, atorvastatin increased the expression of the LDL receptor gene. As determined by reporter gene assays and Northern blot analysis all HMG-CoA reductase inhibitors significantly induced expression of fatty acid synthase (FAS). These data suggest that
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000