- Email: [email protected]
A role of Sp1 in regulation of human serum paraoxonase gene transcription
Ota
positive double action of statins and fibrates through decreased plasma lipid concentration as well as inflammatory cells activity could be set forth on the basis of obtained results. ~
CHYLOMICRON REMNANTS STIMULATE INTERLEUKIN-1II PRODUCTION IN THP-1 MACROPHAGES
T. Okumura, Y. Fujioka, S. Tsuboi, S. Morimoto, M. Masai, Y. Naito, D. Kawasaki, T. Sakoda, T. Tsujino, T. Iwasaki. Hyogo College of Medicine,
Nishinomiya, Hyogo, Japan Accumulating data show that postprandial hyperlipidemia is associated with atherosclerotic diseases and chylomicron remnants (CR) have an important role in this milieu, but it is still unknown how CR can contribute to an inflammatory response in the progression of atherosclerosis in vascular cells. Interleukin 113 (IL-I[3) is one of key cytokines in vascular diseases. We examined the production of IL-I[3 in THP-1 macrophages with the stimulation by human normal LDL (NLDL), human oxidized LDL (OxLDL), and CR. NLDL was prepared with the ultracentrifugation, and OxLDL was made with copper sulphate. CR were prepared from rat lymph. THP-1 cells were stimulated by phorbol ester for 4 h to differentiate to macrophages. After changing medium to RPMI-1640 with 10% fetal bovine serum incubating for 24 h and sequentially with 10% lipoprotein deficient serum incubating for 48 h, cells were stimulated with lipoproteins in RPMI-1640 without serum and phorbol ester. The levels of mRNA of IL-l[3 were examined by Northern blot analysis. The secreted protein mass of IL-I[3 in the culture medium was determined by ELISA. NLDL and OxLDL could not increase mRNA levels or the protein levels of IL-I[3 compared with control. CR increased mRNA levels and the protein levels of IL-I[3 in time and dose dependent manner. 1.0 ~tg/ml CR upregulated both mRNA levels and the protein levels of IL-I[3 about three times greater than control at 24 h. These results suggest that CR could induce inflammatory response in the progression of atherosclerosis.
~PIG
APOLIPOPROTEIN A-IV mRNA HEPATIC EXPRESSION IS COORDINATED INSIDE THE A-I/C-III/A-IV GENE CLUSTER IN DIETARY RESPONSE BUT NOT IN INFLAMMATION
M.A. Navarro 1, S. Acin 1, M. Iturralde 1, L. Calleja 1, R. Carnicer 1, M.A. Guzman-Garcia 1, N. Gonzalez-Ramon 1, R Mata 2, B. Isabel3, C.J. Lopez-Bote 3, E Lampreave 1, A. Pineiro 1, J. Osada 1. IDepartamento
de Bioquimica y Biologia Molecular y Celular, Universidad de Zaragoza," 2Fundacion Jimenez Diaz, Madrid," 3Departamento de Produccion Animal, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain Apolipoprotein (apo) A-IV is a member of the apo A-I/C-III/A-IV gene cluster. To investigate its hypothetical coordinated regulation, we have cloned and sequenced porcine apolipoprotein A-IV cDNA, and addressed the cluster in vivo regulation under several conditions: the amount and saturation of dietary fat and the inflammation process. Mature porcine apo A-IV consists of 362 amino acids and displays a 75.6% sequence identity with human protein. Pig apo A-IV is the smallest reported mammalian apo A-IV because it lacks the repeated motifs of glutamine and glutamic acid at the carboxyl terminus. Diets enriched in fat without cholesterol induced an increase in hepatic apo A-I, A-IV and C-III mRNA levels, these increases being more pronounced with a sunflower oil diet. Plasma apo A-IV also increased in these situations mainly in the lipoprotein-free fraction. In response to turpentine oil induced inflammation a decreased hepatic apo A-IV mRNA expression was observed independent of apos A-I and C-III. Addition of cytokines to pig hepatocyte cultures also decreased apo A-IV mRNA levels. All these results show that: porcine apo A-IV has evolved from a different mammalian branch point, its regulation presents a extraordinary sensitivity to dietary fat saturation and plays a role in the inflammatory response. ~ A
ROLE OF Spl IN REGULATION OF HUMAN SERUM PARAOXONASE GENE TRANSCRIPTION
E Osaki, Y. Ikeda, T. Suehiro, K. Ota, S. Tsuzura, K. Arii, Y. Kumon, K. Hashimoto. Kochi Medical School, Nankoku City, Japan Human serum paraoxonase (PON1) is associated with high density lipoprotein (HI)L), and inhibits oxidative modification of low density lipoprotein (LDL) in vitro. Therefore, PON1 is supposed to protect against atherosclerosis in vivo. We previously demonstrated that the consensus binding site for the transcription factor, Spl in the 5~-upstream region of the PON1
177
gene was crucial for PON1 transcription using deletion analysis. It has been suggested that phosphorylation of the zinc finger region of Spl is required for its activation. In this study, we further investigated a role of Spl in regulation of PON1 transcription. The human hepatoma cells, HepG2, were transfected with luciferase reporter vector cotaining 5~-upstream region of the PON1 gene (-1230/-6), and basal luciferase activity was determined at 48 hrs after transfection. Overexpression of Spl dramatically enhanced PON1 transcriptional activity (5 10-fold). Synergistic promoter activation (>10-fold) was observed when Spl-transfected cells were treated with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) to activate protein kinase C (PKC). These data suggest that Spl induces PON1 promoter activity and that PKC possibly enhances this effect. However, its mechanism of action in this respect remains unclear. It should be clarified whether these effects are mediated by direct interaction between Spl and PKC and which isoform of PKC is responsible for this action. ~
TREATMENT OF FAMILIAL HYPERCHOLESTEROLEMIA(FH) IN CHILDREN
L. Ose. Lipid Clinic, Department of Medicine, Rikshospitalet, Oslo,
Norway Reduced intake of saturated fat and cholesterol and increased intake of unsaturated fat, are the main dietary advises for the treatment of high blood cholesterol in young children with familial hypercholesterolemia (FH). Several studies in children have documented a limited effect in children and the compliance to such diets have been poor. New dietary advises have been proposed and new drugs previously not allowed for the use in children have recently been tested. Resent research do support the favorable effect of naturally occuring plant sterols on blood cholesterol levels in adults. The aim of a study recently performed in our clinic was study the effect of 20 grams of plant sterols ester-enriched spread on serum lipids as well as safety parameters in children with FH. Blood concentration of LDLcholesterol was reduced with 10.2%. Our clinic has recently participated in two studies with statins (atorvastatin and simvastatin) in FH-children, aged 11-16 years. Atorvastatin treatment resulted in a significant reduction in LDL-cholesterol from baseline to week 26 wehen atorvastatin was titrated to 20 mg. The incidence of treatment-related adverse events was low and similar for the atorvastatin and placebo treatment groups. Data from a study using simvastatin in children will also be presented. ~]
PHYTOSTEROLS IN COMMERCIAL CORN OIL REDUCE CHOLESTEROL ABSORPTION
R.E. Ostlund, S.B. Racette, A. Okeke, W.E Stenson. Washington University,
St. Louis, MO, USA Although supplementation of the diet with phytosterols reduces cholesterol absorption and LDL cholesterol, very little is known about the potential effects of phytosterols already present in natural foods. Vegetable oils are the richest dietary source of phytosterols and corn oil contains 0.77% by weight. We tested the hypothesis that removal ofphytosterols from corn oil would increase cholesterol absorption in single-meal tests containing corn oil as a fat source. Free and esterified phytosterols were removed on a kilogram scale by two new processes, competitive saturation adsorption to silica and reversed phase adsorption to charcoal in the presence of ethanol. Healthy subjects (N-25) received an otherwise sterol-free test breakfast on two occasions two weeks apart that contained 35 mg hexadeuterated cholesterol and 3 0 0 5 g of a corn oil preparation. The plasma enrichment of tracer was measured by negative ion mass spectrometry. Cholesterol absorption was 38.0% higher with sterol-free corn oil than with commercial corn oil of identical fatty acid content (p-0.005). When corn oil phytosterols were added back to sterol-free corn oil at a level of 150 mg/test meal, cholesterol absorption was reduced by 12.1% (p-0.03) and by 27.9% after inclusion of 300 mg phytosterols (p-0.01). Trace phytosterols comprising less than 1% of commercial corn oil mass substantially reduce cholesterol absorption and may account for part of the cholesterol-lowering activity of corn oil previously attributed solely to unsaturated fatty acids. F~
EFFECT OF STATIN ON TRANSCRIPTION OF HUMAN SERUM PARAOXONASE 1 GENE
K. Ota, T. Suehiro, K. Arii, Y. Ikeda, Y. Kumon, K. Hashimoto. Kochi
Medical School, Nankoku, Kochi, Japan Statin has pleiotropic effects in addition to decreasing serum cholesterol level. One of them is supposed to be inhibition of lipoprotein oxidation,
73rd EAS Congress
positive double action of statins and fibrates through decreased plasma lipid concentration as well as inflammatory cells activity could be set forth on the basis of obtained results. ~
CHYLOMICRON REMNANTS STIMULATE INTERLEUKIN-1II PRODUCTION IN THP-1 MACROPHAGES
T. Okumura, Y. Fujioka, S. Tsuboi, S. Morimoto, M. Masai, Y. Naito, D. Kawasaki, T. Sakoda, T. Tsujino, T. Iwasaki. Hyogo College of Medicine,
Nishinomiya, Hyogo, Japan Accumulating data show that postprandial hyperlipidemia is associated with atherosclerotic diseases and chylomicron remnants (CR) have an important role in this milieu, but it is still unknown how CR can contribute to an inflammatory response in the progression of atherosclerosis in vascular cells. Interleukin 113 (IL-I[3) is one of key cytokines in vascular diseases. We examined the production of IL-I[3 in THP-1 macrophages with the stimulation by human normal LDL (NLDL), human oxidized LDL (OxLDL), and CR. NLDL was prepared with the ultracentrifugation, and OxLDL was made with copper sulphate. CR were prepared from rat lymph. THP-1 cells were stimulated by phorbol ester for 4 h to differentiate to macrophages. After changing medium to RPMI-1640 with 10% fetal bovine serum incubating for 24 h and sequentially with 10% lipoprotein deficient serum incubating for 48 h, cells were stimulated with lipoproteins in RPMI-1640 without serum and phorbol ester. The levels of mRNA of IL-l[3 were examined by Northern blot analysis. The secreted protein mass of IL-I[3 in the culture medium was determined by ELISA. NLDL and OxLDL could not increase mRNA levels or the protein levels of IL-I[3 compared with control. CR increased mRNA levels and the protein levels of IL-I[3 in time and dose dependent manner. 1.0 ~tg/ml CR upregulated both mRNA levels and the protein levels of IL-I[3 about three times greater than control at 24 h. These results suggest that CR could induce inflammatory response in the progression of atherosclerosis.
~PIG
APOLIPOPROTEIN A-IV mRNA HEPATIC EXPRESSION IS COORDINATED INSIDE THE A-I/C-III/A-IV GENE CLUSTER IN DIETARY RESPONSE BUT NOT IN INFLAMMATION
M.A. Navarro 1, S. Acin 1, M. Iturralde 1, L. Calleja 1, R. Carnicer 1, M.A. Guzman-Garcia 1, N. Gonzalez-Ramon 1, R Mata 2, B. Isabel3, C.J. Lopez-Bote 3, E Lampreave 1, A. Pineiro 1, J. Osada 1. IDepartamento
de Bioquimica y Biologia Molecular y Celular, Universidad de Zaragoza," 2Fundacion Jimenez Diaz, Madrid," 3Departamento de Produccion Animal, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain Apolipoprotein (apo) A-IV is a member of the apo A-I/C-III/A-IV gene cluster. To investigate its hypothetical coordinated regulation, we have cloned and sequenced porcine apolipoprotein A-IV cDNA, and addressed the cluster in vivo regulation under several conditions: the amount and saturation of dietary fat and the inflammation process. Mature porcine apo A-IV consists of 362 amino acids and displays a 75.6% sequence identity with human protein. Pig apo A-IV is the smallest reported mammalian apo A-IV because it lacks the repeated motifs of glutamine and glutamic acid at the carboxyl terminus. Diets enriched in fat without cholesterol induced an increase in hepatic apo A-I, A-IV and C-III mRNA levels, these increases being more pronounced with a sunflower oil diet. Plasma apo A-IV also increased in these situations mainly in the lipoprotein-free fraction. In response to turpentine oil induced inflammation a decreased hepatic apo A-IV mRNA expression was observed independent of apos A-I and C-III. Addition of cytokines to pig hepatocyte cultures also decreased apo A-IV mRNA levels. All these results show that: porcine apo A-IV has evolved from a different mammalian branch point, its regulation presents a extraordinary sensitivity to dietary fat saturation and plays a role in the inflammatory response. ~ A
ROLE OF Spl IN REGULATION OF HUMAN SERUM PARAOXONASE GENE TRANSCRIPTION
E Osaki, Y. Ikeda, T. Suehiro, K. Ota, S. Tsuzura, K. Arii, Y. Kumon, K. Hashimoto. Kochi Medical School, Nankoku City, Japan Human serum paraoxonase (PON1) is associated with high density lipoprotein (HI)L), and inhibits oxidative modification of low density lipoprotein (LDL) in vitro. Therefore, PON1 is supposed to protect against atherosclerosis in vivo. We previously demonstrated that the consensus binding site for the transcription factor, Spl in the 5~-upstream region of the PON1
177
gene was crucial for PON1 transcription using deletion analysis. It has been suggested that phosphorylation of the zinc finger region of Spl is required for its activation. In this study, we further investigated a role of Spl in regulation of PON1 transcription. The human hepatoma cells, HepG2, were transfected with luciferase reporter vector cotaining 5~-upstream region of the PON1 gene (-1230/-6), and basal luciferase activity was determined at 48 hrs after transfection. Overexpression of Spl dramatically enhanced PON1 transcriptional activity (5 10-fold). Synergistic promoter activation (>10-fold) was observed when Spl-transfected cells were treated with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) to activate protein kinase C (PKC). These data suggest that Spl induces PON1 promoter activity and that PKC possibly enhances this effect. However, its mechanism of action in this respect remains unclear. It should be clarified whether these effects are mediated by direct interaction between Spl and PKC and which isoform of PKC is responsible for this action. ~
TREATMENT OF FAMILIAL HYPERCHOLESTEROLEMIA(FH) IN CHILDREN
L. Ose. Lipid Clinic, Department of Medicine, Rikshospitalet, Oslo,
Norway Reduced intake of saturated fat and cholesterol and increased intake of unsaturated fat, are the main dietary advises for the treatment of high blood cholesterol in young children with familial hypercholesterolemia (FH). Several studies in children have documented a limited effect in children and the compliance to such diets have been poor. New dietary advises have been proposed and new drugs previously not allowed for the use in children have recently been tested. Resent research do support the favorable effect of naturally occuring plant sterols on blood cholesterol levels in adults. The aim of a study recently performed in our clinic was study the effect of 20 grams of plant sterols ester-enriched spread on serum lipids as well as safety parameters in children with FH. Blood concentration of LDLcholesterol was reduced with 10.2%. Our clinic has recently participated in two studies with statins (atorvastatin and simvastatin) in FH-children, aged 11-16 years. Atorvastatin treatment resulted in a significant reduction in LDL-cholesterol from baseline to week 26 wehen atorvastatin was titrated to 20 mg. The incidence of treatment-related adverse events was low and similar for the atorvastatin and placebo treatment groups. Data from a study using simvastatin in children will also be presented. ~]
PHYTOSTEROLS IN COMMERCIAL CORN OIL REDUCE CHOLESTEROL ABSORPTION
R.E. Ostlund, S.B. Racette, A. Okeke, W.E Stenson. Washington University,
St. Louis, MO, USA Although supplementation of the diet with phytosterols reduces cholesterol absorption and LDL cholesterol, very little is known about the potential effects of phytosterols already present in natural foods. Vegetable oils are the richest dietary source of phytosterols and corn oil contains 0.77% by weight. We tested the hypothesis that removal ofphytosterols from corn oil would increase cholesterol absorption in single-meal tests containing corn oil as a fat source. Free and esterified phytosterols were removed on a kilogram scale by two new processes, competitive saturation adsorption to silica and reversed phase adsorption to charcoal in the presence of ethanol. Healthy subjects (N-25) received an otherwise sterol-free test breakfast on two occasions two weeks apart that contained 35 mg hexadeuterated cholesterol and 3 0 0 5 g of a corn oil preparation. The plasma enrichment of tracer was measured by negative ion mass spectrometry. Cholesterol absorption was 38.0% higher with sterol-free corn oil than with commercial corn oil of identical fatty acid content (p-0.005). When corn oil phytosterols were added back to sterol-free corn oil at a level of 150 mg/test meal, cholesterol absorption was reduced by 12.1% (p-0.03) and by 27.9% after inclusion of 300 mg phytosterols (p-0.01). Trace phytosterols comprising less than 1% of commercial corn oil mass substantially reduce cholesterol absorption and may account for part of the cholesterol-lowering activity of corn oil previously attributed solely to unsaturated fatty acids. F~
EFFECT OF STATIN ON TRANSCRIPTION OF HUMAN SERUM PARAOXONASE 1 GENE
K. Ota, T. Suehiro, K. Arii, Y. Ikeda, Y. Kumon, K. Hashimoto. Kochi
Medical School, Nankoku, Kochi, Japan Statin has pleiotropic effects in addition to decreasing serum cholesterol level. One of them is supposed to be inhibition of lipoprotein oxidation,
73rd EAS Congress