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4.P.258 Expression of GFP-SREBP fusion protein in CHO cells
350
Thursday 9 October 1997 : Posters Lipoprotein metabolism
in patients with FH . However, HDL size was similar (respectively 115 .4 ± 11 .0 vs 116.1 f 23 .2 A) . We conclude that patients with heterozygous FH up-regulate apo Al production in response to an apo AI-HDL hypercatabolism, which may itself be related to changes in HDL composition .
4 .P257
Moderate alcohol consumption in healthy volunteers reduced plasma clearance of apolipoprotein A-II
F. Gottrand 1 , L . Beghin 1 , N . Duha12 , B . Lacroix 2 , J .-P. Bonte2 , J .-C. Fruchart', G . Luc 1 . ' Department of Atherosclerosis, INSERM U325, Institut Pasteur de Lille, 1, rue du Professeur Calmette, 59019 Lille Cedex ; 2 Centre Universitaire de Mesures et d'Analyses, Faeulte de Pharmacie, Lille, France The present study shows the modifications of the metabolism of apoA-I and apoA-II using an endogenous labeling with cold isotope in normolipidemic healthy volunteers after a period of moderate alcohol consumption . Five non smoking male subjects were studied at the end of 2 consecutive 4-week periods, one without alcohol, the other with an intake of 50 g/day of alcohol, in a random order . The metabolic parameters of the apoA-I and apoA-II in HDL were determined after endogenous labeling using amino acid labeled with stable isotope . Cholesterol and triglycerides did not vary significantly whereas HDLcholesterol increased from 43 .8 to 50 .0 mg/dL (P < 0.05) . ApoA-I and apoA-II increased significantly (20 and 60% respectively) with alcohol . LpA-I :A-II increased from 73 .8 to 101 .6 mg/dL (+32%) (P < 0 .05) while alcohol had no effect on the concentration of LpA-I . The alcohol treatment did not significantly alter the metabolism of apoA-I . Conversely, the fractional catabolic rate of apoA-II decreased significantly by 21% (P < 0 .05) with alcohol while the production rate of apoA-II tended to increase by 18% (p = 0.08) . The decrease in the FCR of apoA-II could lead to the accumulation of apoA-II-containing lipoproteins in plasma and explain the dramatic increase in LpA-I:A-II observed in the plasma of subjects consuming alcohol .
4 .P.258
Expression of GFP-SREBP fusion protein in CHO cells
T. Hamakubo, M. Takaku, H. Shirai, Y. Kawabel, T. Kodama . Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo; 'Chugai Pharmaceutical Co. Ltd. Shizuoka, Japan Purpose : Sterol regulatory element binding proteins (SREBPs) are the transcription factors for cholesterol metabolic enzymes and low density lipoprotein receptor. The sterol-sensitive processing of SREBPs is considered to be crucial for cellular cholesterol regulatory mechanism. The SREBP precursor proteins exist in endoplasmic reticulum (ER) and nuclear membrane, and are sterol dependently processed to generate mature SREBPs . We tried to establish in vivo assay system for the specific SREBP processing enzymes by stable transfection of green fluorescent protein (GFP) labeled SREBP into chinese hamster ovary (CHO) cells . Methods: The eDNA of human SREBP2 (1540-3540) encoding two transmembrane domains and carboxy terminal half was inserted into pEGFP-C1 (Clontech) multi-cloning site . The construct was transfected into either wild type CHO cell-line or Ml9 CHO mutant cell-line (established by M .T. Hasan and T.Y. Chang) with polyamine transfection reagents (Trans-it, PanVera Corp .). Cells were grown in HamF12 medium supplemented with 10% fetal calf serum and neomycine for 10-15 days . Positive colonies were picked up under fluorescent microscopy and analyzed by confocal microscopy . Results and Discussion : Both wild type and M19 cells stably expressed GFP-SREBP fusion protein . The distribution of fluorescence resembled that of ER membrane proteins . The cholesterol depletion from culture medium will result in the liberation of GFP from ER into cytoplasm in wild type CHO cells. On the other hand, in M19 mutant cells, which lack processing enzyme activity, GFP will remain in ER membrane . These stably expressed cells will provide a useful system for the measurement of SREBP processing enzyme activities in vivo.
4 .R259
Disorders of lipoprotein metabolism in active sarcoidosis
I . Hurtado, S . Nomen, P. Caldti, C. Fiol, A. Salazar, A . Castells, X . Pint6, J . Mafia. Ciutat Sanitaria i Universitaria de Bellvitge (URE), Barcelona, Spain
increases in sarcoidosis disease . It has been described that an increase of SAA is associated with a decrease LCAT activity, but its association with CETP, another enzyme implicated in lipid metabolism, has not been studied . In addition, the behaviour of these enzymes on lipoproteins during active phase of sarcoidosis is unknown . Purpose : The aim of the present study was to analyze the effect of both enzymes on lipid metabolism in active and inactive phase of sarcoidosis . Methods : 80 sarcoid patients, 35 in active phase and 45 in inactive phase, was recruited . A lipidic characterization of every subject was done . SAA, LCAT, CETP, apolipoprotein Al (apo Al), and lipoprotein (a) [Lp(a)] were also measured . T-Student was used for comparisons of variables which were normally distributed. Results : In the active phase we found significantly reduced levels of LCAT (246 .3 ± 22 .3 vs 319 .4 f 28 .6 nmol/ml/h, p = 0 .05), CETP (15 .29 ± 0 .36 vs 16 .23 ± 0.24 nmol CE transferred/ml/h, p = 0 .036), HDL-C (1 .24 + 0 .10 vs 1 .53 f 0.09 mM, p = 0 .05), HDL-CE (0 .91 ± 0 .13 vs 1 .85 f 0.09 mM, p = 0.01) and apo Al (1 .37 ± 0.05 vs 1 .62 ± 0.04 g/L, p < 0 .0001) vs the inactive phase . In addition in the active phase we found significantly increased levels of SAA (160 .20 129 .18 vs 86 .07 f 10 .03 mg/ml, p = 0.021), TG (1 .56 ± 0.11 vs 1 .03 f 0.06 mM, p < 0 .0001), IDL-TG (0 .15 ± 0.01 vs 0 .04 ± 0 .01 mM, p = 0.019), VLDL-C (0 .69 ± 0.05 vs 0 .46 ± 0.03 mM, p = 0.001), and IDL-CE (0 .22 f 0 .02 vs 0.06 ± 0 .02 mM, p = 0 .029). We did not found significant differences in CT, CE, Lp(a) and LDL-C between both phases . Conclusions : The displacement of apo Al by apoSAA on HDL in the active phase causes a decrease in LCAT activity . It accounts for the lower level of HDL-CE, which could decreases the effectiveness of CETI? As a result of it IDL becomes enriched in TG. The increase of VLDL and IDL could be due to decreased clearance by liver . All these findings suggest an involvement of sarcoidosis in atherosclerosis .
4 .P.260
Metabolism of cholesterol but not that of fatty acid is regulated in HeLa S3 cells by physiological levels of mature SREBP-2 Y. Kawabe 12 , T. Suzuki t , M. Hayashil, T . Kodama 2 . 'Chugai Pharmaceutical Co ., Ltd. ; 2 RCAST Univ of Tokyo, Japan Intracellular cholesterol concentration is strictly regulated by LDL receptor and cholesterol biosynthetic enzymes . Transcriptional regulation of these factors are thought to be mediated by the sterol-sensitive cleavage of sterolregulated element biding proteins (SREBPs) . Studies using the reporter gene assays have shown that the transient over-expression of SREBPs increases transcriptional activity of LDL receptor (LDLR), HMG CoA reductase (HMGR), squalene synthase (SQS) and fatty acids synthase (FAS) promoters . However, N-acetylleucyl-leucyl-norleucinal, an inhibitor for SREBPs degradation, increases LDLR, HMGR and FAS mRNA levels, but does not change SQS mRNA levels . In order to clarify the manner in which transcriptional regulation occurs under physiological levels of mature SREBP-2, we introduced both lacl and lacl-regulated SREBP-2 (1-481) expression plasmids into HeLa S3 cells so that the mature form of human SREBP-2 (1-481) was induced by a galactose analogue, isopropyl-p-D-thiogalactopyranoside (IPTG) . We subsequently determined mRNA and protein levels in these cells (termed 23-11 cells) before and after the IPTG stimulation . Under similar culture conditions, levels of mRNA and protein for mature SREBP-2 in 23-11 cells were equal to both the wild type and the lacl-introduced HeLa S3 cells. mRNA levels corresponding to mature SREBP-2 in 23-11 cells treated with 0.3 mM IPTG for 4 hours was 2 fold higher compared to non-treated controls. Induced increases in mature SREBP-2 protein were similar either after treatment with IPTG or by culturing under conditions of cholesterol depletion. IPTG treatment to the 23-11 cells, at similar concentrations to those described above, also significantly increased their mRNA levels for LDLR and HMGR, but did not change that of SQS both in the presence or absence of 5 pM 25-hydroxycholesterol . FAS mRNA level was increased only when these cells were stimulated with IPTG in the absence of 25-hydroxycholesterol . IPTG stimulation increased both cellular 125 1-LDL binding and cholesterol biosynthetic activities, but did not change fatty acid biosynthetic activity in the presence of 25-hydroxycholesterol . These results indicate that physiological levels of man ire SREBP-2 regulate metabolism of cholesterol but not that of fatty acids in HeLa S3 cells . Furthermore, it also appears that SQS is a gene that is regulated by sterol and not SREBP-2 .
Introduction : Serum amyloid A (SAA), an apolipoprotein related with HDL, 11th International Symposium on Atherosclerosis, Paris, October 1997
Thursday 9 October 1997 : Posters Lipoprotein metabolism
in patients with FH . However, HDL size was similar (respectively 115 .4 ± 11 .0 vs 116.1 f 23 .2 A) . We conclude that patients with heterozygous FH up-regulate apo Al production in response to an apo AI-HDL hypercatabolism, which may itself be related to changes in HDL composition .
4 .P257
Moderate alcohol consumption in healthy volunteers reduced plasma clearance of apolipoprotein A-II
F. Gottrand 1 , L . Beghin 1 , N . Duha12 , B . Lacroix 2 , J .-P. Bonte2 , J .-C. Fruchart', G . Luc 1 . ' Department of Atherosclerosis, INSERM U325, Institut Pasteur de Lille, 1, rue du Professeur Calmette, 59019 Lille Cedex ; 2 Centre Universitaire de Mesures et d'Analyses, Faeulte de Pharmacie, Lille, France The present study shows the modifications of the metabolism of apoA-I and apoA-II using an endogenous labeling with cold isotope in normolipidemic healthy volunteers after a period of moderate alcohol consumption . Five non smoking male subjects were studied at the end of 2 consecutive 4-week periods, one without alcohol, the other with an intake of 50 g/day of alcohol, in a random order . The metabolic parameters of the apoA-I and apoA-II in HDL were determined after endogenous labeling using amino acid labeled with stable isotope . Cholesterol and triglycerides did not vary significantly whereas HDLcholesterol increased from 43 .8 to 50 .0 mg/dL (P < 0.05) . ApoA-I and apoA-II increased significantly (20 and 60% respectively) with alcohol . LpA-I :A-II increased from 73 .8 to 101 .6 mg/dL (+32%) (P < 0 .05) while alcohol had no effect on the concentration of LpA-I . The alcohol treatment did not significantly alter the metabolism of apoA-I . Conversely, the fractional catabolic rate of apoA-II decreased significantly by 21% (P < 0 .05) with alcohol while the production rate of apoA-II tended to increase by 18% (p = 0.08) . The decrease in the FCR of apoA-II could lead to the accumulation of apoA-II-containing lipoproteins in plasma and explain the dramatic increase in LpA-I:A-II observed in the plasma of subjects consuming alcohol .
4 .P.258
Expression of GFP-SREBP fusion protein in CHO cells
T. Hamakubo, M. Takaku, H. Shirai, Y. Kawabel, T. Kodama . Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo; 'Chugai Pharmaceutical Co. Ltd. Shizuoka, Japan Purpose : Sterol regulatory element binding proteins (SREBPs) are the transcription factors for cholesterol metabolic enzymes and low density lipoprotein receptor. The sterol-sensitive processing of SREBPs is considered to be crucial for cellular cholesterol regulatory mechanism. The SREBP precursor proteins exist in endoplasmic reticulum (ER) and nuclear membrane, and are sterol dependently processed to generate mature SREBPs . We tried to establish in vivo assay system for the specific SREBP processing enzymes by stable transfection of green fluorescent protein (GFP) labeled SREBP into chinese hamster ovary (CHO) cells . Methods: The eDNA of human SREBP2 (1540-3540) encoding two transmembrane domains and carboxy terminal half was inserted into pEGFP-C1 (Clontech) multi-cloning site . The construct was transfected into either wild type CHO cell-line or Ml9 CHO mutant cell-line (established by M .T. Hasan and T.Y. Chang) with polyamine transfection reagents (Trans-it, PanVera Corp .). Cells were grown in HamF12 medium supplemented with 10% fetal calf serum and neomycine for 10-15 days . Positive colonies were picked up under fluorescent microscopy and analyzed by confocal microscopy . Results and Discussion : Both wild type and M19 cells stably expressed GFP-SREBP fusion protein . The distribution of fluorescence resembled that of ER membrane proteins . The cholesterol depletion from culture medium will result in the liberation of GFP from ER into cytoplasm in wild type CHO cells. On the other hand, in M19 mutant cells, which lack processing enzyme activity, GFP will remain in ER membrane . These stably expressed cells will provide a useful system for the measurement of SREBP processing enzyme activities in vivo.
4 .R259
Disorders of lipoprotein metabolism in active sarcoidosis
I . Hurtado, S . Nomen, P. Caldti, C. Fiol, A. Salazar, A . Castells, X . Pint6, J . Mafia. Ciutat Sanitaria i Universitaria de Bellvitge (URE), Barcelona, Spain
increases in sarcoidosis disease . It has been described that an increase of SAA is associated with a decrease LCAT activity, but its association with CETP, another enzyme implicated in lipid metabolism, has not been studied . In addition, the behaviour of these enzymes on lipoproteins during active phase of sarcoidosis is unknown . Purpose : The aim of the present study was to analyze the effect of both enzymes on lipid metabolism in active and inactive phase of sarcoidosis . Methods : 80 sarcoid patients, 35 in active phase and 45 in inactive phase, was recruited . A lipidic characterization of every subject was done . SAA, LCAT, CETP, apolipoprotein Al (apo Al), and lipoprotein (a) [Lp(a)] were also measured . T-Student was used for comparisons of variables which were normally distributed. Results : In the active phase we found significantly reduced levels of LCAT (246 .3 ± 22 .3 vs 319 .4 f 28 .6 nmol/ml/h, p = 0 .05), CETP (15 .29 ± 0 .36 vs 16 .23 ± 0.24 nmol CE transferred/ml/h, p = 0 .036), HDL-C (1 .24 + 0 .10 vs 1 .53 f 0.09 mM, p = 0 .05), HDL-CE (0 .91 ± 0 .13 vs 1 .85 f 0.09 mM, p = 0.01) and apo Al (1 .37 ± 0.05 vs 1 .62 ± 0.04 g/L, p < 0 .0001) vs the inactive phase . In addition in the active phase we found significantly increased levels of SAA (160 .20 129 .18 vs 86 .07 f 10 .03 mg/ml, p = 0.021), TG (1 .56 ± 0.11 vs 1 .03 f 0.06 mM, p < 0 .0001), IDL-TG (0 .15 ± 0.01 vs 0 .04 ± 0 .01 mM, p = 0.019), VLDL-C (0 .69 ± 0.05 vs 0 .46 ± 0.03 mM, p = 0.001), and IDL-CE (0 .22 f 0 .02 vs 0.06 ± 0 .02 mM, p = 0 .029). We did not found significant differences in CT, CE, Lp(a) and LDL-C between both phases . Conclusions : The displacement of apo Al by apoSAA on HDL in the active phase causes a decrease in LCAT activity . It accounts for the lower level of HDL-CE, which could decreases the effectiveness of CETI? As a result of it IDL becomes enriched in TG. The increase of VLDL and IDL could be due to decreased clearance by liver . All these findings suggest an involvement of sarcoidosis in atherosclerosis .
4 .P.260
Metabolism of cholesterol but not that of fatty acid is regulated in HeLa S3 cells by physiological levels of mature SREBP-2 Y. Kawabe 12 , T. Suzuki t , M. Hayashil, T . Kodama 2 . 'Chugai Pharmaceutical Co ., Ltd. ; 2 RCAST Univ of Tokyo, Japan Intracellular cholesterol concentration is strictly regulated by LDL receptor and cholesterol biosynthetic enzymes . Transcriptional regulation of these factors are thought to be mediated by the sterol-sensitive cleavage of sterolregulated element biding proteins (SREBPs) . Studies using the reporter gene assays have shown that the transient over-expression of SREBPs increases transcriptional activity of LDL receptor (LDLR), HMG CoA reductase (HMGR), squalene synthase (SQS) and fatty acids synthase (FAS) promoters . However, N-acetylleucyl-leucyl-norleucinal, an inhibitor for SREBPs degradation, increases LDLR, HMGR and FAS mRNA levels, but does not change SQS mRNA levels . In order to clarify the manner in which transcriptional regulation occurs under physiological levels of mature SREBP-2, we introduced both lacl and lacl-regulated SREBP-2 (1-481) expression plasmids into HeLa S3 cells so that the mature form of human SREBP-2 (1-481) was induced by a galactose analogue, isopropyl-p-D-thiogalactopyranoside (IPTG) . We subsequently determined mRNA and protein levels in these cells (termed 23-11 cells) before and after the IPTG stimulation . Under similar culture conditions, levels of mRNA and protein for mature SREBP-2 in 23-11 cells were equal to both the wild type and the lacl-introduced HeLa S3 cells. mRNA levels corresponding to mature SREBP-2 in 23-11 cells treated with 0.3 mM IPTG for 4 hours was 2 fold higher compared to non-treated controls. Induced increases in mature SREBP-2 protein were similar either after treatment with IPTG or by culturing under conditions of cholesterol depletion. IPTG treatment to the 23-11 cells, at similar concentrations to those described above, also significantly increased their mRNA levels for LDLR and HMGR, but did not change that of SQS both in the presence or absence of 5 pM 25-hydroxycholesterol . FAS mRNA level was increased only when these cells were stimulated with IPTG in the absence of 25-hydroxycholesterol . IPTG stimulation increased both cellular 125 1-LDL binding and cholesterol biosynthetic activities, but did not change fatty acid biosynthetic activity in the presence of 25-hydroxycholesterol . These results indicate that physiological levels of man ire SREBP-2 regulate metabolism of cholesterol but not that of fatty acids in HeLa S3 cells . Furthermore, it also appears that SQS is a gene that is regulated by sterol and not SREBP-2 .
Introduction : Serum amyloid A (SAA), an apolipoprotein related with HDL, 11th International Symposium on Atherosclerosis, Paris, October 1997