- Email: [email protected]
2P-0460 Role of sterol regulatory element binding protein-1 (SREBP-1) in hepatic regeneration after partial hepatectomy
140
Tuesday September 30, 2003: Poster Session Intracellular lipid transport and metabolism
endocytosis pathway mediate LDL uptake. Fluid-phase uptake of LDL as a mechanism of macrophage cholesterol accumulation shows that modification of LDL is not necessary for foam cell formation to occur. In addition, the findings direct attention to macrophage fluid-phase endocytosis pathways as relevant targets for modulating macrophage cholesterol accumulation in atherosclerosis. 2P-0456
Supernatant protein factor (SPF), a cytosolic squalene epoxidase regulator
N. Shibata 1 , M. Tsujimoto 2 , K. Jishage 3 , H. Arai 1 . 1 Dept. of Health Chemistry, Grad. School of Pharma. Sci., Univ. of Tokyo and Laboratory of Cellular Biochemistry, RIKEN; 2 Laboratory of Cellular Biochemistry, RIKEN; 3 Chugai Research Institute For Medical Science, Japan Objective: Squalene epoxidase is a microsomal monooxygenase that catalyzes the conversion of squalene to squalene 2,3-oxide. This conversion is the trigger of steroid skeleton formation in the sterol biosynthetic pathway. SPF (Supernatant Protein Factor) was identified in the 1950s as a cytosolic protein that stimulates microsomal squalene epoxidase activity, but so far, little information is available on the primary structure or the biological function of SPF. Recently, we purified SPF and cloned its cDNA. So far, we made the following observations when analyzing the function of SPF: (1) SPF mRNA is expressed abundantly in the liver and small intestine. (2) Recombinant SPF produced in E. coli promoted intermembrane transfer of squalene and activity of squalene epoxidase in vitro. (3) Transfection of SPF cDNA in hepatoma cells significantly stimulated de novo cholesterol biosynthesis, and mRNA expression of cholesterol biosynthetic enzymes. These data indicate that SPF functions in maintaining cholesterol homeostasis in vivo, and shows characteristics of a novel and tissue-specific cholesterol biosynthesis regulator. Here we examined this hypothesis in SPF-deficient mice. Method: We generated SPF-deficient mice by targeted gene disruption and examined plasma lipids and mRNA levels of cholesterol biosynthetic enzymes in the liver. Results and Conclusion: SPF-deficient mice were normally developed. No apparent phenotype and change of plasma lipids (including cholesterol and triglyceride) was observed under standard conditions. Hepatic HMG-CoA synthase and squalene synthase mRNA expressions significantly increased in SPF-deficient mice. These data indicate that SPF-deficient mice regulate their cholesterol demand and maintain cholesterol homeostasis by this compensatory mechanism. We are analyzing the physiological function of SPF in cholesterol metabolism, and we will present recent observations. 2P-0457
Involvement of phospholipase A2 in the supply of fatty acids required for cholesterol esterification in oxidized LDL-stimulated macrophages
S. Akiba, Y. Yoneda, S. Ohno, T. Sato. Department of Pathological Biochemistry, Kyoto Pharmaceutical University, Japan Objective: In macrophages, the accumulation of cholesterol esters associated with uptake of oxidized LDL (oxLDL) leads to the transformation into lipid-laden foam cells, a key event in the development and progression of atherosclerosis. However, little is known about the pathways involved in the supply of fatty acids, precursors for acyl-CoA required for the cholesterol esterification. We examined the possible involvement of cytosolic phospholipase A2 (cPLA2 ) in the supply of fatty acids using oxLDL-stimulated mouse peritoneal macrophages. Results: In [3 H]oleic acid-labeled cells, oxLDL induced cholesteryl oleate formation with an increase in free oleic acid and a decrease in phosphatidylcholine. The changes in these lipids were suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cPLA2 inhibitor. However, MAFP had no effect on the ACAT activity or the uptake of oxLDL. Stimulation with oxLDL in the presence of [3 H]cholesterol increased [3 H]cholesterol ester. The formation of cholesterol ester under this condition was also inhibited by MAFP, and the inhibitory effect was reversed by adding oleic acid. Under the conditions, oxLDL did not affect the activity or amounts of cPLA2 . When cells were stimulated with oxidized lipids (13-hydroxyoctadecadienoic acid, 7-ketocholesterol, or 25-hydroxycholesterol) in oxLDL, 13-hydroxyoctadecadienoic acid, but not oxysterols, induced oleic acid release, which was sensitive to MAFP. Conclusion: Based on the results, we suggest that oxLDL induces cPLA2 activation, which contributes, at least in part, to the supply of fatty acids required for the cholesterol esterification in macrophages. In the oxLDLinduced acceleration of the catalytic action of cPLA2, oxidized lipids, such as 13-hydroxyoctadecadienoic acid, may be involved.
2P-0458
Microarray analysis of cardiac gene expression in heart-specific hormone-sensitive lipase overexpressing mice
J. Suzuki 1 , H. Kanehara 1 , Y. Zenimaru 1 , S. Takahashi 1 , K. Oida 1 , F. Kraemer 2 , I. Miyamori 1 . 1 Fukui Medical University, Matsuoka, Fukui, Japan; 2 Stanford University, USA Intracellular lipid accumulation is a key in the pathogenesis of cardiomyopathy. We have reported that heart-specific overexpression of hormone-sensitive lipase (HSL) inhibited cardiac lipid accumulation. Here we studied cardiac gene expressions by 2-step comparison analyses to identify gene clusters that respond specifically to fasting and/or HSL-overexpression. Control (Ct) and HSL-transgenic (Tg) mice were fasted for 16h and cardiac gene expressions were screened by Affymetrix Mu11k, followed by RT-PCR or Northern analysis. Analysis 1: Setting the fed Ct as the reference, profiles of fasted Ct, fed Tg and fasted Tg were determined. The resultant profiles were then compared to exclude overlapping genes. 56 genes (pyruvate carboxylase, beta-tropomyosin etc.) were affected exclusively by fasting, indicating their response to stress-hormones. 61 genes (NFk-B, p53 etc.) were affected exclusively by HSL-overexpression, suggesting their response to released FFA. 58 genes (ATP synthase, UCP2 etc.) responded only to the additive effect of fasting and HSL-Tg. Analysis 2: Fasting profiles were determined separately in Ct or Tg with the respective fed Ct or fed Tg as references. Each fasting profile was compared to extract overlapping or non-overlapping genes. Fasting affected 74 genes in Ct while 55 genes were changed in Tg, and only 5 genes (c-fos, N10 etc.) overlapped. In summary, microarray 2-step comparison analyses revealed cardiac gene clusters that specifically respond to fasting or HSL-overexpression, and that HSL-overexpression drastically affected the physiological fasting profile. This approach will be useful to understand the mechanism of cardiomyopathy caused by aberrant regulation of intracellular lipid metabolism. 2P-0459
Cholesterol transport across the intestinal epithelial cells
M. Hussain, J. Iqbal, K. Anwar. SUNY Downstate Medical Center, Depts. of Anatomy and Cell Biology, and Pediatrics, Brooklyn, New York, USA It is generally believed that cholesterol is transported across the intestinal epithelial cells exclusively via chylomicron assembly and secretion. Contrary to the general belief, our data indicate that newly absorbed cholesterol is neither immediately available for secretion with apoB-lipoproteins nor exclusively secreted as part of chylomicrons. Based on our data, cholesterol transport by enterocytes can be broadly classified into two independently modulated apoB-dependent and apoB-independent pathways. Cholesterol secretion by the apoB-dependent pathway is induced by oleic acid, is repressed by microsomal triglyceride transfer protein inhibitors, and occurs only with larger apoB-containing lipoproteins. ApoB-independent pathways do not require microsomal triglyceride transfer protein (MTP) and involve efflux mediated by ABCA1, HDL assembly, and possibly other unknown mechanisms. These pathways show differential specificity toward the free and esterified cholesterol. The apoB-dependent pathway transports the esterified cholesterol, whereas both pathways secrete free cholesterol. Labeling studies indicate for at least two different metabolic pools of cholesterol in enterocytes. The newly absorbed and pre-absorbed cholesterol are preferentially secreted via apoB-independent and apoB-dependent pathways, respectively. In contrast to compartmentalization for secretion, these two metabolic pools are equally accessible for cellular esterification. Thus, enterocytes transport cholesterol by several independently regulated pathways raising the possibility that targeting of apoB-independent pathways may result in selective inhibition of cholesterol transport without affecting triglyceride transport. 2P-0460
Role of sterol regulatory element binding protein-1 (SREBP-1) in hepatic regeneration after partial hepatectomy
K. Hashida, M. Fujita, M. Kinoshita, T. Maeda, Y. Maeda, M. Kudo, T. Suda, D. Hirota, M. Taki, T. Teramoto. Teikyo University School of Medicine, Tokyo, Japan Objective: We had reported that fatty acid synthetase (FAS) activity was increased after partial hepatectomy (PH), and this may be the initial signals for hepatocyte regeneration after PH. In this study, relationship between SREBP-1 mRNA, which is known to be a transcription factor of FAS, and hepatocyte regeneration after PH was examined Methods: Male Sprague-Dawley rat (230-240g) was used for the experiments. Streptozotocin (STZ) induced diabetic rat was made by the injection of STZ from tail vein. Insulin treatment to STZ induced diabetic rat was
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
Tuesday September 30, 2003: Poster Session Adhesion molecules performed by the subcutaneous injection of Humalin N (4units). After 3 day of STZ injection, PH was performed. And 1days or 7 days after PH, liver was removed and used for mRNA measurement. Results: It was revealed that, on the first day after PH, expression of hepatic SREBP-1 mRNA decreased, although FAS activity was increased. STZ induced diabetic rat showed a significant decrease of SREBP-1 mRNA, but the regeneration of hepatocytes were observed as usual. These data indicate that the decrease of SREBP-1 is not be the initiation signal of regeneration of hepatocytes. When STZ induced diabetic rats were treated with insulin, SREBP-1 mRNA increased and hepatic regeneration was occurred more than that of untreated diabetic rats, significantly. This data indicated that SREBP-1 might be the enhancer of hepatic regeneration. Conclusion: SREBP-1 plays not as the initiator but as the enhancer role in the hepatocyte regeneration after partial hepatectomy. 2P-0461
SREBPs are negatively regulated by SUMO-1 modification
R. Sato 1 , Y. Hirano 2 , S. Murata 3 , K. Tanaka 3 , M. Shimizu 2 . 1 Department of Applied Biological Chemistry, University of Tokyo; 2 University of Tokyo; 3 Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
2P-0462
Impact of adipophilin expression on lipid accumulation in human macrophages: Potential role in atherogenesis
G. Larigauderie, C. Furman, C. Lasselin, C. Copin, J.-C. Fruchart, G. Castro, M. Rouis. INSERM U545, Lille Pasteur Institute, Lille, France Macrophage-derived foam cells, which occur in abundance in most stages of the atherosclerotic lesion, play a major role in atheroma plaque rupture. Lipid loading has been reported to modulate expression of several genes such as adipophilin, a protein with a putative role in lipid handling. We have previously shown that adipophilin expression in atherosclerotic lesions was more than 3 fold higher than its expression in healthy areas of the same arteries. In this study, we have investigated the role of adipophilin expression in lipid accumulation in THP-1-derived macrophages. Thus, THP-1 macrophages overexpressing human adipophilin were incubated 48h with acetylated LDL (acLDL). Our data showed 76% and 45% increase of triglycerides (TG) and total cholesterol (TC) respectively in adipophilin transfected cells versus control cells. In addition, following 24h incubation of the lipid loaded cells with apoA-I, cellular cholesterol efflux was significantly reduced from the adipophilin transfected cells by 47% when compared to control cells. Moreover, the impact of adipophilin gene silencing on lipid accumulation and cholesterol efflux, using a specific siRNA directed against adipophilin mRNA, showed a 50% and 44% reduction of TG and TC respectively as well as a 50% decrease in cholesterol efflux. In conclusion, both overexpression and down-regulation of adipophilin reduced cholesterol efflux suggesting a role for adipophilin in the regulation or localisation of the cholesterol pools available for the ABCA1 pathway. Moreover, adipophilin overexpression promoted TC and TG accumulation, whereas the suppression of adipophilin expression reduced both TC and TG content in human THP-1 macrophages. Our results suggested that stimulation of adipophilin expression in macrophages contribute to lipid storage which might lead to plaque rupture. Therefore, adipophilin might constitute a new target gene in atherosclerosis treatment.
Regulation of the LDL receptor gene expression by bile acids
M. Nakahara 1 , Y. Yokoi 1 , H. Fujii 2 , M. Shimizu 1 , R. Sato 1 . 1 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo; 2 Graduate School of Medical and Dental Science, Niigata University, Japan Recent studies have indicated that bile acids regulate the expression of several genes involved in bile acid and lipid metabolism as ligands for farnesoid X receptor (FXR). We here report that bile acids are directly able to govern cholesterol metabolism by a novel mechanism. We show that chenodeoxycholic acid (CDCA) enhances low density lipoprotein (LDL) receptor gene expression in HepG2 cells. The proteolytic activation of sterol regulatory element-binding protein-2 (SREBP-2), a major regulator for LDL receptor gene expression, is not affected by CDCA. Both deoxycholic acid (DCA) and lithocholic acid (LCA) as well as CDCA, but not ursodeoxycholic acid (UDCA), increase the mRNA level for the LDL receptor, even when Hep G2 cells are cultured with 25-hydroxycholesterol, a potent suppressor of gene expression for the LDL receptor. Although it seems possible that FXR might be involved in genetic regulation, both reporter assays with a reporter gene containing the LDL receptor promoter, as well as northern blot analysis reveal that FXR has exerts no genetic effects. On the other hand, inhibition of mitogen-activated protein (MAP) kinase activities, which are found to be induced by CDCA, abolishes the CDCA-mediated up-regulation of LDL receptor gene expression. We further demonstrate that CDCA stabilizes LDL receptor mRNA, and that the MAP kinase inhibitors accelerate its turnover. And we show that this stabilization is mediated by the 3’ untranslated region of the LDL receptor. Taken together, these results indicate that bile acids increase LDL uptake and the intracellular cholesterol levels through the activation of MAP kinase cascades in conjunction with a down-regulation of bile acid biosynthesis by FXR.
ADHESION MOLECULES 2P-0464
Carvedilol, a beta-adrenoceptor antagonist with antioxidant activity, attenuates TNF-alpha stimulated expression of VCAM-1 and E-selectin, and adhesiveness to mononuclear cells in human aortic endothelial cells
J.-W. Chen, F.-Y. Lin, Y.-H. Chen, T.-L. Yang, Y.-L. Chen, S.-J. Lin. National Yang-Ming University School of Medicine, Taiwan Background: The beneficial effects of carvedilol, a beta-adrenoceptor antagonist, in decreasing cardiovascular mortality might be attributed to its antioxidant properties. Since oxidative stress may play an important role in atherogenesis. We then examined the direct effects of carvedilol on endothelial-adhesivenss to human mononuclear cells (MNCs), an in vitro sign of atherogenesis, and the expression of adhesion molecules in human aortic endothelial cells (HAECs) stimulated by tumor necrosis factor-alpha (TNF-α). Methods and Results: Circulating MNCs were isolated from the peripheral blood of healthy subjects. Compared to control condition, pretreatment of carvedilol (10 mM for 18 hours) or probucol (5 mM for 18 hours) but not propanolol significantly decreased TNF-α-stimulated adhesiveness of cultured HAECs to MNCs (51.1±4.0% and 52.1±6.2% inhibition, p<0.05, respectively). Cell ELISA study showed that pretreatment with carvedilol significantly inhibited TNF-α-stimulated VCAM-1 and E-selectin (66.0±2.0% and 55.6±1.0% of control, p<0.05, respectively) but not ICAM-1 expression whereas pretreatment with probucol inhibited only VCAM-1 expression (79.0±5.0% of control, p<0.05) in HAECs. Propranolol did not alter the expression of any adhesion molecules. The above findings were also confirmed by Western blotting study. Further, carvedilol significantly inhibited TNF-α-stimulated intracellular reactive oxygen species (ROS) production and the activation of redox sensitive transcription pathways including both nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) in HAECs. Conclusions: Carvedilol may directly prevent in vitro atherogenesis by inhibiting intracellular ROS production, NF-kB and AP-1 activation, and VCAM-1 as well as E-selectin expression in TNF-α-stimulated HAECs, suggesting the potential implication of carvedilol to clinical atherosclerosis disease.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
TUESDAY
Sterol regulatory element-binding proteins (SREBPs) are major transcription factors that activate the genes involved in cholesterol and fatty acid biosynthesis. We here report that the nuclear forms of SREBPs are modified by the small ubiquitin-related modifier (SUMO)-1. Mutational analyses identified two major sumoylation sites (Lys123 and Lys418) in SREBP-1a and a single site (Lys464) in SREBP-2. Mutant SREBPs lacking one or two sumoylation sites exhibited increased transactivation capacity on an SREBP-responsive promoter. Overexpression of SUMO-1 reduced while its dominant negative form increased mRNA levels of SREBP-responsive genes. Nuclear SREBPs interacted with the SUMO-1-conjugating enzyme Ubc9, and overexpression of a dominant negative form of Ubc9 increased the mRNA levels of SREBPresponsive genes. Pulse-chase experiments revealed that sumoylation did not affect the degradation of SREBPs through the ubiquitin-proteasome pathway. In vitro ubiquitylation assay showed no competition between ubiquitin and SUMO-1 for the same lysine. Considered together, our results indicate that SUMO-1 modification suppresses the transactivation capacity of nuclear SREBPs in a manner different from the negative regulatory mechanism mediated by proteolysis.
2P-0463 ∗
141
Tuesday September 30, 2003: Poster Session Intracellular lipid transport and metabolism
endocytosis pathway mediate LDL uptake. Fluid-phase uptake of LDL as a mechanism of macrophage cholesterol accumulation shows that modification of LDL is not necessary for foam cell formation to occur. In addition, the findings direct attention to macrophage fluid-phase endocytosis pathways as relevant targets for modulating macrophage cholesterol accumulation in atherosclerosis. 2P-0456
Supernatant protein factor (SPF), a cytosolic squalene epoxidase regulator
N. Shibata 1 , M. Tsujimoto 2 , K. Jishage 3 , H. Arai 1 . 1 Dept. of Health Chemistry, Grad. School of Pharma. Sci., Univ. of Tokyo and Laboratory of Cellular Biochemistry, RIKEN; 2 Laboratory of Cellular Biochemistry, RIKEN; 3 Chugai Research Institute For Medical Science, Japan Objective: Squalene epoxidase is a microsomal monooxygenase that catalyzes the conversion of squalene to squalene 2,3-oxide. This conversion is the trigger of steroid skeleton formation in the sterol biosynthetic pathway. SPF (Supernatant Protein Factor) was identified in the 1950s as a cytosolic protein that stimulates microsomal squalene epoxidase activity, but so far, little information is available on the primary structure or the biological function of SPF. Recently, we purified SPF and cloned its cDNA. So far, we made the following observations when analyzing the function of SPF: (1) SPF mRNA is expressed abundantly in the liver and small intestine. (2) Recombinant SPF produced in E. coli promoted intermembrane transfer of squalene and activity of squalene epoxidase in vitro. (3) Transfection of SPF cDNA in hepatoma cells significantly stimulated de novo cholesterol biosynthesis, and mRNA expression of cholesterol biosynthetic enzymes. These data indicate that SPF functions in maintaining cholesterol homeostasis in vivo, and shows characteristics of a novel and tissue-specific cholesterol biosynthesis regulator. Here we examined this hypothesis in SPF-deficient mice. Method: We generated SPF-deficient mice by targeted gene disruption and examined plasma lipids and mRNA levels of cholesterol biosynthetic enzymes in the liver. Results and Conclusion: SPF-deficient mice were normally developed. No apparent phenotype and change of plasma lipids (including cholesterol and triglyceride) was observed under standard conditions. Hepatic HMG-CoA synthase and squalene synthase mRNA expressions significantly increased in SPF-deficient mice. These data indicate that SPF-deficient mice regulate their cholesterol demand and maintain cholesterol homeostasis by this compensatory mechanism. We are analyzing the physiological function of SPF in cholesterol metabolism, and we will present recent observations. 2P-0457
Involvement of phospholipase A2 in the supply of fatty acids required for cholesterol esterification in oxidized LDL-stimulated macrophages
S. Akiba, Y. Yoneda, S. Ohno, T. Sato. Department of Pathological Biochemistry, Kyoto Pharmaceutical University, Japan Objective: In macrophages, the accumulation of cholesterol esters associated with uptake of oxidized LDL (oxLDL) leads to the transformation into lipid-laden foam cells, a key event in the development and progression of atherosclerosis. However, little is known about the pathways involved in the supply of fatty acids, precursors for acyl-CoA required for the cholesterol esterification. We examined the possible involvement of cytosolic phospholipase A2 (cPLA2 ) in the supply of fatty acids using oxLDL-stimulated mouse peritoneal macrophages. Results: In [3 H]oleic acid-labeled cells, oxLDL induced cholesteryl oleate formation with an increase in free oleic acid and a decrease in phosphatidylcholine. The changes in these lipids were suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cPLA2 inhibitor. However, MAFP had no effect on the ACAT activity or the uptake of oxLDL. Stimulation with oxLDL in the presence of [3 H]cholesterol increased [3 H]cholesterol ester. The formation of cholesterol ester under this condition was also inhibited by MAFP, and the inhibitory effect was reversed by adding oleic acid. Under the conditions, oxLDL did not affect the activity or amounts of cPLA2 . When cells were stimulated with oxidized lipids (13-hydroxyoctadecadienoic acid, 7-ketocholesterol, or 25-hydroxycholesterol) in oxLDL, 13-hydroxyoctadecadienoic acid, but not oxysterols, induced oleic acid release, which was sensitive to MAFP. Conclusion: Based on the results, we suggest that oxLDL induces cPLA2 activation, which contributes, at least in part, to the supply of fatty acids required for the cholesterol esterification in macrophages. In the oxLDLinduced acceleration of the catalytic action of cPLA2, oxidized lipids, such as 13-hydroxyoctadecadienoic acid, may be involved.
2P-0458
Microarray analysis of cardiac gene expression in heart-specific hormone-sensitive lipase overexpressing mice
J. Suzuki 1 , H. Kanehara 1 , Y. Zenimaru 1 , S. Takahashi 1 , K. Oida 1 , F. Kraemer 2 , I. Miyamori 1 . 1 Fukui Medical University, Matsuoka, Fukui, Japan; 2 Stanford University, USA Intracellular lipid accumulation is a key in the pathogenesis of cardiomyopathy. We have reported that heart-specific overexpression of hormone-sensitive lipase (HSL) inhibited cardiac lipid accumulation. Here we studied cardiac gene expressions by 2-step comparison analyses to identify gene clusters that respond specifically to fasting and/or HSL-overexpression. Control (Ct) and HSL-transgenic (Tg) mice were fasted for 16h and cardiac gene expressions were screened by Affymetrix Mu11k, followed by RT-PCR or Northern analysis. Analysis 1: Setting the fed Ct as the reference, profiles of fasted Ct, fed Tg and fasted Tg were determined. The resultant profiles were then compared to exclude overlapping genes. 56 genes (pyruvate carboxylase, beta-tropomyosin etc.) were affected exclusively by fasting, indicating their response to stress-hormones. 61 genes (NFk-B, p53 etc.) were affected exclusively by HSL-overexpression, suggesting their response to released FFA. 58 genes (ATP synthase, UCP2 etc.) responded only to the additive effect of fasting and HSL-Tg. Analysis 2: Fasting profiles were determined separately in Ct or Tg with the respective fed Ct or fed Tg as references. Each fasting profile was compared to extract overlapping or non-overlapping genes. Fasting affected 74 genes in Ct while 55 genes were changed in Tg, and only 5 genes (c-fos, N10 etc.) overlapped. In summary, microarray 2-step comparison analyses revealed cardiac gene clusters that specifically respond to fasting or HSL-overexpression, and that HSL-overexpression drastically affected the physiological fasting profile. This approach will be useful to understand the mechanism of cardiomyopathy caused by aberrant regulation of intracellular lipid metabolism. 2P-0459
Cholesterol transport across the intestinal epithelial cells
M. Hussain, J. Iqbal, K. Anwar. SUNY Downstate Medical Center, Depts. of Anatomy and Cell Biology, and Pediatrics, Brooklyn, New York, USA It is generally believed that cholesterol is transported across the intestinal epithelial cells exclusively via chylomicron assembly and secretion. Contrary to the general belief, our data indicate that newly absorbed cholesterol is neither immediately available for secretion with apoB-lipoproteins nor exclusively secreted as part of chylomicrons. Based on our data, cholesterol transport by enterocytes can be broadly classified into two independently modulated apoB-dependent and apoB-independent pathways. Cholesterol secretion by the apoB-dependent pathway is induced by oleic acid, is repressed by microsomal triglyceride transfer protein inhibitors, and occurs only with larger apoB-containing lipoproteins. ApoB-independent pathways do not require microsomal triglyceride transfer protein (MTP) and involve efflux mediated by ABCA1, HDL assembly, and possibly other unknown mechanisms. These pathways show differential specificity toward the free and esterified cholesterol. The apoB-dependent pathway transports the esterified cholesterol, whereas both pathways secrete free cholesterol. Labeling studies indicate for at least two different metabolic pools of cholesterol in enterocytes. The newly absorbed and pre-absorbed cholesterol are preferentially secreted via apoB-independent and apoB-dependent pathways, respectively. In contrast to compartmentalization for secretion, these two metabolic pools are equally accessible for cellular esterification. Thus, enterocytes transport cholesterol by several independently regulated pathways raising the possibility that targeting of apoB-independent pathways may result in selective inhibition of cholesterol transport without affecting triglyceride transport. 2P-0460
Role of sterol regulatory element binding protein-1 (SREBP-1) in hepatic regeneration after partial hepatectomy
K. Hashida, M. Fujita, M. Kinoshita, T. Maeda, Y. Maeda, M. Kudo, T. Suda, D. Hirota, M. Taki, T. Teramoto. Teikyo University School of Medicine, Tokyo, Japan Objective: We had reported that fatty acid synthetase (FAS) activity was increased after partial hepatectomy (PH), and this may be the initial signals for hepatocyte regeneration after PH. In this study, relationship between SREBP-1 mRNA, which is known to be a transcription factor of FAS, and hepatocyte regeneration after PH was examined Methods: Male Sprague-Dawley rat (230-240g) was used for the experiments. Streptozotocin (STZ) induced diabetic rat was made by the injection of STZ from tail vein. Insulin treatment to STZ induced diabetic rat was
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
Tuesday September 30, 2003: Poster Session Adhesion molecules performed by the subcutaneous injection of Humalin N (4units). After 3 day of STZ injection, PH was performed. And 1days or 7 days after PH, liver was removed and used for mRNA measurement. Results: It was revealed that, on the first day after PH, expression of hepatic SREBP-1 mRNA decreased, although FAS activity was increased. STZ induced diabetic rat showed a significant decrease of SREBP-1 mRNA, but the regeneration of hepatocytes were observed as usual. These data indicate that the decrease of SREBP-1 is not be the initiation signal of regeneration of hepatocytes. When STZ induced diabetic rats were treated with insulin, SREBP-1 mRNA increased and hepatic regeneration was occurred more than that of untreated diabetic rats, significantly. This data indicated that SREBP-1 might be the enhancer of hepatic regeneration. Conclusion: SREBP-1 plays not as the initiator but as the enhancer role in the hepatocyte regeneration after partial hepatectomy. 2P-0461
SREBPs are negatively regulated by SUMO-1 modification
R. Sato 1 , Y. Hirano 2 , S. Murata 3 , K. Tanaka 3 , M. Shimizu 2 . 1 Department of Applied Biological Chemistry, University of Tokyo; 2 University of Tokyo; 3 Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
2P-0462
Impact of adipophilin expression on lipid accumulation in human macrophages: Potential role in atherogenesis
G. Larigauderie, C. Furman, C. Lasselin, C. Copin, J.-C. Fruchart, G. Castro, M. Rouis. INSERM U545, Lille Pasteur Institute, Lille, France Macrophage-derived foam cells, which occur in abundance in most stages of the atherosclerotic lesion, play a major role in atheroma plaque rupture. Lipid loading has been reported to modulate expression of several genes such as adipophilin, a protein with a putative role in lipid handling. We have previously shown that adipophilin expression in atherosclerotic lesions was more than 3 fold higher than its expression in healthy areas of the same arteries. In this study, we have investigated the role of adipophilin expression in lipid accumulation in THP-1-derived macrophages. Thus, THP-1 macrophages overexpressing human adipophilin were incubated 48h with acetylated LDL (acLDL). Our data showed 76% and 45% increase of triglycerides (TG) and total cholesterol (TC) respectively in adipophilin transfected cells versus control cells. In addition, following 24h incubation of the lipid loaded cells with apoA-I, cellular cholesterol efflux was significantly reduced from the adipophilin transfected cells by 47% when compared to control cells. Moreover, the impact of adipophilin gene silencing on lipid accumulation and cholesterol efflux, using a specific siRNA directed against adipophilin mRNA, showed a 50% and 44% reduction of TG and TC respectively as well as a 50% decrease in cholesterol efflux. In conclusion, both overexpression and down-regulation of adipophilin reduced cholesterol efflux suggesting a role for adipophilin in the regulation or localisation of the cholesterol pools available for the ABCA1 pathway. Moreover, adipophilin overexpression promoted TC and TG accumulation, whereas the suppression of adipophilin expression reduced both TC and TG content in human THP-1 macrophages. Our results suggested that stimulation of adipophilin expression in macrophages contribute to lipid storage which might lead to plaque rupture. Therefore, adipophilin might constitute a new target gene in atherosclerosis treatment.
Regulation of the LDL receptor gene expression by bile acids
M. Nakahara 1 , Y. Yokoi 1 , H. Fujii 2 , M. Shimizu 1 , R. Sato 1 . 1 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo; 2 Graduate School of Medical and Dental Science, Niigata University, Japan Recent studies have indicated that bile acids regulate the expression of several genes involved in bile acid and lipid metabolism as ligands for farnesoid X receptor (FXR). We here report that bile acids are directly able to govern cholesterol metabolism by a novel mechanism. We show that chenodeoxycholic acid (CDCA) enhances low density lipoprotein (LDL) receptor gene expression in HepG2 cells. The proteolytic activation of sterol regulatory element-binding protein-2 (SREBP-2), a major regulator for LDL receptor gene expression, is not affected by CDCA. Both deoxycholic acid (DCA) and lithocholic acid (LCA) as well as CDCA, but not ursodeoxycholic acid (UDCA), increase the mRNA level for the LDL receptor, even when Hep G2 cells are cultured with 25-hydroxycholesterol, a potent suppressor of gene expression for the LDL receptor. Although it seems possible that FXR might be involved in genetic regulation, both reporter assays with a reporter gene containing the LDL receptor promoter, as well as northern blot analysis reveal that FXR has exerts no genetic effects. On the other hand, inhibition of mitogen-activated protein (MAP) kinase activities, which are found to be induced by CDCA, abolishes the CDCA-mediated up-regulation of LDL receptor gene expression. We further demonstrate that CDCA stabilizes LDL receptor mRNA, and that the MAP kinase inhibitors accelerate its turnover. And we show that this stabilization is mediated by the 3’ untranslated region of the LDL receptor. Taken together, these results indicate that bile acids increase LDL uptake and the intracellular cholesterol levels through the activation of MAP kinase cascades in conjunction with a down-regulation of bile acid biosynthesis by FXR.
ADHESION MOLECULES 2P-0464
Carvedilol, a beta-adrenoceptor antagonist with antioxidant activity, attenuates TNF-alpha stimulated expression of VCAM-1 and E-selectin, and adhesiveness to mononuclear cells in human aortic endothelial cells
J.-W. Chen, F.-Y. Lin, Y.-H. Chen, T.-L. Yang, Y.-L. Chen, S.-J. Lin. National Yang-Ming University School of Medicine, Taiwan Background: The beneficial effects of carvedilol, a beta-adrenoceptor antagonist, in decreasing cardiovascular mortality might be attributed to its antioxidant properties. Since oxidative stress may play an important role in atherogenesis. We then examined the direct effects of carvedilol on endothelial-adhesivenss to human mononuclear cells (MNCs), an in vitro sign of atherogenesis, and the expression of adhesion molecules in human aortic endothelial cells (HAECs) stimulated by tumor necrosis factor-alpha (TNF-α). Methods and Results: Circulating MNCs were isolated from the peripheral blood of healthy subjects. Compared to control condition, pretreatment of carvedilol (10 mM for 18 hours) or probucol (5 mM for 18 hours) but not propanolol significantly decreased TNF-α-stimulated adhesiveness of cultured HAECs to MNCs (51.1±4.0% and 52.1±6.2% inhibition, p<0.05, respectively). Cell ELISA study showed that pretreatment with carvedilol significantly inhibited TNF-α-stimulated VCAM-1 and E-selectin (66.0±2.0% and 55.6±1.0% of control, p<0.05, respectively) but not ICAM-1 expression whereas pretreatment with probucol inhibited only VCAM-1 expression (79.0±5.0% of control, p<0.05) in HAECs. Propranolol did not alter the expression of any adhesion molecules. The above findings were also confirmed by Western blotting study. Further, carvedilol significantly inhibited TNF-α-stimulated intracellular reactive oxygen species (ROS) production and the activation of redox sensitive transcription pathways including both nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) in HAECs. Conclusions: Carvedilol may directly prevent in vitro atherogenesis by inhibiting intracellular ROS production, NF-kB and AP-1 activation, and VCAM-1 as well as E-selectin expression in TNF-α-stimulated HAECs, suggesting the potential implication of carvedilol to clinical atherosclerosis disease.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
TUESDAY
Sterol regulatory element-binding proteins (SREBPs) are major transcription factors that activate the genes involved in cholesterol and fatty acid biosynthesis. We here report that the nuclear forms of SREBPs are modified by the small ubiquitin-related modifier (SUMO)-1. Mutational analyses identified two major sumoylation sites (Lys123 and Lys418) in SREBP-1a and a single site (Lys464) in SREBP-2. Mutant SREBPs lacking one or two sumoylation sites exhibited increased transactivation capacity on an SREBP-responsive promoter. Overexpression of SUMO-1 reduced while its dominant negative form increased mRNA levels of SREBP-responsive genes. Nuclear SREBPs interacted with the SUMO-1-conjugating enzyme Ubc9, and overexpression of a dominant negative form of Ubc9 increased the mRNA levels of SREBPresponsive genes. Pulse-chase experiments revealed that sumoylation did not affect the degradation of SREBPs through the ubiquitin-proteasome pathway. In vitro ubiquitylation assay showed no competition between ubiquitin and SUMO-1 for the same lysine. Considered together, our results indicate that SUMO-1 modification suppresses the transactivation capacity of nuclear SREBPs in a manner different from the negative regulatory mechanism mediated by proteolysis.
2P-0463 ∗
141