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Impaired cholesterol efflux into hdl2 in metabolic syndrome
Abstracts / Atherosclerosis 235 (2014) e84–e191
a
Bioquímica, Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau. Institut d'Investigacions Biomèdiques Sant Pau. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM). Universitat Autònoma de Barcelona, BARCELONA, Spain; b Bioquímica, Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau. Institut d'Investigacions Biomèdiques Sant Pau. Universitat Autònoma de Barcelona, BARCELONA, Spain; c Bioquímica, CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), BARCELONA, Spain Objectives: The biological action of HDL is known to be impaired in diabetes mellitus (DM). DM could presumably modify two main HDL atheroprotective functions, macrophage-to-feces reverse cholesterol transport (RCT) and HDL antioxidant properties. In the present study, we evaluated the effect of DM on macrophage-specific RCT process and HDL ability to protect against LDL oxidation in a mouse model of DM. Methods: Ten-weeks-old diabetic (db/db) and wild-type mice in the same genetic background (C57Bl/6J) were injected intraperitoneally with [3H]cholesterol-labeled J774 macrophages, and the presence of [3H] in plasma, liver and feces was determined. The degree of HDL oxidation and the protection of oxidative modification of LDL by the HDL of each group of mice were assessed by monitoring the conjugated diene kinetics over time. Results: Plasma concentration of HDL lipoproteins and the two main HDL-associated enzymatic activities (ie., paraoxonase-1 and lipoprotein-associated phospholipase A2) were increased in the diabetic mice. The diabetic mice displayed increased levels of radiolabeled [3H]cholesterol in the plasma HDL and in the liver after 48h of the label injection. However, the content of macrophage-derived [3H]-cholesterol in feces was decreased in the diabetic mice. The relative decrease in observed [3H] in feces was associated with a decrease in liver-specific gene expression of ABCG5/G8 transporters. The HDL from diabetic mice displayed a similar susceptibility to be oxidised and ability to protect LDL from oxidation compared to the HDL derived from wildtype mice. Conclusion: Only the macrophage-specific RCT was impaired in the diabetic mice and this was in part attributed to a decrease in the liver expression of the ABCG5/G8 transporters which are involved in the cholesterol trafficking into feces. 33 - Cholesterol efflux and reverse cholesterol transport EAS-0228. LIPID ACCUMULATION ENHANCES CELL-SURFACE ATP-BINDING CASSETTE TRANSPORTER A1 (ABCA1) DEGRADATION IN UBLYSOSOME DEGRADATION PATHWAY T. Mizuno, H. Hayashi, H. Kusuhara Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Japan Objectives: ATP-binding cassette transporter A1 (ABCA1) plays an essential role in the biogenesis of high-density lipoprotein in the liver and in the prevention of foam cell formation in macrophages by mediating the efflux of cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I). Previously, we have revealed that the cell surface-resident ABCA1 was ubiquitinated and degraded in lysosome via the Endosomal Sorting Complex Required for Transport (ESCRT) pathway. In order to investigate the relevance of the degradation pathway to lipid disorders, further analyses were conducted. Methods: Mouse peritoneal macrophages (MPM) were isolated from thioglycollate-injected mice and employed in the study. The cells were treated with acetylated LDL (acLDL) to induce foam cell formation. In the studies using HepG2 cells, GW3965, an LXR agonist, was employed to mimic the state of lipid accumulation. Results: Co-immunoprecipitation studies and cell surface biotinylation studies showed that the amount of ubiquitinated ABCA1 and the degradation of cell-surface ABCA1 was increased in foam cells compared
e183
with normal MPM. The enhanced degradation was repressed by a wellknown lysosome inhibitor, Bafilomycin. GW3965 brought about the similar effect in MPM as well as in HepG2 cells. The disruption of the ESCRT pathway mediated by the knockdown of tumor susceptibility gene 101 (TSG101) also repressed the enhanced degradation induced by the LXR agonist. Conclusion: The current study suggests that the degradation of cell-surface ABCA1 in Ub-Lysosome degradation pathway is activated by lipid accumulation, which suggests that the degradation pathway works in lipid disorders. Therefore, the modulation of ABCA1 ubiquitination could be a potential new therapeutic target for atherosclerosis. 33 - Cholesterol efflux and reverse cholesterol transport EAS-0578. IMPAIRED CHOLESTEROL SYNDROME
EFFLUX
INTO
HDL2
IN
METABOLIC
T. Paavolaa, S.M. Kuusistoa, T. Kangas-Kontioa, J. Metsob, M. Jauhiainenb, M.L. Hannukselaa, M.J. Savolainena, S. Kakkoa, T. Salonurmia a Department of Internal Medicine and Biocenter Oulu and Medical Research Centre, University of Oulu and Oulu University Hospital, Oulu, Finland; b Public Health Genomics Unit, National Institute for Health and Welfare and Biomedicum, Helsinki, Finland
Objectives: Cholesterol efflux from peripheral macrophages into highdensity lipoprotein (HDL) is a key atheroprotective function of HDL. The capacity of human serum or total HDL to accept cholesterol has been linked with cardiovascular morbidity. Our aim was to study 1) how cholesterol efflux into total HDL, HDL2 and HDL3 (total HDL-, HDL2- and HDL3-efflux, respectively) is associated with a phenotype of low HDLcholesterol (HDL-C) and early coronary heart disease (CHD) 2) how the efflux parameters are related to each other 3) which cardiometabolic factors predict efflux. Methods: Serum samples were derived from Finnish families with low HDL-C as the main risk factor for early CHD; non-related spouses of the family members were also included (total n ¼ 132). HDL-fractions were isolated by sequential ultracentrifugation. Cholesterol efflux from THP1-macrophages loaded with 3H-labeled-acetylated-LDL into subjects' total HDL-, HDL2-, HDL3-fractions and serum was assayed and expressed as percentage of 3H-cholesterol in medium from 3H-cholesterol in a well. Results: Low HDL2-efflux was associated with metabolic syndrome (MetS). HDL2-efflux also correlated with the parameters of MetS, as HDL-C. Males with CHD had lower HDL2-efflux than males without CHD; in females the difference was non-significant. Total HDL-efflux showed no linear correlation with HDL2- or HDL3-efflux; HDL2- and HDL3-efflux were weakly positively correlated. The results were independent of age, sex and confirmed in non-related subjects. Conclusion: Impaired HDL2-efflux may be a significant mechanistic link leading to low HDL-C and the associated cardiometabolic morbidity.
Ă
a
Bioquímica, Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau. Institut d'Investigacions Biomèdiques Sant Pau. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM). Universitat Autònoma de Barcelona, BARCELONA, Spain; b Bioquímica, Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau. Institut d'Investigacions Biomèdiques Sant Pau. Universitat Autònoma de Barcelona, BARCELONA, Spain; c Bioquímica, CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), BARCELONA, Spain Objectives: The biological action of HDL is known to be impaired in diabetes mellitus (DM). DM could presumably modify two main HDL atheroprotective functions, macrophage-to-feces reverse cholesterol transport (RCT) and HDL antioxidant properties. In the present study, we evaluated the effect of DM on macrophage-specific RCT process and HDL ability to protect against LDL oxidation in a mouse model of DM. Methods: Ten-weeks-old diabetic (db/db) and wild-type mice in the same genetic background (C57Bl/6J) were injected intraperitoneally with [3H]cholesterol-labeled J774 macrophages, and the presence of [3H] in plasma, liver and feces was determined. The degree of HDL oxidation and the protection of oxidative modification of LDL by the HDL of each group of mice were assessed by monitoring the conjugated diene kinetics over time. Results: Plasma concentration of HDL lipoproteins and the two main HDL-associated enzymatic activities (ie., paraoxonase-1 and lipoprotein-associated phospholipase A2) were increased in the diabetic mice. The diabetic mice displayed increased levels of radiolabeled [3H]cholesterol in the plasma HDL and in the liver after 48h of the label injection. However, the content of macrophage-derived [3H]-cholesterol in feces was decreased in the diabetic mice. The relative decrease in observed [3H] in feces was associated with a decrease in liver-specific gene expression of ABCG5/G8 transporters. The HDL from diabetic mice displayed a similar susceptibility to be oxidised and ability to protect LDL from oxidation compared to the HDL derived from wildtype mice. Conclusion: Only the macrophage-specific RCT was impaired in the diabetic mice and this was in part attributed to a decrease in the liver expression of the ABCG5/G8 transporters which are involved in the cholesterol trafficking into feces. 33 - Cholesterol efflux and reverse cholesterol transport EAS-0228. LIPID ACCUMULATION ENHANCES CELL-SURFACE ATP-BINDING CASSETTE TRANSPORTER A1 (ABCA1) DEGRADATION IN UBLYSOSOME DEGRADATION PATHWAY T. Mizuno, H. Hayashi, H. Kusuhara Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Japan Objectives: ATP-binding cassette transporter A1 (ABCA1) plays an essential role in the biogenesis of high-density lipoprotein in the liver and in the prevention of foam cell formation in macrophages by mediating the efflux of cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I). Previously, we have revealed that the cell surface-resident ABCA1 was ubiquitinated and degraded in lysosome via the Endosomal Sorting Complex Required for Transport (ESCRT) pathway. In order to investigate the relevance of the degradation pathway to lipid disorders, further analyses were conducted. Methods: Mouse peritoneal macrophages (MPM) were isolated from thioglycollate-injected mice and employed in the study. The cells were treated with acetylated LDL (acLDL) to induce foam cell formation. In the studies using HepG2 cells, GW3965, an LXR agonist, was employed to mimic the state of lipid accumulation. Results: Co-immunoprecipitation studies and cell surface biotinylation studies showed that the amount of ubiquitinated ABCA1 and the degradation of cell-surface ABCA1 was increased in foam cells compared
e183
with normal MPM. The enhanced degradation was repressed by a wellknown lysosome inhibitor, Bafilomycin. GW3965 brought about the similar effect in MPM as well as in HepG2 cells. The disruption of the ESCRT pathway mediated by the knockdown of tumor susceptibility gene 101 (TSG101) also repressed the enhanced degradation induced by the LXR agonist. Conclusion: The current study suggests that the degradation of cell-surface ABCA1 in Ub-Lysosome degradation pathway is activated by lipid accumulation, which suggests that the degradation pathway works in lipid disorders. Therefore, the modulation of ABCA1 ubiquitination could be a potential new therapeutic target for atherosclerosis. 33 - Cholesterol efflux and reverse cholesterol transport EAS-0578. IMPAIRED CHOLESTEROL SYNDROME
EFFLUX
INTO
HDL2
IN
METABOLIC
T. Paavolaa, S.M. Kuusistoa, T. Kangas-Kontioa, J. Metsob, M. Jauhiainenb, M.L. Hannukselaa, M.J. Savolainena, S. Kakkoa, T. Salonurmia a Department of Internal Medicine and Biocenter Oulu and Medical Research Centre, University of Oulu and Oulu University Hospital, Oulu, Finland; b Public Health Genomics Unit, National Institute for Health and Welfare and Biomedicum, Helsinki, Finland
Objectives: Cholesterol efflux from peripheral macrophages into highdensity lipoprotein (HDL) is a key atheroprotective function of HDL. The capacity of human serum or total HDL to accept cholesterol has been linked with cardiovascular morbidity. Our aim was to study 1) how cholesterol efflux into total HDL, HDL2 and HDL3 (total HDL-, HDL2- and HDL3-efflux, respectively) is associated with a phenotype of low HDLcholesterol (HDL-C) and early coronary heart disease (CHD) 2) how the efflux parameters are related to each other 3) which cardiometabolic factors predict efflux. Methods: Serum samples were derived from Finnish families with low HDL-C as the main risk factor for early CHD; non-related spouses of the family members were also included (total n ¼ 132). HDL-fractions were isolated by sequential ultracentrifugation. Cholesterol efflux from THP1-macrophages loaded with 3H-labeled-acetylated-LDL into subjects' total HDL-, HDL2-, HDL3-fractions and serum was assayed and expressed as percentage of 3H-cholesterol in medium from 3H-cholesterol in a well. Results: Low HDL2-efflux was associated with metabolic syndrome (MetS). HDL2-efflux also correlated with the parameters of MetS, as HDL-C. Males with CHD had lower HDL2-efflux than males without CHD; in females the difference was non-significant. Total HDL-efflux showed no linear correlation with HDL2- or HDL3-efflux; HDL2- and HDL3-efflux were weakly positively correlated. The results were independent of age, sex and confirmed in non-related subjects. Conclusion: Impaired HDL2-efflux may be a significant mechanistic link leading to low HDL-C and the associated cardiometabolic morbidity.
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