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ADIPONECTIN DEFICIENCY SUPPRESSES ABCA1 EXPRESSION AND APOA-I SYNTHESIS IN THE LIVER
24 PO4-25
Poster Sessions PO5 Hepatic lipid metabolism CAPACITY OF THE HIGH DENSITY LIPOPROTEIN TO RECEIVE LIPIDS IN AGE GROUPS: A STUDY USING AN ARTIFICIAL NANOEMULSION
C.H. Azevedo 1,3 , A.C. Lo Prete 1,3 , C. Puk 1 , M. Wajngarten 2 , J. Diament 2 , R.C. Maranhao 1,3 . 1 Lipid Metabolism Laboratory, the Heart Institute (InCor), Medical School Hospital of University of Sao Paulo, Sao Paulo,SP, Brazil; 2 Cardiology, the Heart Institute (InCor), Medical School Hospital of University of Sao Paulo, Sao Paulo, SP, Brazil; 3 Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil Background and aims: The shift of lipids to HDL may be altered by the aging process and be related with coronary artery disease (CAD). Inside of these aspects, the HDL size and its capacity to receive lipids was determined in different age groups. Methods: It was studied 25 younger, 25 middle-age and 36 elderly subjects with a cineocoronariography (11 with CAD, 74±5 yo; and 25 non-CAD, 75±6 yo). An artificial nanoemulsion (LDE) labeled with 3H-Triglycerides (TG) and 14C-Free Cholesterol (FC) or 3H-Cholesteryl Ester (CE) and 14C-Phospholipids (PL) was incubated with plasma for 1h. After chemical precipitation, the supernatant containing HDL was counted for radioactivity. The HDL diameter was measured by laser-light-scattering. Results: Transfer of CE and PL to HDL was smaller in young subjects than in the elderly non-CAD patients, but the transfer of the others lipids was similar (CE: young= 3.7±1.0%, middle-age=4.1±0.7%, elderly non-CAD= 5.3±1.8%, p=0.024 and PL: young= 18.7±4.6%, middle-age= 18.3±4.0%, elderly non-CAD=20.6±5.3, p=0.0368). The HDL size was greater in elderly non-CAD group (9.7± 1.6nm) than in younger (8.9±0.3nm) and middle-age subjects (8.9±0.3nm); p=0,0444. With intention to know if the results found in the present work suggest some protection, was carried through the comparison with an aged CAD group. CE and FL transfer as well as HDL size was smaller in the CAD group (CE=3.1±2.3 and TG=5.1±1.6; 8.7±0.7nm). Conclusions: A decreased lipid transfer is associated with the CAD. Due to HDL important antiatherogenic roles, this result can be relevant to establish new mechanisms and risk factors in CAD.
PO5 HEPATIC LIPID METABOLISM PO5-27
A. Laatsch, T. Hesper, U. Beisiegel, J. Heeren. Department of Biochemistry and Molecular Biology II: Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Background and aims: The physiological relevance of hepatic cell models often suffers from dedifferentiation due to their tumour background resulting in an untypical phenotype. This problem can be addressed by using cell lines which are differentiated in vitro (e. g. adipocytes derived from progenitor cells, hMSC-TERT, as we reported earlier). HepaRG cells can be differentiated into a hepatic phenotype (Gripon et al., PNAS 2002). In contrast to all other human hepatic cell models, this one is susceptible to an infection with hepatitis B virus. On the level of the host cell, this process depends on complex intracellular processes, which are obviously functional in HepaRG. This prompted us to characterise HepaRG in the context of lipoprotein metabolism. Methods: HepaRG cells differentiate in a time frame of four weeks. Different stages were compared to HuH7 and FAO cells. After western blot analysis, cell biological characterisation by fluorescence microscopy was carried out. Results: After differentiation the cells present a hepatocyte-like morphology and express all tested proteins connected to lipoprotein metabolism including LDLR, LRP1, apoB-100, apoCs and apoE. They efficiently take up CR, RAP and a2M*, all of which are subsequently stored in different intracellular compartments. HDL is present in the area of the cell surface. Unlike many other cell models, the microscopic overall picture is very clear, homogeneous and undisturbed. Conclusions: HepaRG cells present themselves as a balanced and robust hepatic cell system. Due to their conclusive features we consider them as a promising cell biological model and propose them for future experiments. PO5-28
PO4-26
COMPOSITIONAL PROTEIN PROFILING OF HDL BY SELDI-TOF MS DURING EXPERIMENTAL ENDOTOXEMIA
J. Levels 1 , R. Maree 2 , J. Kuivenhoven 1 , L. Wehenkel 2 , J. Kastelein 3 , J. Meijers 1 . 1 Department of Experimental Vascular Medicine, Academic Medical Center, Amsterdam, the Netherlands; 2 Department of Electrical Engineering and Computer Science & GIGA Research, University Liege, Liege, Belgium; 3 Department of Vascular Medicine, Academic Medical Center, Amsterdam, the Netherlands Objective: High Density Lipoprotein (HDL), one of the main plasma lipoproteins, serves as a docking station for a variety of factors involved in inflammation, coagulation, protective properties against lipid-oxidation and lipid metabolism. We assessed SELDI-TOF mass spectrometry as a high-throughput proteomic tool to study the dynamics of the HDL protein composition in a human low dose experimental endotoxemia model system. Methods: Ten healthy men with genetically determined isolated low HDL cholesterol (0.7±0.1 mmol/L) and 10 age- and body weight-matched healthy men with normal/high HDL cholesterol levels (1.9±0.4 mmol/L) were challenged with endotoxin intravenously (1 ng/kg bodyweight). Antibodies, against apo A-I, were covalently bound to a PS20 protein-chip and HDL was immuno-captured from the sequential plasma samples followed by mass spectrometry measurement. The relative changes in HDL associated proteins were evaluated by an univariate process and a multivariate statistical method based on multiple decision trees algorithms. Results: The most detailed fingerprint was observed up to 50 kDa, 15 to 20 separate proteins were detected in the 5-50 kDa molecular mass range. Between 50 and 160 kDa 5 to 10 more proteins were detected. Dynamic changes in the fingerprint were observed in the low and high HDL cholesterol group upon the LPS challenge. Conclusions: SELDI-TOF mass spectrometry of HDL proved to be a suitable candidate in the search for potential (new) biomarkers with regard to this inflammatory model. This approach may be a welcome contribution to the investigation of the underlying mechanisms that lead to increased atherothrombotic risk.
A NOVEL HUMAN HEPATIC CELL MODEL FOR POSTPRANDIAL LIPOPROTEIN METABOLISM
ADIPONECTIN DEFICIENCY SUPPRESSES ABCA1 EXPRESSION AND APOA-I SYNTHESIS IN THE LIVER
F. Matsuura 1 , H. Oku 1 , M. Koseki 1 , K. Yamamoto 1 , J.C. Sandoval 1 , M. Kawase 1 , D. Masuda 1 , T. Ohama 1 , N. Maeda 2 , M. Ishigami 3 , M. Nishida 4 , K. Hirano 1 , S. Kihara 2 , M. Hori 1 , I. Shimomura 2 , S. Yamashita 1 . 1 Department of Cardiovascular Medicine, Osaka University, Suita, Osaka, Japan; 2 Department of Metabolic Medicine, Osaka University, Suita, Osaka, Japan; 3 Department of Biomedical Informatics, Division of Health Sciences, Osaka University, Suita, Osaka, Japan; 4 Health Care Center, Osaka University, Suita, Osaka, Japan Background: Plasma high density lipoprotein (HDL)-cholesterol levels are inversely correlated with the incidence of atherosclerotic cardiovascular diseases. HDL is mainly assembled in the liver through the ATP-binding cassette transporters (ABCA1 and ABCG1) pathways. In humans, plasma HDL-cholesterol levels are positively correlated with plasma concentrations of adiponectin (APN). Recently, we reported that APN enhanced apolipoprotein A-I (apoA-I) secretion and ABCA1 expression in HepG2 cells, suggesting that APN might increase HDL assembly in the liver. [Aim] In the present study, we investigated the lipid metabolism, in particular HDL assembly, in APN-knockout mice. Methods: The profiles of plasma lipids in APN-knockout (APN-KO) and wild type (WT) mice, 8-weeks old, male were studied by HPLC analysis. The expression levels of apoA-I and ABCA1 in the liver were examined by western blot or real time-quantitative PCR. Results: Although there was no significant difference in plasma HDLcholesterol levels between APN-KO and WT mice fed a normal chow diet, the protein levels of apoA-I in plasma and the liver were definitely reduced in APN-KO mice. Furthermore, the expression levels of ABCA1 in the liver were also significantly decreased in APN-KO mice. Conclusions: These data suggest that APN deficiency might cause the impaired HDL assembly by decreasing ABCA1 expression and apoA-I synthesis in the liver.
77th Congress of the European Atherosclerosis Society, April 26–29, 2008, Istanbul, Turkey
Poster Sessions PO5 Hepatic lipid metabolism CAPACITY OF THE HIGH DENSITY LIPOPROTEIN TO RECEIVE LIPIDS IN AGE GROUPS: A STUDY USING AN ARTIFICIAL NANOEMULSION
C.H. Azevedo 1,3 , A.C. Lo Prete 1,3 , C. Puk 1 , M. Wajngarten 2 , J. Diament 2 , R.C. Maranhao 1,3 . 1 Lipid Metabolism Laboratory, the Heart Institute (InCor), Medical School Hospital of University of Sao Paulo, Sao Paulo,SP, Brazil; 2 Cardiology, the Heart Institute (InCor), Medical School Hospital of University of Sao Paulo, Sao Paulo, SP, Brazil; 3 Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil Background and aims: The shift of lipids to HDL may be altered by the aging process and be related with coronary artery disease (CAD). Inside of these aspects, the HDL size and its capacity to receive lipids was determined in different age groups. Methods: It was studied 25 younger, 25 middle-age and 36 elderly subjects with a cineocoronariography (11 with CAD, 74±5 yo; and 25 non-CAD, 75±6 yo). An artificial nanoemulsion (LDE) labeled with 3H-Triglycerides (TG) and 14C-Free Cholesterol (FC) or 3H-Cholesteryl Ester (CE) and 14C-Phospholipids (PL) was incubated with plasma for 1h. After chemical precipitation, the supernatant containing HDL was counted for radioactivity. The HDL diameter was measured by laser-light-scattering. Results: Transfer of CE and PL to HDL was smaller in young subjects than in the elderly non-CAD patients, but the transfer of the others lipids was similar (CE: young= 3.7±1.0%, middle-age=4.1±0.7%, elderly non-CAD= 5.3±1.8%, p=0.024 and PL: young= 18.7±4.6%, middle-age= 18.3±4.0%, elderly non-CAD=20.6±5.3, p=0.0368). The HDL size was greater in elderly non-CAD group (9.7± 1.6nm) than in younger (8.9±0.3nm) and middle-age subjects (8.9±0.3nm); p=0,0444. With intention to know if the results found in the present work suggest some protection, was carried through the comparison with an aged CAD group. CE and FL transfer as well as HDL size was smaller in the CAD group (CE=3.1±2.3 and TG=5.1±1.6; 8.7±0.7nm). Conclusions: A decreased lipid transfer is associated with the CAD. Due to HDL important antiatherogenic roles, this result can be relevant to establish new mechanisms and risk factors in CAD.
PO5 HEPATIC LIPID METABOLISM PO5-27
A. Laatsch, T. Hesper, U. Beisiegel, J. Heeren. Department of Biochemistry and Molecular Biology II: Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Background and aims: The physiological relevance of hepatic cell models often suffers from dedifferentiation due to their tumour background resulting in an untypical phenotype. This problem can be addressed by using cell lines which are differentiated in vitro (e. g. adipocytes derived from progenitor cells, hMSC-TERT, as we reported earlier). HepaRG cells can be differentiated into a hepatic phenotype (Gripon et al., PNAS 2002). In contrast to all other human hepatic cell models, this one is susceptible to an infection with hepatitis B virus. On the level of the host cell, this process depends on complex intracellular processes, which are obviously functional in HepaRG. This prompted us to characterise HepaRG in the context of lipoprotein metabolism. Methods: HepaRG cells differentiate in a time frame of four weeks. Different stages were compared to HuH7 and FAO cells. After western blot analysis, cell biological characterisation by fluorescence microscopy was carried out. Results: After differentiation the cells present a hepatocyte-like morphology and express all tested proteins connected to lipoprotein metabolism including LDLR, LRP1, apoB-100, apoCs and apoE. They efficiently take up CR, RAP and a2M*, all of which are subsequently stored in different intracellular compartments. HDL is present in the area of the cell surface. Unlike many other cell models, the microscopic overall picture is very clear, homogeneous and undisturbed. Conclusions: HepaRG cells present themselves as a balanced and robust hepatic cell system. Due to their conclusive features we consider them as a promising cell biological model and propose them for future experiments. PO5-28
PO4-26
COMPOSITIONAL PROTEIN PROFILING OF HDL BY SELDI-TOF MS DURING EXPERIMENTAL ENDOTOXEMIA
J. Levels 1 , R. Maree 2 , J. Kuivenhoven 1 , L. Wehenkel 2 , J. Kastelein 3 , J. Meijers 1 . 1 Department of Experimental Vascular Medicine, Academic Medical Center, Amsterdam, the Netherlands; 2 Department of Electrical Engineering and Computer Science & GIGA Research, University Liege, Liege, Belgium; 3 Department of Vascular Medicine, Academic Medical Center, Amsterdam, the Netherlands Objective: High Density Lipoprotein (HDL), one of the main plasma lipoproteins, serves as a docking station for a variety of factors involved in inflammation, coagulation, protective properties against lipid-oxidation and lipid metabolism. We assessed SELDI-TOF mass spectrometry as a high-throughput proteomic tool to study the dynamics of the HDL protein composition in a human low dose experimental endotoxemia model system. Methods: Ten healthy men with genetically determined isolated low HDL cholesterol (0.7±0.1 mmol/L) and 10 age- and body weight-matched healthy men with normal/high HDL cholesterol levels (1.9±0.4 mmol/L) were challenged with endotoxin intravenously (1 ng/kg bodyweight). Antibodies, against apo A-I, were covalently bound to a PS20 protein-chip and HDL was immuno-captured from the sequential plasma samples followed by mass spectrometry measurement. The relative changes in HDL associated proteins were evaluated by an univariate process and a multivariate statistical method based on multiple decision trees algorithms. Results: The most detailed fingerprint was observed up to 50 kDa, 15 to 20 separate proteins were detected in the 5-50 kDa molecular mass range. Between 50 and 160 kDa 5 to 10 more proteins were detected. Dynamic changes in the fingerprint were observed in the low and high HDL cholesterol group upon the LPS challenge. Conclusions: SELDI-TOF mass spectrometry of HDL proved to be a suitable candidate in the search for potential (new) biomarkers with regard to this inflammatory model. This approach may be a welcome contribution to the investigation of the underlying mechanisms that lead to increased atherothrombotic risk.
A NOVEL HUMAN HEPATIC CELL MODEL FOR POSTPRANDIAL LIPOPROTEIN METABOLISM
ADIPONECTIN DEFICIENCY SUPPRESSES ABCA1 EXPRESSION AND APOA-I SYNTHESIS IN THE LIVER
F. Matsuura 1 , H. Oku 1 , M. Koseki 1 , K. Yamamoto 1 , J.C. Sandoval 1 , M. Kawase 1 , D. Masuda 1 , T. Ohama 1 , N. Maeda 2 , M. Ishigami 3 , M. Nishida 4 , K. Hirano 1 , S. Kihara 2 , M. Hori 1 , I. Shimomura 2 , S. Yamashita 1 . 1 Department of Cardiovascular Medicine, Osaka University, Suita, Osaka, Japan; 2 Department of Metabolic Medicine, Osaka University, Suita, Osaka, Japan; 3 Department of Biomedical Informatics, Division of Health Sciences, Osaka University, Suita, Osaka, Japan; 4 Health Care Center, Osaka University, Suita, Osaka, Japan Background: Plasma high density lipoprotein (HDL)-cholesterol levels are inversely correlated with the incidence of atherosclerotic cardiovascular diseases. HDL is mainly assembled in the liver through the ATP-binding cassette transporters (ABCA1 and ABCG1) pathways. In humans, plasma HDL-cholesterol levels are positively correlated with plasma concentrations of adiponectin (APN). Recently, we reported that APN enhanced apolipoprotein A-I (apoA-I) secretion and ABCA1 expression in HepG2 cells, suggesting that APN might increase HDL assembly in the liver. [Aim] In the present study, we investigated the lipid metabolism, in particular HDL assembly, in APN-knockout mice. Methods: The profiles of plasma lipids in APN-knockout (APN-KO) and wild type (WT) mice, 8-weeks old, male were studied by HPLC analysis. The expression levels of apoA-I and ABCA1 in the liver were examined by western blot or real time-quantitative PCR. Results: Although there was no significant difference in plasma HDLcholesterol levels between APN-KO and WT mice fed a normal chow diet, the protein levels of apoA-I in plasma and the liver were definitely reduced in APN-KO mice. Furthermore, the expression levels of ABCA1 in the liver were also significantly decreased in APN-KO mice. Conclusions: These data suggest that APN deficiency might cause the impaired HDL assembly by decreasing ABCA1 expression and apoA-I synthesis in the liver.
77th Congress of the European Atherosclerosis Society, April 26–29, 2008, Istanbul, Turkey