- Email: [email protected]
PO1-4 ADIPONECTIN ACCELERATES REVERSE CHOLESTEROL TRANSPORT BY INCREASING HDL ASSEMBLY THROUGH UPREGULATION OF ABCA1 PATHWAY AND APOA-I SYNTHESIS IN THE LIVER
19
Poster Sessions PO1 HEPATIC AND ADIPOSE TISSUE LIPID METABOLISM PO1-1
DOSE-DEPENDENT EFFECTS OF ROSUVASTATIN ON GENE EXPRESSION IN PRIMARY CULTURES OF HUMAN AND RAT HEPATOCYTES
R.W. James, M.C. Brulhart-Meynet. Dept. of Internal Medicine, University Hospital, Geneva, Switzerland
Relative mRNA levels in human hepatocytes treated with rosuvastatin.
mRNA levels standardised to cyclophilin and control mRNA levels. Control, non-treated; LDL-R, LDL-receptor; SREBP-1, sterol regulatory element binding protein-1; Mev, mevalonate (0.5mM). Conclusions: The study confirms the impact of rosuvastatin on mRNA levels in primary human and rat hepatocytes and, by implication, expression of genes implicated in cholesterol metabolism. Rosuvastatin is effective at concentrations compatible with the known kinetics of HMGCoA reductase inhibition. PO1-2
EFFECT OF ROSUVASTATIN ON HDL SECRETION BY LIVER CELLS
F. Turrini 1 , S. Bertolini 2 , S. Calandra 1 . 1 Department of Biomedical Sciences, University of Modena & Reggio Emilia, Modena, Italy; 2 Department of Internal Medicine, University of Genova, Genova, Italy Rosuvastatin (RSV) treatment is associated with a substantial increase of plasma HDL. To clarify the mechanism underlying this effect Chang human liver cells were incubated for 24 hours with 0.1,1,5 and 10 μM of RSV and apo A-I and apo B-100 secreted into the medium were measured. Apo A-I secreted by RSV (5 μM) treated cells was increased by 55% while apo B was reduced by 70%. To investigate whether apo A-I was secreted as constituent of lipoproteins, cells were pulsed with labelled methionine for 30 minutes, chased for 2 hours and medium lipoproteins were isolated by ultracentrifugation. In RSV treated cells the peak of medium HDL was increased by 35% while that of VLD/LDL was reduced by 70%. In RSV treated cells the level of ApoA-I and ABCA1 mRNA was increased by 1.5 fold and 35% respectively. These changes were abolished by mevalonate. To investigate whether these changes were due to reduced availability of non steroidal isoprenoids derived from mevalonate, cells were incubated with RSV (5 ±M) in the presence of 10 μM of farnesyl pyrophosphate (Fpp) or geranylgeranyl pyrophosphate (GGpp). GGpp partly prevented the increase
PO1-3
MICROARRAY GENE EXPRESSION PROFILE OF ATGL -/- MICE
M. Pinent 1 , H. Hackl 1 , G. Haemmerle 2 , R. Zechner 2 , J.G. Strauss 1 , Z. Trajanoski 1 . 1 Institute for Genomics and Bioinformatics, Graz University of Technology, Graz, Austria; 2 Institue of Molecular Biosciences, University of Graz, Graz, Austria Background: Adipose triglyceride lipase (ATGL) is a recently described lipase with an important role on the catabolism of triacylglycerides. ATGL deficient mice show increased adipose mass and triacylglycerol deposition in multiple tissues. Furthermore, due to its inability to mobilize fat stores, they exhibit energy starvation, resulting in reduced energy expenditure, a decline in body temperature, and premature death due to cardiac insufficiency. Methods: In the present work we show the gene expression profile of ATGL -/- mice, obtained by microarray technology. Several tissues were analyzed: heart, skeletal muscle, white and brown adipose tissue (BAT), liver and kidney. Results: Among all those tissues, those which presented more changes were the BAT and the heart, with a total of 1110 and 384 differentially expressed EST respectively. In those tissues we found a significant downregulation of many genes involved in fatty acid β-oxidation, tricarboxylic acid cycle and electron transport. This demonstrates the lack of fatty acid catabolism that leads to a greater accumulation of triacylglycerides in those tissues. In BAT, some proteins involved in fatty acid transport were also down-regulated, suggesting a reduction in energy fuel uptake that together with the down-regulated catabolic pathways agree with the defective cold adaptation that those mice exhibited. Conclusion: We show that ATGL-/- mice have a highly modified gene expression profile that reinforces the important role of this enzyme on the hydrolysis of fat stores. PO1-4
ADIPONECTIN ACCELERATES REVERSE CHOLESTEROL TRANSPORT BY INCREASING HDL ASSEMBLY THROUGH UPREGULATION OF ABCA1 PATHWAY AND APOA-I SYNTHESIS IN THE LIVER
F. Matsuura 1 , M. Koseki 1 , Y. Oku 1 , C. Ikegami 1 , M. Kawase 1 , K. Yamamoto 1 , D. Masuda 1 , M. Nishida 2 , M. Ishigami 3 , K. Hirano 1 , S. Kihara 2 , 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, Suita, Osaka, Japan Background: Plasma high density lipoprotein (HDL) -cholesterol levels are negatively correlated with the incidence of coronary heart disease. The ATP-binding cassette transporter (ABCA1) and scavenger receptor, class B, type I (SR-BI) are thought to be rate-limiting factors to generate HDL in the liver. Adiponectin (APN) secreted from adipocytes is also one of the important molecules to inhibit the development of atherosclerosis. Recently it has been reported that plasma HDL-cholesterol levels are positively correlated with plasma APN concentrations in humans. Therefore, to investigate the role of APN on HDL assembly in the liver, we examined the effects of APN on apolipoprotein A-I (apoA-I) secretion from human hepatoma HepG2 cells and cellular expressions of ABCA1 and SR-BI. Methods: HepG2 cells were incubated in Dulbecco’s modified Eagle’s medium containing 0.5% fetal bovine serum with the indicated concentrations of recombinant APN for 24 hours. The secretion of apoA-I from HepG2 cells to culture medium and the cellular expression levels of ABCA1 and SR-BI were examined by western blot analysis or quantitative real-time PCR.
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
POSTER SESSIONS
Backgrounds and Aims: There is little published data on gene expression by primary hepatocytes in response to statins. This study examined the impact of rosuvastatin on expression of genes linked to cholesterol metabolism in primary human and rat hepatocyte. Methods: Primary cultures were established by collagenase digestion. Cells were analysed for mRNA by quantitative RT-PCR and proteins by immunoblotting. Results: Rosuvastatin caused dose-dependent increases (2-4 fold) in mRNA for LDL-receptor, HMGCoA reductase, HMGCoA synthase and convertase, NARC-1 (PCSK9) genes (Fig) in human hepatocytes. Increases in expression occurred between 10-9M/10-8M rosuvastatin. The SREBP-2 gene increased modestly (1.3fold). mRNA levels for SREBP-1 and FXR genes were dose-dependently down-regulated (Fig). Co-incubation with mevalonate abrogated the above changes. Immunoblots revealed an increase in the concentration of activated SREBP-2 by rosuvastatin, again abrogated by mevalonate. Comparable results were obtained with rat hepatocytes.
of apoA-I and ABCA1 mRNA induced by RSV. These observations suggest that RSV increases the secretion of HDL by stimulating the production of apoA-I and facilitating its lipidation by ABCA1. This effect may be related to the inhibition of the prenylation of some cell protein.
20
Poster Sessions PO1 Hepatic and adipose tissue lipid metabolism
Results: APN increased the expression of ABCA1 protein, but not SR-BI, in HepG2 cells. Furthermore, APN enhanced the mRNA level of apoA-1 in HepG2 cells and increased apoA-I secretion from the cells in a dose-dependent manner. Conclusions: APN might protect against atherosclerosis by increasing the HDL assembly through enhancing ABCA1 pathway and apoA-1 synthesis in the liver. PO1-5
THE HEPATO-OOCYTE-EMBRYO AXIS: LIPOLYTIC ENZYMES IN THE CHICKEN
J. Saarela, G. Jung, W.J. Schneider. Max F. Perutz Laboratories, Department of Medical Biochemistry, Medical University of Vienna, Vienna, Austria In mature hens of the domesticated chicken (Gallus gallus), fully-grown oocytes contain up to 5 g triacylglycerols and 240 mg cholesterol, and ovulate every 25 hr. In parallel to this oocyte-targeted flow, lipid homeostasis of somatic tissues including the follicular cell layers is maintained. This is achieved through regulation of systemic lipoprotein metabolism involving somatic-cell-specific genes including genes for lipases, lipid transfer proteins, and LDL receptor relatives. Thus, the laying hen is a prime model for delineating molecular mechanisms underlying both unidirectional (hepato-oocyte-embryo) mass lipid transfer and fine-tuned regulation of systemic lipid metabolism. We have previously shown that estrogen induces a unique apolipoprotein, apoVLDL-II, which inhibits VLDL-lipolysis in the circulation, thereby assuring efficient transport of triglycerides into the oocyte for subsequent use by the embryo. Currently, we are molecularly characterizing other lipolytic enzymes which must function towards the efficient lipolysis of lipoproteins by the embryo. So far, 6 novel lipase genes have been fully cloned, and preliminary expression analysis of 9 lipases has been studied using quantitative PCR. In addition, a specific lipase-activator has been cloned and characterized. The cloning of the remaining (patatin-type) lipases has been initiated. The enzymes are being produced as recombinant proteins for further studies including the analysis of their substrate specificities using fluorescent suicide lipase inhibitors. We have begun to investigate the regulation of the novel lipases, their possible interactions with apoVLDL-II, and their roles in the transfer of lipid components from yolk to the developing embryo. (Supported by Project GEN-AU). PO1-6
INSULIN STIMULATES THE HEPATIC LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN 1 (LRP1) IN VIVO
A. Laatsch 1 , M. Merkel 2 , U. Beisiegel 1 , J. Heeren 1 . 1 University Medical Center Hamburg-Eppendorf, IBM II: Molecular Cell Biology, Hamburg, Germany; 2 University Medical Center Hamburg-Eppendorf, Department for Internal Medicine, Hamburg, Germany Background and Aims: The hepatic low density lipoprotein receptorrelated protein 1 (LRP1) plays an important role for the postprandial clearance of pro-atherogenic chylomicron remnants. As we demonstrated earlier, insulin can activate LRP1 by translocating the receptor from intracellular vesicles to the plasma membrane in vitro. In a scenario of insulin resistance, we expect an impaired postprandial activation of LRP1, which would lead to a postprandial hyperlipoproteinaemia, which is a cardiovascular risk factor. The aim of this study was to challenge this in vitro mechanism in vivo. Methods: Turnover experiments were carried out in mice using radiolabelled receptor associated protein and activated alpha-2-macroglobuline as LRP1-specific ligands. An insulin response was provoked by i. p. injection of glucose prior to tail vein injection of the labelled ligand. Leptin-deficient ob/ob-mice served as a model of hepatic insulin resistance. Results: Glucose-injected animals showed a significantly higher uptake of LRP1-specific ligands into the liver compared to saline-injected animals. In insulin-resistant animals, this effect was abolished. Conclusions: Hepatic LRP1 is activated by insulin in vivo and leads to an accelerated clearance of LRP1 ligands. This activation does not occur in insulin resistance indicating a major role of LRP1 for the development of a postprandial hyperlipidaemia in diabetic patients.
PO1-7
RNA DIFFERENTIAL EXPRESSION IN SUBCUTANEOUS ADIPOSE TISSUE OF PATIENTS WITH FAMILIAL COMBINED HYPERLIPIDEMIA
J. Salazar, R. Ferre, M. Guardiola, J.C. Vallve, L. Masana, J. Ribalta. IRCIS-Universitat Rovira I Virgili, Reus, Spain Background and Aims: Clinical, biochemical and genetic data suggests that an impaired function of the adipose tissue underlies the metabolic alterations characteristic of familial combined hyperlipidemia (FCHL). However, our understanding of the nature of such impairment is incomplete. We have aimed to obtain a global view of the disturbances of the adipose tissue of FCHL patients by using whole genome transcriptomics. Methods: Total RNA from subcutaneous adipose tissue from five FCHL male patients was compared with a pool of five healthy male controls that had comparable age and BMI. Microarray analysis was used together with real time RT-PCR of a series of selected genes. The set of genes either up or down-regulated were analyzed using the PANTHER program. Results: We identified a total of 348 probes that had a differential expression. By identifying sets of genes belonging to the same biological process and which were collectively up or down regulated we identified the following alterations: 1) Down-regulation of Beta oxidation of FFA (HADHSC) and of its re-esterification (PEPCK, GPD1L), both leading to increased FFA release; 2) Up-regulation of monocyte, macrophage and neutrophil activation (integrins). There was an overall down-regulation of the PPARg and AQP7 genes by a 50% in FCHL patients. Of note, one of these patients presented a 100-fold up-regulation of the CNR-1. Conclusions: Collectively, all these genetic changes indicate that in adipose tissue the release of FFA is stimulated. This situation is consistent with a scenario of lipotoxicity due to a limited ability to differentiate, as suggested by low PPARg expression. PO1-8
EFFECTS OF FATTY ACIDS DELIVERED IN CHYLOMICRON REMNANTS ON THE HEPATIC EXPRESSION OF GENES REGULATING SYNTHESIS AND SECRETION OF VLDL
I. Lopez-Soldado, M. Avella, K.M. Botham. Royal Veterinary College, London, UK Background and Aims: Previous work has shown that the fatty acid composition of chylomicron remnants influences hepatic VLDL secretion. The aim of the study is to test the influence of the fatty acid composition of chylomicron remnant-like particles (CRLPs) on the expression of mRNA for five genes involved in the regulation of the synthesis, assembly and secretion of very-low-density lipoprotein (VLDL) in the liver. The five genes investigated were those encoding apolipoprotein B (apoB), the microsomal triaclyglycerol transfer protein (MTP) and the enzymes acyl coenzyme A:cholesterol acyltransferase 2 (ACAT-2), 3hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA Reductase), and glycerol-3-phosphate acyltransferase mitochondrial (GPAT) which play a role in the regulation of cholesterol ester, cholesterol and triacylglycerol respectively. Methods: Hepatocytes were isolated from rat livers by perfusion with collagenase. CRLPs containing triacylglycerol enriched in saturated, monounsaturated or n-6 polyunsaturated fatty acids derived from palm, olive and corn, respectively, were used. Expression of mRNA was estimated by real time-PCR. Results: mRNA levels for apoB, MTP and GPAT were unaffected by either corn, olive or palm CRLPs, but the relative amount of HMG-CoA reductase mRNA was significantly reduced after incubation of the hepatocytes for 5 hr with olive CRLPs compared with corn-CRLPs. The levels of mRNA for ACAT-2 were significantly lower in hepatocytes incubated with corn, olive and palm CRLPs as compared to without remnats. Conclusions: These findings indicate that the delivery of MUFA to hepatocytes in CRLPs downregulate the expression of mRNA HMG-CoA reductase, and this may play a role in their inhibition of VLDL secretion.
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
Poster Sessions PO1 HEPATIC AND ADIPOSE TISSUE LIPID METABOLISM PO1-1
DOSE-DEPENDENT EFFECTS OF ROSUVASTATIN ON GENE EXPRESSION IN PRIMARY CULTURES OF HUMAN AND RAT HEPATOCYTES
R.W. James, M.C. Brulhart-Meynet. Dept. of Internal Medicine, University Hospital, Geneva, Switzerland
Relative mRNA levels in human hepatocytes treated with rosuvastatin.
mRNA levels standardised to cyclophilin and control mRNA levels. Control, non-treated; LDL-R, LDL-receptor; SREBP-1, sterol regulatory element binding protein-1; Mev, mevalonate (0.5mM). Conclusions: The study confirms the impact of rosuvastatin on mRNA levels in primary human and rat hepatocytes and, by implication, expression of genes implicated in cholesterol metabolism. Rosuvastatin is effective at concentrations compatible with the known kinetics of HMGCoA reductase inhibition. PO1-2
EFFECT OF ROSUVASTATIN ON HDL SECRETION BY LIVER CELLS
F. Turrini 1 , S. Bertolini 2 , S. Calandra 1 . 1 Department of Biomedical Sciences, University of Modena & Reggio Emilia, Modena, Italy; 2 Department of Internal Medicine, University of Genova, Genova, Italy Rosuvastatin (RSV) treatment is associated with a substantial increase of plasma HDL. To clarify the mechanism underlying this effect Chang human liver cells were incubated for 24 hours with 0.1,1,5 and 10 μM of RSV and apo A-I and apo B-100 secreted into the medium were measured. Apo A-I secreted by RSV (5 μM) treated cells was increased by 55% while apo B was reduced by 70%. To investigate whether apo A-I was secreted as constituent of lipoproteins, cells were pulsed with labelled methionine for 30 minutes, chased for 2 hours and medium lipoproteins were isolated by ultracentrifugation. In RSV treated cells the peak of medium HDL was increased by 35% while that of VLD/LDL was reduced by 70%. In RSV treated cells the level of ApoA-I and ABCA1 mRNA was increased by 1.5 fold and 35% respectively. These changes were abolished by mevalonate. To investigate whether these changes were due to reduced availability of non steroidal isoprenoids derived from mevalonate, cells were incubated with RSV (5 ±M) in the presence of 10 μM of farnesyl pyrophosphate (Fpp) or geranylgeranyl pyrophosphate (GGpp). GGpp partly prevented the increase
PO1-3
MICROARRAY GENE EXPRESSION PROFILE OF ATGL -/- MICE
M. Pinent 1 , H. Hackl 1 , G. Haemmerle 2 , R. Zechner 2 , J.G. Strauss 1 , Z. Trajanoski 1 . 1 Institute for Genomics and Bioinformatics, Graz University of Technology, Graz, Austria; 2 Institue of Molecular Biosciences, University of Graz, Graz, Austria Background: Adipose triglyceride lipase (ATGL) is a recently described lipase with an important role on the catabolism of triacylglycerides. ATGL deficient mice show increased adipose mass and triacylglycerol deposition in multiple tissues. Furthermore, due to its inability to mobilize fat stores, they exhibit energy starvation, resulting in reduced energy expenditure, a decline in body temperature, and premature death due to cardiac insufficiency. Methods: In the present work we show the gene expression profile of ATGL -/- mice, obtained by microarray technology. Several tissues were analyzed: heart, skeletal muscle, white and brown adipose tissue (BAT), liver and kidney. Results: Among all those tissues, those which presented more changes were the BAT and the heart, with a total of 1110 and 384 differentially expressed EST respectively. In those tissues we found a significant downregulation of many genes involved in fatty acid β-oxidation, tricarboxylic acid cycle and electron transport. This demonstrates the lack of fatty acid catabolism that leads to a greater accumulation of triacylglycerides in those tissues. In BAT, some proteins involved in fatty acid transport were also down-regulated, suggesting a reduction in energy fuel uptake that together with the down-regulated catabolic pathways agree with the defective cold adaptation that those mice exhibited. Conclusion: We show that ATGL-/- mice have a highly modified gene expression profile that reinforces the important role of this enzyme on the hydrolysis of fat stores. PO1-4
ADIPONECTIN ACCELERATES REVERSE CHOLESTEROL TRANSPORT BY INCREASING HDL ASSEMBLY THROUGH UPREGULATION OF ABCA1 PATHWAY AND APOA-I SYNTHESIS IN THE LIVER
F. Matsuura 1 , M. Koseki 1 , Y. Oku 1 , C. Ikegami 1 , M. Kawase 1 , K. Yamamoto 1 , D. Masuda 1 , M. Nishida 2 , M. Ishigami 3 , K. Hirano 1 , S. Kihara 2 , 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, Suita, Osaka, Japan Background: Plasma high density lipoprotein (HDL) -cholesterol levels are negatively correlated with the incidence of coronary heart disease. The ATP-binding cassette transporter (ABCA1) and scavenger receptor, class B, type I (SR-BI) are thought to be rate-limiting factors to generate HDL in the liver. Adiponectin (APN) secreted from adipocytes is also one of the important molecules to inhibit the development of atherosclerosis. Recently it has been reported that plasma HDL-cholesterol levels are positively correlated with plasma APN concentrations in humans. Therefore, to investigate the role of APN on HDL assembly in the liver, we examined the effects of APN on apolipoprotein A-I (apoA-I) secretion from human hepatoma HepG2 cells and cellular expressions of ABCA1 and SR-BI. Methods: HepG2 cells were incubated in Dulbecco’s modified Eagle’s medium containing 0.5% fetal bovine serum with the indicated concentrations of recombinant APN for 24 hours. The secretion of apoA-I from HepG2 cells to culture medium and the cellular expression levels of ABCA1 and SR-BI were examined by western blot analysis or quantitative real-time PCR.
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
POSTER SESSIONS
Backgrounds and Aims: There is little published data on gene expression by primary hepatocytes in response to statins. This study examined the impact of rosuvastatin on expression of genes linked to cholesterol metabolism in primary human and rat hepatocyte. Methods: Primary cultures were established by collagenase digestion. Cells were analysed for mRNA by quantitative RT-PCR and proteins by immunoblotting. Results: Rosuvastatin caused dose-dependent increases (2-4 fold) in mRNA for LDL-receptor, HMGCoA reductase, HMGCoA synthase and convertase, NARC-1 (PCSK9) genes (Fig) in human hepatocytes. Increases in expression occurred between 10-9M/10-8M rosuvastatin. The SREBP-2 gene increased modestly (1.3fold). mRNA levels for SREBP-1 and FXR genes were dose-dependently down-regulated (Fig). Co-incubation with mevalonate abrogated the above changes. Immunoblots revealed an increase in the concentration of activated SREBP-2 by rosuvastatin, again abrogated by mevalonate. Comparable results were obtained with rat hepatocytes.
of apoA-I and ABCA1 mRNA induced by RSV. These observations suggest that RSV increases the secretion of HDL by stimulating the production of apoA-I and facilitating its lipidation by ABCA1. This effect may be related to the inhibition of the prenylation of some cell protein.
20
Poster Sessions PO1 Hepatic and adipose tissue lipid metabolism
Results: APN increased the expression of ABCA1 protein, but not SR-BI, in HepG2 cells. Furthermore, APN enhanced the mRNA level of apoA-1 in HepG2 cells and increased apoA-I secretion from the cells in a dose-dependent manner. Conclusions: APN might protect against atherosclerosis by increasing the HDL assembly through enhancing ABCA1 pathway and apoA-1 synthesis in the liver. PO1-5
THE HEPATO-OOCYTE-EMBRYO AXIS: LIPOLYTIC ENZYMES IN THE CHICKEN
J. Saarela, G. Jung, W.J. Schneider. Max F. Perutz Laboratories, Department of Medical Biochemistry, Medical University of Vienna, Vienna, Austria In mature hens of the domesticated chicken (Gallus gallus), fully-grown oocytes contain up to 5 g triacylglycerols and 240 mg cholesterol, and ovulate every 25 hr. In parallel to this oocyte-targeted flow, lipid homeostasis of somatic tissues including the follicular cell layers is maintained. This is achieved through regulation of systemic lipoprotein metabolism involving somatic-cell-specific genes including genes for lipases, lipid transfer proteins, and LDL receptor relatives. Thus, the laying hen is a prime model for delineating molecular mechanisms underlying both unidirectional (hepato-oocyte-embryo) mass lipid transfer and fine-tuned regulation of systemic lipid metabolism. We have previously shown that estrogen induces a unique apolipoprotein, apoVLDL-II, which inhibits VLDL-lipolysis in the circulation, thereby assuring efficient transport of triglycerides into the oocyte for subsequent use by the embryo. Currently, we are molecularly characterizing other lipolytic enzymes which must function towards the efficient lipolysis of lipoproteins by the embryo. So far, 6 novel lipase genes have been fully cloned, and preliminary expression analysis of 9 lipases has been studied using quantitative PCR. In addition, a specific lipase-activator has been cloned and characterized. The cloning of the remaining (patatin-type) lipases has been initiated. The enzymes are being produced as recombinant proteins for further studies including the analysis of their substrate specificities using fluorescent suicide lipase inhibitors. We have begun to investigate the regulation of the novel lipases, their possible interactions with apoVLDL-II, and their roles in the transfer of lipid components from yolk to the developing embryo. (Supported by Project GEN-AU). PO1-6
INSULIN STIMULATES THE HEPATIC LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN 1 (LRP1) IN VIVO
A. Laatsch 1 , M. Merkel 2 , U. Beisiegel 1 , J. Heeren 1 . 1 University Medical Center Hamburg-Eppendorf, IBM II: Molecular Cell Biology, Hamburg, Germany; 2 University Medical Center Hamburg-Eppendorf, Department for Internal Medicine, Hamburg, Germany Background and Aims: The hepatic low density lipoprotein receptorrelated protein 1 (LRP1) plays an important role for the postprandial clearance of pro-atherogenic chylomicron remnants. As we demonstrated earlier, insulin can activate LRP1 by translocating the receptor from intracellular vesicles to the plasma membrane in vitro. In a scenario of insulin resistance, we expect an impaired postprandial activation of LRP1, which would lead to a postprandial hyperlipoproteinaemia, which is a cardiovascular risk factor. The aim of this study was to challenge this in vitro mechanism in vivo. Methods: Turnover experiments were carried out in mice using radiolabelled receptor associated protein and activated alpha-2-macroglobuline as LRP1-specific ligands. An insulin response was provoked by i. p. injection of glucose prior to tail vein injection of the labelled ligand. Leptin-deficient ob/ob-mice served as a model of hepatic insulin resistance. Results: Glucose-injected animals showed a significantly higher uptake of LRP1-specific ligands into the liver compared to saline-injected animals. In insulin-resistant animals, this effect was abolished. Conclusions: Hepatic LRP1 is activated by insulin in vivo and leads to an accelerated clearance of LRP1 ligands. This activation does not occur in insulin resistance indicating a major role of LRP1 for the development of a postprandial hyperlipidaemia in diabetic patients.
PO1-7
RNA DIFFERENTIAL EXPRESSION IN SUBCUTANEOUS ADIPOSE TISSUE OF PATIENTS WITH FAMILIAL COMBINED HYPERLIPIDEMIA
J. Salazar, R. Ferre, M. Guardiola, J.C. Vallve, L. Masana, J. Ribalta. IRCIS-Universitat Rovira I Virgili, Reus, Spain Background and Aims: Clinical, biochemical and genetic data suggests that an impaired function of the adipose tissue underlies the metabolic alterations characteristic of familial combined hyperlipidemia (FCHL). However, our understanding of the nature of such impairment is incomplete. We have aimed to obtain a global view of the disturbances of the adipose tissue of FCHL patients by using whole genome transcriptomics. Methods: Total RNA from subcutaneous adipose tissue from five FCHL male patients was compared with a pool of five healthy male controls that had comparable age and BMI. Microarray analysis was used together with real time RT-PCR of a series of selected genes. The set of genes either up or down-regulated were analyzed using the PANTHER program. Results: We identified a total of 348 probes that had a differential expression. By identifying sets of genes belonging to the same biological process and which were collectively up or down regulated we identified the following alterations: 1) Down-regulation of Beta oxidation of FFA (HADHSC) and of its re-esterification (PEPCK, GPD1L), both leading to increased FFA release; 2) Up-regulation of monocyte, macrophage and neutrophil activation (integrins). There was an overall down-regulation of the PPARg and AQP7 genes by a 50% in FCHL patients. Of note, one of these patients presented a 100-fold up-regulation of the CNR-1. Conclusions: Collectively, all these genetic changes indicate that in adipose tissue the release of FFA is stimulated. This situation is consistent with a scenario of lipotoxicity due to a limited ability to differentiate, as suggested by low PPARg expression. PO1-8
EFFECTS OF FATTY ACIDS DELIVERED IN CHYLOMICRON REMNANTS ON THE HEPATIC EXPRESSION OF GENES REGULATING SYNTHESIS AND SECRETION OF VLDL
I. Lopez-Soldado, M. Avella, K.M. Botham. Royal Veterinary College, London, UK Background and Aims: Previous work has shown that the fatty acid composition of chylomicron remnants influences hepatic VLDL secretion. The aim of the study is to test the influence of the fatty acid composition of chylomicron remnant-like particles (CRLPs) on the expression of mRNA for five genes involved in the regulation of the synthesis, assembly and secretion of very-low-density lipoprotein (VLDL) in the liver. The five genes investigated were those encoding apolipoprotein B (apoB), the microsomal triaclyglycerol transfer protein (MTP) and the enzymes acyl coenzyme A:cholesterol acyltransferase 2 (ACAT-2), 3hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA Reductase), and glycerol-3-phosphate acyltransferase mitochondrial (GPAT) which play a role in the regulation of cholesterol ester, cholesterol and triacylglycerol respectively. Methods: Hepatocytes were isolated from rat livers by perfusion with collagenase. CRLPs containing triacylglycerol enriched in saturated, monounsaturated or n-6 polyunsaturated fatty acids derived from palm, olive and corn, respectively, were used. Expression of mRNA was estimated by real time-PCR. Results: mRNA levels for apoB, MTP and GPAT were unaffected by either corn, olive or palm CRLPs, but the relative amount of HMG-CoA reductase mRNA was significantly reduced after incubation of the hepatocytes for 5 hr with olive CRLPs compared with corn-CRLPs. The levels of mRNA for ACAT-2 were significantly lower in hepatocytes incubated with corn, olive and palm CRLPs as compared to without remnats. Conclusions: These findings indicate that the delivery of MUFA to hepatocytes in CRLPs downregulate the expression of mRNA HMG-CoA reductase, and this may play a role in their inhibition of VLDL secretion.
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland