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PO2-38 DIFFERENTIAL INTESTINAL MUCOSAL PROTEIN EXPRESSION IN HYPERCHOLESTEROLEMIC MICE FED A PHYTOSTEROL-ENRICHED DIET
28 PO2-38
Poster Sessions PO2 Lipoprotein and cholesterol metabolism DIFFERENTIAL INTESTINAL MUCOSAL PROTEIN EXPRESSION IN HYPERCHOLESTEROLEMIC MICE FED A PHYTOSTEROL-ENRICHED DIET
L. Calpe-Berdiel 1 , J.C. Escola-Gil 1 , J. Julve 1 , E. Zapico-Muniz 1 , F. Canals 2 , F. Blanco-Vaca 1 . 1 Servei de Bioquimica I Institut de Recerca, Hospital de La Santa Creu I Sant Pau, Barcelona, Spain; 2 Proteomics Laboratory. Medical Oncology Research Program, Vall D’Hebron University Hospital Research Institute, Barcelona, Spain Background: The molecular mechanisms involved in the phytosterolinduced decrease in intestinal cholesterol absorption remain unclear. Other biological properties such as immunomodulatory activity and protection against cancer have also been ascribed to these plant compounds. Methods: To gain insight into the mechanisms underlying phytosterol actions, we conducted a proteomic study in intestinal mucosa of phytosterol-fed apolipoprotein E-deficient (apoE-/-) hypercholesterolemic mice. The main results were re-tested in normocholesterolemic C57BL/6J mice. Results: With respect to control-fed apoE-/- mice, 18 differentially expressed proteins were identified in whole-enterocyte homogenates using 2-D DIGE and MALDI-TOF mass spectrometry. These proteins are involved in plasma membrane stabilization, cytoskeleton assembly network and cholesterol and carbohydrate metabolism. Four of these proteins were selected for further study since they showed the highest abundance change or had a potential functional relationship with known effects of phytosterols. Annexin A2 and beta-actin decrease and annexin A4 and annexin A5 increase were confirmed by Western blot analysis. Intestinal gene expression of annexin A2 and A5 and beta-actin was reduced whereas that of annexin A4 was unchanged. Annexin A4 protein upregulation and annexin A2 and beta-actin downregulation were reproduced in normolipemic animals. No changes in gene expression were found in C57BL/6J mice in either of the four proteins selected. Conclusions: Annexins A2, A4 and A5 and beta-actin are proteins of special interest given their pleiotropic functions that include cholesterolester transport from caveolae, apoptosis and anti-inflammatory properties. Therefore, the protein expression changes identified in this study might be involved in the biological effects of phystosterols. PO2-39
RELATIONSHIP OF ENDOGENOUS HYPERLEPTINEMIA TO SERUM PARAOXONASE 1, CHOLESTERYL ESTER TRANSFER PROTEIN AND LECITHIN CHOLESTEROL ACYL-TRANSFERASE IN OBESE INDIVIDUALS
L. Bajnok 1 , I. Seres 2 , Z.S. Varga 2 , S. Jeges 3 , A. Peti 4 , Z.S. Karanyi 2 , A. Juhasz 2 , E. Csongradi 2 , E. Mezosi 1 , E.V. Nagy 2 , G.Y. Paragh 2 . 1 1st Department of Medicine, School of Medicine, University of Pecs, Pecs, Hungary; 2 1st Department of Medicine, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary; 3 Institute of Bioanalysis, School of Medicine and Applied Health Sciences, Faculty of Health Sciences, University of Pecs, Pecs, Hungary; 4 Institute of Laboratory Medicine, Faculty of Medicine, University of Pecs, Pecs, Hungary Inverse associations of PON1 with obesity and serum leptin levels have been demonstrated. Our aims were to test (i) the independence of this relationship from other variables, and (ii) the association of leptin with other HDL-associated proteins, CETP and LCAT. Subjects: 3 age-, and sex-matched groups: non-diabetic obese with BMI below (n= 25), or at least 40 kg/m2 (n=25), and 24 healthy, normal weight controls. PON1 correlated inversely with BMI (r = -0.39, P < 0.01), waist circumferences (r = -0.42, P < 0.001), and leptin levels (r = -0.38, P < 0.001). However, in a multiple regression model neither these variables, nor others, e.g. age, gender, blood pressure, HOMA-IR, HDL-C, LDL-C and TBARS were independent predictors of PON1. LCAT correlated negatively with BMI (r = -0.40, P < 0.01), waist circumferences (r = -0.42, P < 0.001), and leptin levels (r = -0.40, P < 0.01). During multiple regression analyses, BMI was an independent predictor of LCAT after adjustments for age, gender, HOMA-IR, and HDL-C. However, this was replaced by leptin and HOMA-IR when leptin was also included into the model. CETP activities were correlated with HOMA-IR (r = 0.33, P < 0.01), TBARS (r = 0.45, P < 0.001), and leptin (r = 0.36, P < 0.01) in univariate, but not in multivariate models. Conclusions: Elevated leptin level is an independent predictor of low LCAT, but not PON activity. In a population with broad range of BMI, LCAT correlates inversely with obesity and CETP directly with insulin resistance.
PO2-40
PHOSPHATIDYLINOSITOL 4-5 BISPHOSPHATE IS IMPORTANT FOR LIPID DROPLET FORMATION
L. Li, M. Rutberg, L. Andersson, J. Ericson, B. Magnusson, H. Peilot, S-O. Olofsson. Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska Universityhospital, Goteborg University, Goteborg, Sweden Phospholipase D1 (PLD1) and ERK2 are important for the assembly of lipid droplets. ERK2 phosphorylates dynein which is essential for the growth of the droplets by fusion. The downstream effectors of PLD1 are unknown but possible mechanisms involves the formation of lysophosphatidic acid and the activation of mTOR. However our results do not support these two possibilities. A third possibility is that the phophatidic acid formed is the precursor for the formation of phosphatidylinositol 4-5 bisphosphate (PIP2). PIP2 is known to play important roles in vesicle fusion and signal transduction by binding specific PH domain proteins. Indeed PIP2 is even important for the PLD1 activity. To address the importance of PIP2 in the assembly of lipid droplets, we developed a semi-intact cell system. The permeabilized NIH 3T3 cells start to generate lipid droplets after being substituted by cytosol and an energy regenerating system. Both components being equally important. The system was stable for at least 12 hours and generated droplets with a maximum size of 0.9 μm already after 3 hours. Compared to control antibodies, an antibody to PIP2 gave rise to a more than 50% decrease in the formation of lipid droplets indicating that PIP2 has a central function in the assembly of the droplets. These results demonstrate that PIP2 is important for the assembly of droplets and points to a downstream mediator of PLD1. Moreover it indicates a mechanism by which the activity of PLD1 can be amplified. PO2-41
CETP ACTIVITY IS INCREASED IN PERSONS WITH IMPAIRED GLUCOSE TOLERANCE
U.A. Julius 1 , M. Jauhiainen 2 , C. Ehnholm 2 , J. Pietzsch 3 . 1 Medizinische Klinik III, Universitätsklinikum Dresden, Dresden, Germany; 2 Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland; 3 Pathological Biochemistry Group, Department of Radiopharmaceutical Biology, Institute of Radiopharmacy, Research Center Rossendorf, Rossendorf, Germany Background and Aims: An impaired glucose tolerance (IGT) is associated with an increased risk for atherosclerosis that may be in part due to dyslipidemia. The purpose of this study was to assess the regulatory role of lipid transfer proteins in the development of this dyslipidemia. Methods: Activities of cholesterol ester transfer protein (CETP) and phospholipid transfer protein (PLTP) as well as lipid and protein components of the major lipoprotein fractions were evaluated in probands with IGT (N 30) and were compared with those in subjects with normal glucose tolerance (N 80). The effect of a fat-rich meal on these variables has also been investigated. Results: IGT probands had a higher triglyceride content in subfractions of LDL and HDL. IGT patients have increased fasting CETP activity. The latter was positively correlated with HDL2 triglycerides and negatively with HDL3 total cholesterol. PLTP activity and mass were not elevated in IGT patients. However, PLTP activity correlated with components of VLDL and LDL and was influenced by the type of obesity. Neither CETP and PLTP activities nor PLTP mass were altered by a fat-rich meal. PLTP and CETP activities correlated in both fasting and postprandial conditions. Conclusions: Increased fasting CETP activity may contribute to the increased risk for atherosclerosis in subjects with IGT. PO2-42
DEVELOPMENT AND APPLICATION OF A NOVEL MOUSE APOE ELISA METHOD
M.J. Muilu 1 , S. Nuutinen 1 , J. Metso 1 , P. Pussinen 2 , C. Enholm 1 , M. Jauhiainen 1 . 1 Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland; 2 Institute of Dentistry, University of Helsinki, Helsinki, Finland Background and Aims: ApoE is a important modulator in the development of atherosclerosis. Recent data suggests novel function for apoE in reverse cholesterol transport process. Our aim was to 1) create a new immunochemical method for mouse apoE and 2) determine plasma apoE levels in different mouse models. Methods: Polyclonal rabbit anti-apoE IgG was used as a primary and secondary antibody. The secondary antibody was labeled with horseradish peroxidase. Purified mouse plasma apoE was used as a primary standard
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
Poster Sessions PO2 Lipoprotein and cholesterol metabolism DIFFERENTIAL INTESTINAL MUCOSAL PROTEIN EXPRESSION IN HYPERCHOLESTEROLEMIC MICE FED A PHYTOSTEROL-ENRICHED DIET
L. Calpe-Berdiel 1 , J.C. Escola-Gil 1 , J. Julve 1 , E. Zapico-Muniz 1 , F. Canals 2 , F. Blanco-Vaca 1 . 1 Servei de Bioquimica I Institut de Recerca, Hospital de La Santa Creu I Sant Pau, Barcelona, Spain; 2 Proteomics Laboratory. Medical Oncology Research Program, Vall D’Hebron University Hospital Research Institute, Barcelona, Spain Background: The molecular mechanisms involved in the phytosterolinduced decrease in intestinal cholesterol absorption remain unclear. Other biological properties such as immunomodulatory activity and protection against cancer have also been ascribed to these plant compounds. Methods: To gain insight into the mechanisms underlying phytosterol actions, we conducted a proteomic study in intestinal mucosa of phytosterol-fed apolipoprotein E-deficient (apoE-/-) hypercholesterolemic mice. The main results were re-tested in normocholesterolemic C57BL/6J mice. Results: With respect to control-fed apoE-/- mice, 18 differentially expressed proteins were identified in whole-enterocyte homogenates using 2-D DIGE and MALDI-TOF mass spectrometry. These proteins are involved in plasma membrane stabilization, cytoskeleton assembly network and cholesterol and carbohydrate metabolism. Four of these proteins were selected for further study since they showed the highest abundance change or had a potential functional relationship with known effects of phytosterols. Annexin A2 and beta-actin decrease and annexin A4 and annexin A5 increase were confirmed by Western blot analysis. Intestinal gene expression of annexin A2 and A5 and beta-actin was reduced whereas that of annexin A4 was unchanged. Annexin A4 protein upregulation and annexin A2 and beta-actin downregulation were reproduced in normolipemic animals. No changes in gene expression were found in C57BL/6J mice in either of the four proteins selected. Conclusions: Annexins A2, A4 and A5 and beta-actin are proteins of special interest given their pleiotropic functions that include cholesterolester transport from caveolae, apoptosis and anti-inflammatory properties. Therefore, the protein expression changes identified in this study might be involved in the biological effects of phystosterols. PO2-39
RELATIONSHIP OF ENDOGENOUS HYPERLEPTINEMIA TO SERUM PARAOXONASE 1, CHOLESTERYL ESTER TRANSFER PROTEIN AND LECITHIN CHOLESTEROL ACYL-TRANSFERASE IN OBESE INDIVIDUALS
L. Bajnok 1 , I. Seres 2 , Z.S. Varga 2 , S. Jeges 3 , A. Peti 4 , Z.S. Karanyi 2 , A. Juhasz 2 , E. Csongradi 2 , E. Mezosi 1 , E.V. Nagy 2 , G.Y. Paragh 2 . 1 1st Department of Medicine, School of Medicine, University of Pecs, Pecs, Hungary; 2 1st Department of Medicine, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary; 3 Institute of Bioanalysis, School of Medicine and Applied Health Sciences, Faculty of Health Sciences, University of Pecs, Pecs, Hungary; 4 Institute of Laboratory Medicine, Faculty of Medicine, University of Pecs, Pecs, Hungary Inverse associations of PON1 with obesity and serum leptin levels have been demonstrated. Our aims were to test (i) the independence of this relationship from other variables, and (ii) the association of leptin with other HDL-associated proteins, CETP and LCAT. Subjects: 3 age-, and sex-matched groups: non-diabetic obese with BMI below (n= 25), or at least 40 kg/m2 (n=25), and 24 healthy, normal weight controls. PON1 correlated inversely with BMI (r = -0.39, P < 0.01), waist circumferences (r = -0.42, P < 0.001), and leptin levels (r = -0.38, P < 0.001). However, in a multiple regression model neither these variables, nor others, e.g. age, gender, blood pressure, HOMA-IR, HDL-C, LDL-C and TBARS were independent predictors of PON1. LCAT correlated negatively with BMI (r = -0.40, P < 0.01), waist circumferences (r = -0.42, P < 0.001), and leptin levels (r = -0.40, P < 0.01). During multiple regression analyses, BMI was an independent predictor of LCAT after adjustments for age, gender, HOMA-IR, and HDL-C. However, this was replaced by leptin and HOMA-IR when leptin was also included into the model. CETP activities were correlated with HOMA-IR (r = 0.33, P < 0.01), TBARS (r = 0.45, P < 0.001), and leptin (r = 0.36, P < 0.01) in univariate, but not in multivariate models. Conclusions: Elevated leptin level is an independent predictor of low LCAT, but not PON activity. In a population with broad range of BMI, LCAT correlates inversely with obesity and CETP directly with insulin resistance.
PO2-40
PHOSPHATIDYLINOSITOL 4-5 BISPHOSPHATE IS IMPORTANT FOR LIPID DROPLET FORMATION
L. Li, M. Rutberg, L. Andersson, J. Ericson, B. Magnusson, H. Peilot, S-O. Olofsson. Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska Universityhospital, Goteborg University, Goteborg, Sweden Phospholipase D1 (PLD1) and ERK2 are important for the assembly of lipid droplets. ERK2 phosphorylates dynein which is essential for the growth of the droplets by fusion. The downstream effectors of PLD1 are unknown but possible mechanisms involves the formation of lysophosphatidic acid and the activation of mTOR. However our results do not support these two possibilities. A third possibility is that the phophatidic acid formed is the precursor for the formation of phosphatidylinositol 4-5 bisphosphate (PIP2). PIP2 is known to play important roles in vesicle fusion and signal transduction by binding specific PH domain proteins. Indeed PIP2 is even important for the PLD1 activity. To address the importance of PIP2 in the assembly of lipid droplets, we developed a semi-intact cell system. The permeabilized NIH 3T3 cells start to generate lipid droplets after being substituted by cytosol and an energy regenerating system. Both components being equally important. The system was stable for at least 12 hours and generated droplets with a maximum size of 0.9 μm already after 3 hours. Compared to control antibodies, an antibody to PIP2 gave rise to a more than 50% decrease in the formation of lipid droplets indicating that PIP2 has a central function in the assembly of the droplets. These results demonstrate that PIP2 is important for the assembly of droplets and points to a downstream mediator of PLD1. Moreover it indicates a mechanism by which the activity of PLD1 can be amplified. PO2-41
CETP ACTIVITY IS INCREASED IN PERSONS WITH IMPAIRED GLUCOSE TOLERANCE
U.A. Julius 1 , M. Jauhiainen 2 , C. Ehnholm 2 , J. Pietzsch 3 . 1 Medizinische Klinik III, Universitätsklinikum Dresden, Dresden, Germany; 2 Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland; 3 Pathological Biochemistry Group, Department of Radiopharmaceutical Biology, Institute of Radiopharmacy, Research Center Rossendorf, Rossendorf, Germany Background and Aims: An impaired glucose tolerance (IGT) is associated with an increased risk for atherosclerosis that may be in part due to dyslipidemia. The purpose of this study was to assess the regulatory role of lipid transfer proteins in the development of this dyslipidemia. Methods: Activities of cholesterol ester transfer protein (CETP) and phospholipid transfer protein (PLTP) as well as lipid and protein components of the major lipoprotein fractions were evaluated in probands with IGT (N 30) and were compared with those in subjects with normal glucose tolerance (N 80). The effect of a fat-rich meal on these variables has also been investigated. Results: IGT probands had a higher triglyceride content in subfractions of LDL and HDL. IGT patients have increased fasting CETP activity. The latter was positively correlated with HDL2 triglycerides and negatively with HDL3 total cholesterol. PLTP activity and mass were not elevated in IGT patients. However, PLTP activity correlated with components of VLDL and LDL and was influenced by the type of obesity. Neither CETP and PLTP activities nor PLTP mass were altered by a fat-rich meal. PLTP and CETP activities correlated in both fasting and postprandial conditions. Conclusions: Increased fasting CETP activity may contribute to the increased risk for atherosclerosis in subjects with IGT. PO2-42
DEVELOPMENT AND APPLICATION OF A NOVEL MOUSE APOE ELISA METHOD
M.J. Muilu 1 , S. Nuutinen 1 , J. Metso 1 , P. Pussinen 2 , C. Enholm 1 , M. Jauhiainen 1 . 1 Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland; 2 Institute of Dentistry, University of Helsinki, Helsinki, Finland Background and Aims: ApoE is a important modulator in the development of atherosclerosis. Recent data suggests novel function for apoE in reverse cholesterol transport process. Our aim was to 1) create a new immunochemical method for mouse apoE and 2) determine plasma apoE levels in different mouse models. Methods: Polyclonal rabbit anti-apoE IgG was used as a primary and secondary antibody. The secondary antibody was labeled with horseradish peroxidase. Purified mouse plasma apoE was used as a primary standard
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland