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PO2-52 AMINO ACIDS 140–148 OF APOAV PLAY A CRUCIAL ROLE IN LPL ACTIVATION
Poster Sessions PO2 Lipoprotein and cholesterol metabolism PO2-51
EFFECTS ON CHYLOMICRONS METABOLISM IN PATIENTS WITH TYPE 2 DIABETES
V.M. Silva, C.G.C. Vinagre, R.C. Maranhao. Lipid Metabolism Laboratory and Lipid Clinic, São Paulo, Brazil; 2 The Heart Institute (InCor), São Paulo, Brazil; 3 Faculty of Pharmaceutical Sciences, São Paulo, Brazil; 4 University of São Paulo, Medical School Hospital, São Paulo, Brazil
PO2-52
AMINO ACIDS 140-148 OF APOAV PLAY A CRUCIAL ROLE IN LPL ACTIVATION
B. Dorfmeister 1 , J. Heeren 2 , M. Merkel 3 , P.J. Talmud 1 , S.E. Humphries 1 . 1 Centre for Cardiovascular Genetics, Department of Medicine, University College London, London, UK; 2 IBM II, Molecular Cell Biology, University Hospital Hamburg-Eppendorf, Hamburg, Germany; 3 Department of Internal Medicine I, University Hospital Hamburg, Eppendorf, Hamburg, Germany Background: Animal models and human association studies have identified apolipoprotein AV (apoAV) as a key player in triglyceride (TG) metabolism. Two premature truncations in APOA5, Q148X and Q139X, associated with severe hypertriglyceridemia (HTG) have been reported. Post-heparin plasma from the Q148X homozygous patient had 40% residual LPL activity. Q139X heterozygous patients showed a dramatic reduction in LPL activity by 79%. Aim: To test the effects on LPL activation in vitro of X139, X148 and a deletion construct ( 140-148). Methods: ApoAV wildtype, X139, X148 and 140-148 were expressed in a pET20b vector and purified by affinity chromatography and FPLC. Bovine LPL was immobilized onto HSPG coated plates and then exposed to the TG-rich lipoproteins, which were prior incubated with the apoAV proteins. LPL activity was measured by FFA release. Results: Preliminary data showed that compared to WT (0.70 ± 0.00 mmol/l), Q148X (0.59 ± 0.01 mmol/l) and Q139X (0.56 ± 0.01 mmol/l) had an impaired stimulatory effect on LPL activity, while the 140-148 (0.42 ± 0.02 mmol/l) mutant did not stimulate LPL activity.
ments with VLDL isolated from apoa5 -/- mice are underway to confirm and extend the current findings. PO2-53
APOLIPOPROTEIN J (CLUSTERIN) BINDS TO AND REDUCES CYTOTOXICITY OF ENZYMATICALLY HYDROLYZED LOW DENSITY LIPOPROTEIN
M. Schwarz 1 , L. Spath 2 , K. Paprotka 2 , M. Torzewski 3 , K. Dersch 2 , C. Koch-Brandt 1 , M. Husmann 2 , S. Bhakdi 2 . 1 Institute of Biochemistry, Johannes Gutenberg-University, Mainz, Germany; 2 Institute of Medical Microbiology and Hygiene, Johannes Gutenberg-University, Mainz, Germany; 3 Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg-University, Mainz, Germany Following entrapment in the arterial intima, low density lipoprotein can be modified by hydrolytic enzymes to yield a lipoprotein derivative that binds C-reactive protein, activates complement, and is rapidly taken up by monocytes/macrophages. Free fatty acids contained in enzymatically modified LDL (E-LDL) render the lipoprotein cytotoxic due to their capacity to trigger programmed cell death. Apolipoprotein J alias clusterin, a multifunctional protein that has the propensity to interact with diverse substrates, is present in the intima and the media of arteries with atherosclerotic lesions, and is also synthesized by smooth muscle cells during development of atherosclerosis. We report that Apo J binds to E-LDL but not to native LDL. Binding resulted in marked reduction of cytotoxicity of E-LDL on smooth muscle cells, as revealed by determination of caspase activity, annexin binding, and cellular ATP. In atherosclerotic lesions, Apo J may thus subserve protective functions through its capacity to inactivate C5b-9 complement complexes and by reducing the cytotoxic effects of modified LDL on smooth muscle cells that gain contact with the lipoprotein. PO2-54
A CORRELATION BETWEEN HYPERLIPIDAEMIA AND TESTICULAR FUNCTION IN ALBINO RATS
A. Purohit. Department of Zoology, Jai Narain Vyas University, Jodhpur, India A considerable work has been done on hyperlipidaemia in relation to body metabolism and function status in animal kingdom but the relationship between hyperlipidaemia and testicular function remained unexplored. The present work deal with effect of hyperlipidaemia on testicular function in albino rats. Hyperlipidaemia was induced by cholesterol feeding at the dose 400mg, 800 mg and 1000 mg/kg body wt. to male albino rats altered the testicular cell population dynamics. Cholesterol treatment resulted in severe degeneration in spermatogonia, primary and secondary spermatocytes and spermatids showing 28.92, 31.24, 71.97 and 93.69% reduction respectively with 400 mg group;43.01, 58.85,53.52,75.25% reduction respectively with 800 mg group and 83.88,91.77, 94.88 and 99.09% reduction respectively with 1000 mg group, as compared to control group. The number of interstitial cell types i.e., fibroblasts, immature and mature Leydig cells also showed significant reduction in all the treated groups. While there was a significant increase in the percent degenerating Leydig cells in all the cholesterol treated groups showing 73.8, 79.15 and 82.85 percent respectively. The result indicate that cholesterol diet may have significant effect on the intratesticular androgen levels either by inhibiting Leydig cell function or by inhibiting hypothalamo-pituitary axis. PO2-55
APOE/LDL RECEPTOR-DOUBLE-KNOCKOUT MICE: AN ANIMAL MODEL FOR THE STUDY OF STATINS EFFECTS IN ATHEROGENESIS
N. Pospisilova 1 , P. Nachtigal 1 , G. Jamborova 1 , K. Pospechova 1 , D. Solichova 2 , C. Andrys 3 , P. Zdansky 2 , V. Semecky 1 . 1 Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles University, Hradec Kralove, Czech Republic; 2 Department of Metabolic Care and Gerontology, Charles University Medical School and Teaching Hospital, Hradec Kralove, Czech Republic; 3 Institute of Clinical Immunology and Allergology, Charles University Medical School and Teaching Hospital, Hradec Kralove, Czech Republic Fig. 1. VLDL hydrolysis with recombinant apoAV by HSPG-bound LPL in vitro.
Conclusion: These preliminary data suggest that amino acids 140-148 could play a crucial role in apoAV/HSPG/LPL interaction. Further experi-
The statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) represent golden standard in dyslipidemia treatment in humans. However, its effects in animal models of atherosclerosis are very inconsistent. Thus, the aim of this study was to evaluate whether atorvastatin has hypolipidemic and inflammatory effects in apoE/LDLR deficient mice.
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
POSTER SESSIONS
Type 2 diabetes is usually accompanied by fasting hypertrigliceridemia and post-prandial hyperlipidemia. Chylomicron metabolism is constituted by lipolysis by lipoprotein lipase on the endothelium and removal of the resulting remnants by the liver and delay in those processes is associated with coronary artery disease (CAD) and worse evolution of CAD patients. Chylomicron metabolism has not been evaluated in diabetes patients with normal plasma lipids and this study aims to clarify this issue. The chylomicron-like emulsion method was used. The emulsion labeled with 3 H-triolein (TO) and 14C-cholesteryl olate (CO) was injected I.V. in a bolus into 12 patients diabetes and 10 controls without the disease. Both groups had total cholesterol <200, LDL-c < 130 and triglycerides < 200 mg/dl. The plasma kinetics of the labels was determined from blood samples taken over 60 min. Fractional clearance rates (FCR) were calculated by compartmental analysis. TO FCR estimates lipolysis while CO FCR estimates the remnant removal. In diabetes patients CO-FCR was diminished compared to controls (0.0053±0.032 and 0.0316±0.0061 min-1 , respectively, p=0.009) while TO-FCR was not different (0.0322±0.0182 and 0.0316 min-1 , p=0.94). In conclusion, despite normal plasma lipids, diabetes patients show decreased removal of chylomicron remnants, a feature associated with CAD. Because hypolipidemic drugs such as statins and fibrates can accelerate remnant removal, this finding has implications for the treatment of the disease.
31
EFFECTS ON CHYLOMICRONS METABOLISM IN PATIENTS WITH TYPE 2 DIABETES
V.M. Silva, C.G.C. Vinagre, R.C. Maranhao. Lipid Metabolism Laboratory and Lipid Clinic, São Paulo, Brazil; 2 The Heart Institute (InCor), São Paulo, Brazil; 3 Faculty of Pharmaceutical Sciences, São Paulo, Brazil; 4 University of São Paulo, Medical School Hospital, São Paulo, Brazil
PO2-52
AMINO ACIDS 140-148 OF APOAV PLAY A CRUCIAL ROLE IN LPL ACTIVATION
B. Dorfmeister 1 , J. Heeren 2 , M. Merkel 3 , P.J. Talmud 1 , S.E. Humphries 1 . 1 Centre for Cardiovascular Genetics, Department of Medicine, University College London, London, UK; 2 IBM II, Molecular Cell Biology, University Hospital Hamburg-Eppendorf, Hamburg, Germany; 3 Department of Internal Medicine I, University Hospital Hamburg, Eppendorf, Hamburg, Germany Background: Animal models and human association studies have identified apolipoprotein AV (apoAV) as a key player in triglyceride (TG) metabolism. Two premature truncations in APOA5, Q148X and Q139X, associated with severe hypertriglyceridemia (HTG) have been reported. Post-heparin plasma from the Q148X homozygous patient had 40% residual LPL activity. Q139X heterozygous patients showed a dramatic reduction in LPL activity by 79%. Aim: To test the effects on LPL activation in vitro of X139, X148 and a deletion construct ( 140-148). Methods: ApoAV wildtype, X139, X148 and 140-148 were expressed in a pET20b vector and purified by affinity chromatography and FPLC. Bovine LPL was immobilized onto HSPG coated plates and then exposed to the TG-rich lipoproteins, which were prior incubated with the apoAV proteins. LPL activity was measured by FFA release. Results: Preliminary data showed that compared to WT (0.70 ± 0.00 mmol/l), Q148X (0.59 ± 0.01 mmol/l) and Q139X (0.56 ± 0.01 mmol/l) had an impaired stimulatory effect on LPL activity, while the 140-148 (0.42 ± 0.02 mmol/l) mutant did not stimulate LPL activity.
ments with VLDL isolated from apoa5 -/- mice are underway to confirm and extend the current findings. PO2-53
APOLIPOPROTEIN J (CLUSTERIN) BINDS TO AND REDUCES CYTOTOXICITY OF ENZYMATICALLY HYDROLYZED LOW DENSITY LIPOPROTEIN
M. Schwarz 1 , L. Spath 2 , K. Paprotka 2 , M. Torzewski 3 , K. Dersch 2 , C. Koch-Brandt 1 , M. Husmann 2 , S. Bhakdi 2 . 1 Institute of Biochemistry, Johannes Gutenberg-University, Mainz, Germany; 2 Institute of Medical Microbiology and Hygiene, Johannes Gutenberg-University, Mainz, Germany; 3 Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg-University, Mainz, Germany Following entrapment in the arterial intima, low density lipoprotein can be modified by hydrolytic enzymes to yield a lipoprotein derivative that binds C-reactive protein, activates complement, and is rapidly taken up by monocytes/macrophages. Free fatty acids contained in enzymatically modified LDL (E-LDL) render the lipoprotein cytotoxic due to their capacity to trigger programmed cell death. Apolipoprotein J alias clusterin, a multifunctional protein that has the propensity to interact with diverse substrates, is present in the intima and the media of arteries with atherosclerotic lesions, and is also synthesized by smooth muscle cells during development of atherosclerosis. We report that Apo J binds to E-LDL but not to native LDL. Binding resulted in marked reduction of cytotoxicity of E-LDL on smooth muscle cells, as revealed by determination of caspase activity, annexin binding, and cellular ATP. In atherosclerotic lesions, Apo J may thus subserve protective functions through its capacity to inactivate C5b-9 complement complexes and by reducing the cytotoxic effects of modified LDL on smooth muscle cells that gain contact with the lipoprotein. PO2-54
A CORRELATION BETWEEN HYPERLIPIDAEMIA AND TESTICULAR FUNCTION IN ALBINO RATS
A. Purohit. Department of Zoology, Jai Narain Vyas University, Jodhpur, India A considerable work has been done on hyperlipidaemia in relation to body metabolism and function status in animal kingdom but the relationship between hyperlipidaemia and testicular function remained unexplored. The present work deal with effect of hyperlipidaemia on testicular function in albino rats. Hyperlipidaemia was induced by cholesterol feeding at the dose 400mg, 800 mg and 1000 mg/kg body wt. to male albino rats altered the testicular cell population dynamics. Cholesterol treatment resulted in severe degeneration in spermatogonia, primary and secondary spermatocytes and spermatids showing 28.92, 31.24, 71.97 and 93.69% reduction respectively with 400 mg group;43.01, 58.85,53.52,75.25% reduction respectively with 800 mg group and 83.88,91.77, 94.88 and 99.09% reduction respectively with 1000 mg group, as compared to control group. The number of interstitial cell types i.e., fibroblasts, immature and mature Leydig cells also showed significant reduction in all the treated groups. While there was a significant increase in the percent degenerating Leydig cells in all the cholesterol treated groups showing 73.8, 79.15 and 82.85 percent respectively. The result indicate that cholesterol diet may have significant effect on the intratesticular androgen levels either by inhibiting Leydig cell function or by inhibiting hypothalamo-pituitary axis. PO2-55
APOE/LDL RECEPTOR-DOUBLE-KNOCKOUT MICE: AN ANIMAL MODEL FOR THE STUDY OF STATINS EFFECTS IN ATHEROGENESIS
N. Pospisilova 1 , P. Nachtigal 1 , G. Jamborova 1 , K. Pospechova 1 , D. Solichova 2 , C. Andrys 3 , P. Zdansky 2 , V. Semecky 1 . 1 Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles University, Hradec Kralove, Czech Republic; 2 Department of Metabolic Care and Gerontology, Charles University Medical School and Teaching Hospital, Hradec Kralove, Czech Republic; 3 Institute of Clinical Immunology and Allergology, Charles University Medical School and Teaching Hospital, Hradec Kralove, Czech Republic Fig. 1. VLDL hydrolysis with recombinant apoAV by HSPG-bound LPL in vitro.
Conclusion: These preliminary data suggest that amino acids 140-148 could play a crucial role in apoAV/HSPG/LPL interaction. Further experi-
The statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) represent golden standard in dyslipidemia treatment in humans. However, its effects in animal models of atherosclerosis are very inconsistent. Thus, the aim of this study was to evaluate whether atorvastatin has hypolipidemic and inflammatory effects in apoE/LDLR deficient mice.
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
POSTER SESSIONS
Type 2 diabetes is usually accompanied by fasting hypertrigliceridemia and post-prandial hyperlipidemia. Chylomicron metabolism is constituted by lipolysis by lipoprotein lipase on the endothelium and removal of the resulting remnants by the liver and delay in those processes is associated with coronary artery disease (CAD) and worse evolution of CAD patients. Chylomicron metabolism has not been evaluated in diabetes patients with normal plasma lipids and this study aims to clarify this issue. The chylomicron-like emulsion method was used. The emulsion labeled with 3 H-triolein (TO) and 14C-cholesteryl olate (CO) was injected I.V. in a bolus into 12 patients diabetes and 10 controls without the disease. Both groups had total cholesterol <200, LDL-c < 130 and triglycerides < 200 mg/dl. The plasma kinetics of the labels was determined from blood samples taken over 60 min. Fractional clearance rates (FCR) were calculated by compartmental analysis. TO FCR estimates lipolysis while CO FCR estimates the remnant removal. In diabetes patients CO-FCR was diminished compared to controls (0.0053±0.032 and 0.0316±0.0061 min-1 , respectively, p=0.009) while TO-FCR was not different (0.0322±0.0182 and 0.0316 min-1 , p=0.94). In conclusion, despite normal plasma lipids, diabetes patients show decreased removal of chylomicron remnants, a feature associated with CAD. Because hypolipidemic drugs such as statins and fibrates can accelerate remnant removal, this finding has implications for the treatment of the disease.
31