- Email: [email protected]
Plasma phospholipid transfer protein activity, high density lipoprotein subfractions and atherogenesis: Studies in mice and man
84
Friday, 28 May 1999 Workshop: Enz3,mes and transfer proteins inuolued in intrauascular modeling of lipoproteins
Workshop: Enzymes and transfer proteins involved in intravascular modeling of lipoproteins
the local environment of the vessel wall lipoprotein lipase might aggravate the atherosclerotic process by stimulating foam cell formation.
THE ROLE OF PHOSPHOLIPID TRANSFER PROTEIN IN LIPOPROTEIN METABOLISM C. Ehnholm. National Public Health Institute. Department of Biochemistr3; Helsinki, Finland Epidemiological studies have provided strong evidence for an inverse relationship between plasma high density lipoprotein (HDL) levels and coronary heart disease (CHD). The protective role of HDL against cardiovascular disease is commonly attributed to its ability to remove excess cholesterol from cells in the arterial wall and transport it to the liver for disposal, a process called reverse cholesterol transport. HDL in human plasma is heterogenous and consists of several distinct subpopulations of particles that differ in their composition, size and function. The initial acceptor of cell-derived cholesterol in the reverse cholesterol transport process is a minor subpopulation of HDL that contains apoA-I as its sole apoprotein and has prel~-electrophoretic mobility. In vitro and in vivo evidence indicates that PLTP, in additional to its function in phospholipid transfer, plays an important role in the remodelling of lipoproteins and the formation of pre~-HDL. PLTP facilitates conversion ofHDL a process whereby HDL particles with increased size and prel~-HDL are found. PLTP-mediated HDL conversion is regulated by the composition of HDL. ApoA-II is inhibitory whereas elevated levels of core triglycerides increase HDL conversion. Pre0-HDL particles produced by the function of PLTP can function as primary acceptors o f cell membrane cholesterol. The physiological function of PLTP is not well known, although in vitro studies and findings in genetically manipulated animals suggest a central role for PLTP in HDL metabolism. Adenovirus mediated overexpression of PLTP causes a dramatic decrease of plasma lipid levels. Wild type mice overexpressing human PLTP showed an almost complete absence of plasma cholesterol and triglycerides while human apoAI transgenic mice overexpressing PLTP had also a clear decrease in plasma cholesterol, apoA-I and triglycerides. Even though these studies do not give conclusive answers to the question of PLTP function in vivo, they indicate that PLTP is closely connected to the metabolism of HDL. LIPOPROTEIN LIPASE AS A LIGAND FOR BINDING TO CELL SURFACES AND RECEPTORS. GOOD OR BAD?
G. Olivecrona,E. Makoveichuk,A. Lookene,S.-Q. Xiang. Dept. of Medical Biochemistry and Biophysics, limed University. SE-901 87 Umed, Sweden Lipoprotein lipase has high affinity for heparan sulfate proteoglycans, as well as for lipoproteins and for lipoprotein receptors. Therefore the enzyme is able to bridge between lipoproteins and cell surfaces. We have studied this property by use of the biosensor technique (BIAcore0 with immobilized proteoglycans to which binding of lipoprotein lipase and lipoproteins can be studied in real time. Dissociation constants in the nM range were determined between dimeric lipoprotein lipase and heparan sulfate proteoglycans. The binding affinity of inactive lipase monomers was more than thousand-fold lower. Chylomicrons, VLDL and LDL bound with high affinity to surfaces covered by heparan sulfate and lipoprotein lipase, but HDL did not bind at all. Mildly oxidized LDL bound more efficiently than did non-oxidized LDL. We used THP-I monocytes and macrophages to study binding and uptake o f normal and modified LDL mediated by lipoprotein lipase. Binding of LDL in the absence of the lipase was low and occurred rather slow, while binding in the presence of lipoprotein lipase occurred rapidly and was 20100 fold higher than in the absence of lipase. Uptake in the cells was also much increased by the presence of lipoprotein lipase in both monocytes and stimulated macrophages leading to lipid accumulation within the cells. The bridging function of the lipase was dependent on the presence of heparan sulfate proteoglycans on the cell surfaces, but it was not inhibited by receptor associated protein (RAP), indicating that it was not dependent on specific lipoprotein receptors. The effect of lipoprotein lipase was not dependent on catalytic activity of the lipase, but was dependent on the dimeric structure of the enzyme molecule. Internalization was not inhibited by eytochalasine, indicating that it did not occur mainly through phagocytosis of aggregated lipoproteins. Lipase-mediated binding/uptake was not prevented by chemical modification of LDL (by oxidation, cyclohexanedione treatment or acetylation). We conclude that binding/uptake of LDL by monocytes and macrophagas is much stimulated by the presence of lipoprotein lipase and that the binding is not likely to occur by specific protein/protein interaction but rather directly to the lipid constituents of the lipoprotein. In
PLASMA PHOSPHOLIPID TRANSFER PROTEIN ACTIVITY, HIGH DENSITY LIPOPROTEIN SUBFRACTIONS AND ATHEROGENESIS: STUDIES IN MICE AND MAN A. van Tol I , M.J. van Haperen 2, ES. VermeulenI , M. Jauhiainen 3, "i2 van Gent 1. P. van den Berg 1, L.M. Scheek 1, EG. Grosveld 2, A.W.M. van der Kamp 2, R. de Crom 2, S.C. Riemens4, W.J. Sluiter4, R.EE Dullaart a.
t Dept. Biochemistry, Cardiouascular Research Institute COEUR, Erasmus University Rotterdam: ?MGC-Dept. Cell Biology & Genetics, Rotterdam: 4Dept. EndocrinoloKv, Academic Hospital Groningen. The Netherlands: JDept. Biochemistry, National Public Health Institute, Helsinki. Finland Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters and triglycerides (TG) between high density lipoproteins (HDL) and very low + low density lipoproteins (VLDL + LDL). In addition, CETP is able to transfer phospholipids. Plasma phospholipid transfer protein (PLTP) also transfers phospholipids between lipoprotein particles. Both lipid transfer proteins are part of a protein family, together with bactericidal/permeabilityincreasing protein (BPI). A very interesting observation is that PLTP promotes HDL conversion, resulting in larger and smaller particles, e.g. in the generation of prel~,-HDL. We studied HDL conversion using plasma from transgenic mice that overexpress human PLTP. High PLTP activity results in decreased plasma HDL levels and increased potential to generate pre['LHDL. It was also found that plasma from transgenic mice prevents cholesterol accumulation in macrophages. We concluded from these animal studies that PLTP may act as an anti-atherogenic factor. The physiological function of PLTP in man is largely unknown. Plasma PLTP activity levels correlate with plasma triglycerides (TG) and are downregulated by insulin, together with the TG-rich lipoproteins (VLDL). Lately we examined the effects of 24 h hyperinsulinemia (30 mU/kg/h) and 24 h Acipimox (250 mg/4 h) on plasma lipids as well as PLTP activities (measured with exogenous substrates) in 8 healthy controls and in 8 type 2 diabetic subjects. Both treatments decreased free fatty acids (FFA), HDL-cholesterol and plasma apo A-I levels. The decrease in plasma TG was significant after Acipimox. Insulin decreased plasma PLTP activity by 17.6% at 24 h in healthy subjects and by 10.2% in diabetic patients (p < 0.05 from baseline, p < 0.05 from healthy subjects). When insulin was infused for 3 h after Acipimox, a further decrease was seen only in healthy subjects. These findings support the hypothesis that there is a metabolic link between the regulation of plasma PLTP and FFA. It is concluded that the PLTP response to insulin is blunted in type 2 diabetes mellitus. These effects on PLTP may have consequences for HDL metabolism and reverse cholesterol transport.
ROLE OF HEPATIC LIPASE IN LIPOPROTEIN METABOLISM AND ATHEROSCLEROSIS S. Santamarina-Fojo, M. Amar, C. Handenschild, K. Dugi, H.B. Brewer, Jr.. Molecular Disease Branch, NHLBL NIH, Bethesda. MD, USA We have investigated the role that hepatic lipase (HL) plays in remnant lipoprotein and HDL metabolism as well as in the development ofatherosclerosis in oioo in different transgene and knockout mouse models. Adenovirusmediated expression o f either native or catalytically inactive HL in apoEdeficient and HL-deficient mice resulted in significant reductions in the baseline plasma lipids establishing the importance of the role of HL in both remnant lipoprotein and HDL metabolism independent of lipolysis. Catalytic activity was important for part of the effects of ilL on HDL but not remnant lipoprotein metabolism. Expression of HL mutated in the proposed LRP binding site (HL-433Q) demonstrated the importance of this residue for remnant but not HDL metabolism in oioo. Metabolic studies performed after virus infusion utilizing [1251] apoB-VLDL and [3H]cholesteryl etherVLDL in apoE-def mice and [1251]apoA-I HDL, [131 l]apoA-ll HDL and [3H]cholesteryl ether-HDL in HL-deficient mice established a role for HL in mediating the selective uptake of cholesterol from remnant lipoproteins. In oitro 3H-CE HDL uptake studies confirmed that part of the HL effect is mediated by SR-BI. These studies provide in vioo evidence for a role of the ligand-binding function o f HL in the metabolism of both HDL and apoB-containing lipoproteins.
71st EAS Congress and Satellite Symposia
Friday, 28 May 1999 Workshop: Enz3,mes and transfer proteins inuolued in intrauascular modeling of lipoproteins
Workshop: Enzymes and transfer proteins involved in intravascular modeling of lipoproteins
the local environment of the vessel wall lipoprotein lipase might aggravate the atherosclerotic process by stimulating foam cell formation.
THE ROLE OF PHOSPHOLIPID TRANSFER PROTEIN IN LIPOPROTEIN METABOLISM C. Ehnholm. National Public Health Institute. Department of Biochemistr3; Helsinki, Finland Epidemiological studies have provided strong evidence for an inverse relationship between plasma high density lipoprotein (HDL) levels and coronary heart disease (CHD). The protective role of HDL against cardiovascular disease is commonly attributed to its ability to remove excess cholesterol from cells in the arterial wall and transport it to the liver for disposal, a process called reverse cholesterol transport. HDL in human plasma is heterogenous and consists of several distinct subpopulations of particles that differ in their composition, size and function. The initial acceptor of cell-derived cholesterol in the reverse cholesterol transport process is a minor subpopulation of HDL that contains apoA-I as its sole apoprotein and has prel~-electrophoretic mobility. In vitro and in vivo evidence indicates that PLTP, in additional to its function in phospholipid transfer, plays an important role in the remodelling of lipoproteins and the formation of pre~-HDL. PLTP facilitates conversion ofHDL a process whereby HDL particles with increased size and prel~-HDL are found. PLTP-mediated HDL conversion is regulated by the composition of HDL. ApoA-II is inhibitory whereas elevated levels of core triglycerides increase HDL conversion. Pre0-HDL particles produced by the function of PLTP can function as primary acceptors o f cell membrane cholesterol. The physiological function of PLTP is not well known, although in vitro studies and findings in genetically manipulated animals suggest a central role for PLTP in HDL metabolism. Adenovirus mediated overexpression of PLTP causes a dramatic decrease of plasma lipid levels. Wild type mice overexpressing human PLTP showed an almost complete absence of plasma cholesterol and triglycerides while human apoAI transgenic mice overexpressing PLTP had also a clear decrease in plasma cholesterol, apoA-I and triglycerides. Even though these studies do not give conclusive answers to the question of PLTP function in vivo, they indicate that PLTP is closely connected to the metabolism of HDL. LIPOPROTEIN LIPASE AS A LIGAND FOR BINDING TO CELL SURFACES AND RECEPTORS. GOOD OR BAD?
G. Olivecrona,E. Makoveichuk,A. Lookene,S.-Q. Xiang. Dept. of Medical Biochemistry and Biophysics, limed University. SE-901 87 Umed, Sweden Lipoprotein lipase has high affinity for heparan sulfate proteoglycans, as well as for lipoproteins and for lipoprotein receptors. Therefore the enzyme is able to bridge between lipoproteins and cell surfaces. We have studied this property by use of the biosensor technique (BIAcore0 with immobilized proteoglycans to which binding of lipoprotein lipase and lipoproteins can be studied in real time. Dissociation constants in the nM range were determined between dimeric lipoprotein lipase and heparan sulfate proteoglycans. The binding affinity of inactive lipase monomers was more than thousand-fold lower. Chylomicrons, VLDL and LDL bound with high affinity to surfaces covered by heparan sulfate and lipoprotein lipase, but HDL did not bind at all. Mildly oxidized LDL bound more efficiently than did non-oxidized LDL. We used THP-I monocytes and macrophages to study binding and uptake o f normal and modified LDL mediated by lipoprotein lipase. Binding of LDL in the absence of the lipase was low and occurred rather slow, while binding in the presence of lipoprotein lipase occurred rapidly and was 20100 fold higher than in the absence of lipase. Uptake in the cells was also much increased by the presence of lipoprotein lipase in both monocytes and stimulated macrophages leading to lipid accumulation within the cells. The bridging function of the lipase was dependent on the presence of heparan sulfate proteoglycans on the cell surfaces, but it was not inhibited by receptor associated protein (RAP), indicating that it was not dependent on specific lipoprotein receptors. The effect of lipoprotein lipase was not dependent on catalytic activity of the lipase, but was dependent on the dimeric structure of the enzyme molecule. Internalization was not inhibited by eytochalasine, indicating that it did not occur mainly through phagocytosis of aggregated lipoproteins. Lipase-mediated binding/uptake was not prevented by chemical modification of LDL (by oxidation, cyclohexanedione treatment or acetylation). We conclude that binding/uptake of LDL by monocytes and macrophagas is much stimulated by the presence of lipoprotein lipase and that the binding is not likely to occur by specific protein/protein interaction but rather directly to the lipid constituents of the lipoprotein. In
PLASMA PHOSPHOLIPID TRANSFER PROTEIN ACTIVITY, HIGH DENSITY LIPOPROTEIN SUBFRACTIONS AND ATHEROGENESIS: STUDIES IN MICE AND MAN A. van Tol I , M.J. van Haperen 2, ES. VermeulenI , M. Jauhiainen 3, "i2 van Gent 1. P. van den Berg 1, L.M. Scheek 1, EG. Grosveld 2, A.W.M. van der Kamp 2, R. de Crom 2, S.C. Riemens4, W.J. Sluiter4, R.EE Dullaart a.
t Dept. Biochemistry, Cardiouascular Research Institute COEUR, Erasmus University Rotterdam: ?MGC-Dept. Cell Biology & Genetics, Rotterdam: 4Dept. EndocrinoloKv, Academic Hospital Groningen. The Netherlands: JDept. Biochemistry, National Public Health Institute, Helsinki. Finland Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters and triglycerides (TG) between high density lipoproteins (HDL) and very low + low density lipoproteins (VLDL + LDL). In addition, CETP is able to transfer phospholipids. Plasma phospholipid transfer protein (PLTP) also transfers phospholipids between lipoprotein particles. Both lipid transfer proteins are part of a protein family, together with bactericidal/permeabilityincreasing protein (BPI). A very interesting observation is that PLTP promotes HDL conversion, resulting in larger and smaller particles, e.g. in the generation of prel~,-HDL. We studied HDL conversion using plasma from transgenic mice that overexpress human PLTP. High PLTP activity results in decreased plasma HDL levels and increased potential to generate pre['LHDL. It was also found that plasma from transgenic mice prevents cholesterol accumulation in macrophages. We concluded from these animal studies that PLTP may act as an anti-atherogenic factor. The physiological function of PLTP in man is largely unknown. Plasma PLTP activity levels correlate with plasma triglycerides (TG) and are downregulated by insulin, together with the TG-rich lipoproteins (VLDL). Lately we examined the effects of 24 h hyperinsulinemia (30 mU/kg/h) and 24 h Acipimox (250 mg/4 h) on plasma lipids as well as PLTP activities (measured with exogenous substrates) in 8 healthy controls and in 8 type 2 diabetic subjects. Both treatments decreased free fatty acids (FFA), HDL-cholesterol and plasma apo A-I levels. The decrease in plasma TG was significant after Acipimox. Insulin decreased plasma PLTP activity by 17.6% at 24 h in healthy subjects and by 10.2% in diabetic patients (p < 0.05 from baseline, p < 0.05 from healthy subjects). When insulin was infused for 3 h after Acipimox, a further decrease was seen only in healthy subjects. These findings support the hypothesis that there is a metabolic link between the regulation of plasma PLTP and FFA. It is concluded that the PLTP response to insulin is blunted in type 2 diabetes mellitus. These effects on PLTP may have consequences for HDL metabolism and reverse cholesterol transport.
ROLE OF HEPATIC LIPASE IN LIPOPROTEIN METABOLISM AND ATHEROSCLEROSIS S. Santamarina-Fojo, M. Amar, C. Handenschild, K. Dugi, H.B. Brewer, Jr.. Molecular Disease Branch, NHLBL NIH, Bethesda. MD, USA We have investigated the role that hepatic lipase (HL) plays in remnant lipoprotein and HDL metabolism as well as in the development ofatherosclerosis in oioo in different transgene and knockout mouse models. Adenovirusmediated expression o f either native or catalytically inactive HL in apoEdeficient and HL-deficient mice resulted in significant reductions in the baseline plasma lipids establishing the importance of the role of HL in both remnant lipoprotein and HDL metabolism independent of lipolysis. Catalytic activity was important for part of the effects of ilL on HDL but not remnant lipoprotein metabolism. Expression of HL mutated in the proposed LRP binding site (HL-433Q) demonstrated the importance of this residue for remnant but not HDL metabolism in oioo. Metabolic studies performed after virus infusion utilizing [1251] apoB-VLDL and [3H]cholesteryl etherVLDL in apoE-def mice and [1251]apoA-I HDL, [131 l]apoA-ll HDL and [3H]cholesteryl ether-HDL in HL-deficient mice established a role for HL in mediating the selective uptake of cholesterol from remnant lipoproteins. In oitro 3H-CE HDL uptake studies confirmed that part of the HL effect is mediated by SR-BI. These studies provide in vioo evidence for a role of the ligand-binding function o f HL in the metabolism of both HDL and apoB-containing lipoproteins.
71st EAS Congress and Satellite Symposia