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Regulation of apoprotein E secretion
82
Monday 10 October 1994: Workshop Abstracts W3 Remnant metabolism
regulator of LRP activity, in the livers of normal and LDLR-/mice using adenoviral gene transfer. In the LDLR-/- animals, RAP overexpression resulted in massive accumulation of chylomicron remnants. Wild-type mice were largely protected from this RAP-induced hyperlipemia. These findings suggest that both LDL receptor and LRP participate in the endocytic uptake of remnants into the hepatocytes. Role of lipases and LDL receptor-related protein in human chylomicron remnant catabolism .. Belsleeel Weber W, Krapp A, Meyer N, Olivecrona G, Gliemann J, Mldical Clinic, Univ. Hosp, Hamburg, Martinistr. 52,
reduced and vesicular staining was observed in the cytoplasm, suggesting that the cell-surface apo E was used for hepatic uptake of chylomicrons and remnants. These results provide strong evidence for the secretion-recapture process of apo E whereby chylomicron remnants enter the sinusoidal space, acquire apo E molecules, and subsequently are endocytosed. Data from experiments with very low density lipoprotein and LDL showed that this system is specific for chylomicron remnants Regulation of apoprotein E secretion Getz GS, Ye S, Thurberg B, Reardon-Alulis C, Dept. of Pathol., Univ. of Chicago, USA
20251 Hamburg, Germany
The LDL receptor-related protein (LRP) has been proposed as the chylomicron remnant receptor and to mediate the catabolism of these lipoproteins. LRP also functions as receptor for protease/protease inhibitor complexes such as a-Zmacroglobulin and therefore must be considered a multifunctional protein. As ligands in lipoprotein catabolism, apolipoprotein E and lipoprotein lipase (LPL) have been shown to bind to the LRP [1,2]. LPL can travel with the remnant particle after its hydrolysis at the endothelium and function as a recognition site for the receptor. Structural studies revealed that the C-terminus of the LPL contains the binding site for LRP [3]. Since this nonenzymatic role of LPL in remnant catabolism is new, we analyzed the structure of human chylomicron and chylomicron remnants, particular in respect to lipase content. As there is high sequence homology between LPL and hepatic lipase (HL) and because of the hepatic origin and function of HL, we considered a role for HL in remnant catabolism. HL was produced in HuH7 cells and isolated by hepaxin columns, or isolated from human postheparin plasma. In studies of the binding of radiolabelled human chylomicrons and rabbit pVLDL to several cell lines (hepatoma cells, fibroblasts and CHO cells) we showed that HL, like LPL, increases the binding and uptake of lipoproteins to cells. This binding of HL to the cell surface might be in part due to interaction with proteoglycans. However, crosslinking studies revealed that HL can bind directly to LRP. Currently we are analyzing the binding site for LRP in HL analogous to the C-terminal binding site in LPL. We conclude that HL might be next to apo E and LPL in importance for the recognition of chylomicron remnants by hepatocytes via LRP. [l] Beisiegel et al. Nature 1989; 341: 162-164. [2] Beisiegel et al. Proc Nat1 Acad Sci USA 1991; 88: 8342-8346. [3] Nykjaer et al. J Biol Chem 1994, submitted. Role of apo E on the surface of hepatocytes in chylomicron remnant catabolism wN, Shimano H, 3rd Dept. of Int. Med., Faculty of Med., Univ. Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo, Japan, 113
Apolipoprotein E (apo E) plays a crucial role in the metabolism of plasma lipoproteins, mainly through its interaction with lipoprotein receptors in the liver. Chylomicrons synthesized by the intestine enter the mesenteric and thoracic duct lymph, where they acquire apo E. After hydrolysis of triglycerides in chylomicrons by lipoprotein lipase, the chylomicrons become smaller and more enriched in cholesterol and are referred to as chylomicron remnants. Chylomicron remnants are rapidly cleared from the plasma by the liver, and apo E functions as a specific ligand for a putative hepatic chylomicron remnant receptor. To investigate the role of apo E in hepatic uptake of chylomicron remnants, we studied chylomicron metabolism in transgenic mice overexpressing apo E in the liver. Immunohistochemistry demonstrated that apo E was specifically localized at the basolateral surface of hepatocytes of fasted transgenic mice. After injection of a large amount of chylomicrons, the density of the cell-surface apo E was markedly
When intestinal lipoprotein particles (chylomicrons) are first released from the intestinal enterocytes they do not contain their complement of apoprotein E, which is acquired during the metabolism and circulation of these particles. It is not clear that this apoprotein E is acquired from the pool of circulating apoprotein. Rather it may be possible that remnants become enriched with apoprotein E as they are concentrated on or near the hepatocyte surface. We have developed biochemical evidence for a pool of cell surface apoprotein, which can be released at 4°C by a variety of agents. Hepatoma cells, either HepG2 or McA-RH 7777, were pulse-labeled with [35S]methionine at 37°C after which cells were shifted to 4°C and washed thoroughly. Apoprotein E is releasable from such cells at low temperature by LDL (a lipoprotein indicator), phospholipid vesicles or heparin. Two cell-surface pools are suggested by the fact that the amount of apoprotein E released by phospholipid vesicles and heparin is additive over what is released by each alone. These pools may be lipid membraneassociated and proteoglycan-bound respectively, with the apoprotein E thus available to refashion the surface of lipoprotein particles, including remnants. Such behavior is observed with other apoproteins such as apoprotein A-I, apoprotein C-III, chimeric molecules containing portions of apoprotein A-I and apoprotein E, and several apoprotein E mutants, but not with albumin produced in hepatoma cells. Further studies will define the nature of the apoprotein association with cell surface. Remnants and coronary artery disease progression Karpe F, Steiner G, Uffelman K, Olivecrona T, Hamsten A, King Gustaf V Research Inst., Dept. of Med., Karolinska Hosp.. Stockholm, Sweden (FK, AH), Dept. of Med. Chem. and Biophysics, Universig of Umed, Umed, Sweden (TO), Division of Endocrinology and Metabolism, Toronto General Hospital, Toronto, Ontario, Canada (GS, KU)
The relations between triglyceride-rich lipoproteins, alimentary lipemia and coronary heart disease (CHD) have remained obscure and much debated. We studied the basal and postprandial plasma levels of chylomicron remnants and very low density lipoproteins (VLDL) of varying particle size in 32 male postinfarction patients (mean (SD) age 48.8 (3.2) years) and in 10 age-matched control men. Postprandial intestinal and hepatic lipoproteins were selectively quantified by determining apolipoproteins B-48 and B-100 in lipoprotein subfractions of Sf rates >12 before and 3, 6 and 12 h after an oral fat load. Since all patients had undergone two coronary angiographies with an intervening time interval of around 5 years, lipoprotein fractions were examined in relation to the global severity as well as the rate of progression of coronary lesions. The postprandial plasma levels of small chylomicron remnants (Sf 20-60, apo B-48) were found to correlate with the rate of progression of coronary lesions between the angiographies (r= 0.51, p = 0.01). Adjustment for the possible confounding effect of HDL-cholesterol and dense LDL apo B concentrations did not substantially alter the strength of this association. Neither the increment of plasma triglyceride during the postprandial period nor the concentrations of other lipoprotein fractions closely
Atherosclerosis X, Montreal, October 1994
Monday 10 October 1994: Workshop Abstracts W3 Remnant metabolism
regulator of LRP activity, in the livers of normal and LDLR-/mice using adenoviral gene transfer. In the LDLR-/- animals, RAP overexpression resulted in massive accumulation of chylomicron remnants. Wild-type mice were largely protected from this RAP-induced hyperlipemia. These findings suggest that both LDL receptor and LRP participate in the endocytic uptake of remnants into the hepatocytes. Role of lipases and LDL receptor-related protein in human chylomicron remnant catabolism .. Belsleeel Weber W, Krapp A, Meyer N, Olivecrona G, Gliemann J, Mldical Clinic, Univ. Hosp, Hamburg, Martinistr. 52,
reduced and vesicular staining was observed in the cytoplasm, suggesting that the cell-surface apo E was used for hepatic uptake of chylomicrons and remnants. These results provide strong evidence for the secretion-recapture process of apo E whereby chylomicron remnants enter the sinusoidal space, acquire apo E molecules, and subsequently are endocytosed. Data from experiments with very low density lipoprotein and LDL showed that this system is specific for chylomicron remnants Regulation of apoprotein E secretion Getz GS, Ye S, Thurberg B, Reardon-Alulis C, Dept. of Pathol., Univ. of Chicago, USA
20251 Hamburg, Germany
The LDL receptor-related protein (LRP) has been proposed as the chylomicron remnant receptor and to mediate the catabolism of these lipoproteins. LRP also functions as receptor for protease/protease inhibitor complexes such as a-Zmacroglobulin and therefore must be considered a multifunctional protein. As ligands in lipoprotein catabolism, apolipoprotein E and lipoprotein lipase (LPL) have been shown to bind to the LRP [1,2]. LPL can travel with the remnant particle after its hydrolysis at the endothelium and function as a recognition site for the receptor. Structural studies revealed that the C-terminus of the LPL contains the binding site for LRP [3]. Since this nonenzymatic role of LPL in remnant catabolism is new, we analyzed the structure of human chylomicron and chylomicron remnants, particular in respect to lipase content. As there is high sequence homology between LPL and hepatic lipase (HL) and because of the hepatic origin and function of HL, we considered a role for HL in remnant catabolism. HL was produced in HuH7 cells and isolated by hepaxin columns, or isolated from human postheparin plasma. In studies of the binding of radiolabelled human chylomicrons and rabbit pVLDL to several cell lines (hepatoma cells, fibroblasts and CHO cells) we showed that HL, like LPL, increases the binding and uptake of lipoproteins to cells. This binding of HL to the cell surface might be in part due to interaction with proteoglycans. However, crosslinking studies revealed that HL can bind directly to LRP. Currently we are analyzing the binding site for LRP in HL analogous to the C-terminal binding site in LPL. We conclude that HL might be next to apo E and LPL in importance for the recognition of chylomicron remnants by hepatocytes via LRP. [l] Beisiegel et al. Nature 1989; 341: 162-164. [2] Beisiegel et al. Proc Nat1 Acad Sci USA 1991; 88: 8342-8346. [3] Nykjaer et al. J Biol Chem 1994, submitted. Role of apo E on the surface of hepatocytes in chylomicron remnant catabolism wN, Shimano H, 3rd Dept. of Int. Med., Faculty of Med., Univ. Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo, Japan, 113
Apolipoprotein E (apo E) plays a crucial role in the metabolism of plasma lipoproteins, mainly through its interaction with lipoprotein receptors in the liver. Chylomicrons synthesized by the intestine enter the mesenteric and thoracic duct lymph, where they acquire apo E. After hydrolysis of triglycerides in chylomicrons by lipoprotein lipase, the chylomicrons become smaller and more enriched in cholesterol and are referred to as chylomicron remnants. Chylomicron remnants are rapidly cleared from the plasma by the liver, and apo E functions as a specific ligand for a putative hepatic chylomicron remnant receptor. To investigate the role of apo E in hepatic uptake of chylomicron remnants, we studied chylomicron metabolism in transgenic mice overexpressing apo E in the liver. Immunohistochemistry demonstrated that apo E was specifically localized at the basolateral surface of hepatocytes of fasted transgenic mice. After injection of a large amount of chylomicrons, the density of the cell-surface apo E was markedly
When intestinal lipoprotein particles (chylomicrons) are first released from the intestinal enterocytes they do not contain their complement of apoprotein E, which is acquired during the metabolism and circulation of these particles. It is not clear that this apoprotein E is acquired from the pool of circulating apoprotein. Rather it may be possible that remnants become enriched with apoprotein E as they are concentrated on or near the hepatocyte surface. We have developed biochemical evidence for a pool of cell surface apoprotein, which can be released at 4°C by a variety of agents. Hepatoma cells, either HepG2 or McA-RH 7777, were pulse-labeled with [35S]methionine at 37°C after which cells were shifted to 4°C and washed thoroughly. Apoprotein E is releasable from such cells at low temperature by LDL (a lipoprotein indicator), phospholipid vesicles or heparin. Two cell-surface pools are suggested by the fact that the amount of apoprotein E released by phospholipid vesicles and heparin is additive over what is released by each alone. These pools may be lipid membraneassociated and proteoglycan-bound respectively, with the apoprotein E thus available to refashion the surface of lipoprotein particles, including remnants. Such behavior is observed with other apoproteins such as apoprotein A-I, apoprotein C-III, chimeric molecules containing portions of apoprotein A-I and apoprotein E, and several apoprotein E mutants, but not with albumin produced in hepatoma cells. Further studies will define the nature of the apoprotein association with cell surface. Remnants and coronary artery disease progression Karpe F, Steiner G, Uffelman K, Olivecrona T, Hamsten A, King Gustaf V Research Inst., Dept. of Med., Karolinska Hosp.. Stockholm, Sweden (FK, AH), Dept. of Med. Chem. and Biophysics, Universig of Umed, Umed, Sweden (TO), Division of Endocrinology and Metabolism, Toronto General Hospital, Toronto, Ontario, Canada (GS, KU)
The relations between triglyceride-rich lipoproteins, alimentary lipemia and coronary heart disease (CHD) have remained obscure and much debated. We studied the basal and postprandial plasma levels of chylomicron remnants and very low density lipoproteins (VLDL) of varying particle size in 32 male postinfarction patients (mean (SD) age 48.8 (3.2) years) and in 10 age-matched control men. Postprandial intestinal and hepatic lipoproteins were selectively quantified by determining apolipoproteins B-48 and B-100 in lipoprotein subfractions of Sf rates >12 before and 3, 6 and 12 h after an oral fat load. Since all patients had undergone two coronary angiographies with an intervening time interval of around 5 years, lipoprotein fractions were examined in relation to the global severity as well as the rate of progression of coronary lesions. The postprandial plasma levels of small chylomicron remnants (Sf 20-60, apo B-48) were found to correlate with the rate of progression of coronary lesions between the angiographies (r= 0.51, p = 0.01). Adjustment for the possible confounding effect of HDL-cholesterol and dense LDL apo B concentrations did not substantially alter the strength of this association. Neither the increment of plasma triglyceride during the postprandial period nor the concentrations of other lipoprotein fractions closely
Atherosclerosis X, Montreal, October 1994