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Endosomal and extraendosomal intracellular trafficking of lipoproteins
Tuesday I1 October: Workshop Abstracts WI0 Intracellular cholesterol transport
WlO INTRACELLULAR
CHOLESTEROL
Regulation of hepatie secretion of lipoproteins &lg&QI& Wu X, S&rata N, Dixon JL, Dept. ofMed., Colt. of Physicians and Surgeons, l&32, USA -
630 West 168th St., New York. NY
173
TRANSPORT
Endosomal and extraendosomal intracellular trafficking of lipoproteins JLkle, Rinninger F, Beisiegel U, Block A, Greten H, Windler E, Medizinische Klinik, Univ.-Krankenhaus Eppendot$ Martinistr. 52, D-20246 Hamburg, Germany
The secretion of apo B-containing lipoproteins from hepatocytes requires the assembly of newly synthesized apo B with core lipids, triglyceride and cholesterol, and surface lipids, particularly phospholipids. Regulation of secretion can, therefore, reside at several points. Regulation of apo B synthesis at the transcriptional level does not appear to play a major role in determining secretion of apo B-containing lipoproteins. On the other hand, posttranslational degradation of apo B in the endoplasmic reticulum (ER) has a significant role in regulating secretion In cultured hepatocytes, a significant proportion of newly synthesized apo B is rapidly degraded in the ER. Studies from several laboratories indicate that newly synthesized apo B associates with the ER membrane either cotranslationally or soon after translation. The availability of the core lipid triglyceride, and possibly cholesteryl ester, then determines if rapid degradation of nascent apo B occurs or if translocation of the protein is completed. A number of ER chaperone proteins am probably involved in this process. Oleic acid protects apo B from degradation, but this protection requires the synthesis of triglyceride. Microsomal transfer protein may play a critical role in delivery newly synthesized triglyceride to nascent apo B. Phospholipid availability appears to limit assembly of apo B-containing lipoproteins only in extreme circumstances. Apo B degradation seems to involve a cysteine-protease that is associated with the ER; little degradation occurs in the Golgi or the lysosomes. In HepG2 cells, limited triglyceride availability results in secretion of mostly low density lipoproteins; in rat hepatocytes, a ‘bolus’ of core lipids is added to the nascent apo B-containing lipoprotein, possibly in the smooth ER, resulting in secretion of typical very low density lipoproteins. Nascent VLDL assembly occurs by two steps in the endoplasmic reticulum (RR) of hepatocytes &J&&&, Erickson SK, Have1 RJ, Cardiovascular Research Inst. and Depts. of Anatomy and Med., UCSF Sch. of Med., San Francisco, CA 94143-0130 USA
Core lipidation of apo B in the ER is essential for assembly of nascent VLDL. To study this, we developed a procedure for isolating large amounts of ribosome-rich membranes from rat liver. Short strips of rough (R)ER predominate. in this fraction, enriched many-fold in RER markers and apo B. Markers for other organelles are low. RER contents, released at pH 11.2, are separated from membranes by ultracentrifugation. Most apo B remains membrane-bound, but measurable amounts a~ released. Density gradient ultracentrifugation isolates spherical HDULDL-like particles, -125-250 A in diameter, that contain mom triglycerides (TG) than cholesteryl esters (CE) together with apo B48 and BlOO. Another particle, the size of nascent Golgi VLDL (-450 A), has a similar ratio of TG to CE. Remarkably, these TGrich particles lack detectable apo B, which is consistent with the observation of VLDL-size particles in rat hepatocytes that do not immunostain for apo B (Alexander et al. J Cell Rio1 1976; 69: 241-263). These results are consistent with the two-step model of nascent VLDL assembly: the smaller particles are produced as apo B is sequestered in the RER lumen by a process requiring neutral lipid transfer protein; subsequently this particle coalesces with a larger TG-rich particle sequestered in the absence of ape B in the lumen of the SER. Small particles produced in the RER are sometimes secreted; apo B-deficient TG-rich particles probably are not.
Multivesicular bodies (MVB), the compartment of uncoupling of receptors and ligands (CURL) and the receptor-recycling compartment (RRC) were isolated from rat livers to investigate the intracellular trafficking of different lipoprotein components, These three endosomal fractions are characterized by high concentrations of annexin VI, which has recently been identified as the main constituent of hepatocytic endosomes. Annexin VI is virtually absent from lysosomes and plasma membranes. It is localized exclusively on the cytoplasmatic leaflet of the endosoma1 membrane and may participate in interactions of endosomes with the cytoskeleton. LDL, j3-VLDL, small chylomicron remnants and HDLparticles are taken up first into CURL and am subsequently transferred to the prelysosomal compartment of MVB. Finally these lipoproteins are degraded in lysosomes. Although large chylomicrons, prepared from lymph of lipidfed rats, and their chylomicron remants were rapidly taken up by the liver, large chylomicrons and large chylomicron remnants accumulate in endosomes at a lower rate than small chylomicrons and small chylomicron remnants. These findings suggest that large chylomicron remnants interact with binding sites that do not primarily lead to endocytosis or that extracellular processing precedes endocytosis of large chylomicron remnants. HDL-associated cholesteryl esters in the whole liver exceeded HDL-associated apolipoprotein A-I two- to three-fold, which is compatible with selective uptake of HDL-cholesteryl esters in addition to uptake of intact HDL-particles. The excess cholesteryl esters accumulated in a non-endosomal fraction, whose major proteins differed from the integral proteins of endosomes. These data suggest two distinct intracellular routes of hepatocytic HDL trafficking in vivo: HDL-particles are predominantly transported to endosomes and are finally subjected to lysosomal degradation, while HDL-cholesteryl esters accumulate in a non-endosomal, non-lysosomal compartment. Mechanisms regulating hepatic chylomicron remnant uptake and metabolism Windle& Greeve J, Jlckle S, Rinninger F, Greten H, Medizinische Kernklinik und Poliklinik, Universitiits-Krankenhaus Eppendot$ Martinistr. 52, D-20246, Hamburg, Germany
Exogenous lipids am transported in chylomicron remnants to the liver. Several aspects of the regulation of their hepatic uptake and metabolism have been investigated. A quantitative reverse transcription PCR using an external standard has been established to determine LDL-receptor mRNA. In the rat, hepatic LDL-receptor mRNA is suppressed by exogenous cholesterol, saturated fatty acids and bile acids and by uptake of cholesterol-rich /I-VLDL. Removal of small chylomicron remnants by perfused rat livers closely correlates with the degree of suppression of LDL-receptor mRNA. In contrast, removal of remnants of large chylomicrons is not appreciably influenced by the level of LDL-receptor mRNA. The asialoglycoprotein receptor may serve as an alternative mechanism of remnant removal, Transient and stable transfection of the cDNAs of the two subunits of the human asialoglycoprotein receptor markedly increased the binding capacity for chylomicron remnants. ln rats with permanent venous catheters and biliodigestive anastomosis the catabolism of cholesterol from chylomicron rem-
Atherosclerosis X, Montreal, October 1994
WlO INTRACELLULAR
CHOLESTEROL
Regulation of hepatie secretion of lipoproteins &lg&QI& Wu X, S&rata N, Dixon JL, Dept. ofMed., Colt. of Physicians and Surgeons, l&32, USA -
630 West 168th St., New York. NY
173
TRANSPORT
Endosomal and extraendosomal intracellular trafficking of lipoproteins JLkle, Rinninger F, Beisiegel U, Block A, Greten H, Windler E, Medizinische Klinik, Univ.-Krankenhaus Eppendot$ Martinistr. 52, D-20246 Hamburg, Germany
The secretion of apo B-containing lipoproteins from hepatocytes requires the assembly of newly synthesized apo B with core lipids, triglyceride and cholesterol, and surface lipids, particularly phospholipids. Regulation of secretion can, therefore, reside at several points. Regulation of apo B synthesis at the transcriptional level does not appear to play a major role in determining secretion of apo B-containing lipoproteins. On the other hand, posttranslational degradation of apo B in the endoplasmic reticulum (ER) has a significant role in regulating secretion In cultured hepatocytes, a significant proportion of newly synthesized apo B is rapidly degraded in the ER. Studies from several laboratories indicate that newly synthesized apo B associates with the ER membrane either cotranslationally or soon after translation. The availability of the core lipid triglyceride, and possibly cholesteryl ester, then determines if rapid degradation of nascent apo B occurs or if translocation of the protein is completed. A number of ER chaperone proteins am probably involved in this process. Oleic acid protects apo B from degradation, but this protection requires the synthesis of triglyceride. Microsomal transfer protein may play a critical role in delivery newly synthesized triglyceride to nascent apo B. Phospholipid availability appears to limit assembly of apo B-containing lipoproteins only in extreme circumstances. Apo B degradation seems to involve a cysteine-protease that is associated with the ER; little degradation occurs in the Golgi or the lysosomes. In HepG2 cells, limited triglyceride availability results in secretion of mostly low density lipoproteins; in rat hepatocytes, a ‘bolus’ of core lipids is added to the nascent apo B-containing lipoprotein, possibly in the smooth ER, resulting in secretion of typical very low density lipoproteins. Nascent VLDL assembly occurs by two steps in the endoplasmic reticulum (RR) of hepatocytes &J&&&, Erickson SK, Have1 RJ, Cardiovascular Research Inst. and Depts. of Anatomy and Med., UCSF Sch. of Med., San Francisco, CA 94143-0130 USA
Core lipidation of apo B in the ER is essential for assembly of nascent VLDL. To study this, we developed a procedure for isolating large amounts of ribosome-rich membranes from rat liver. Short strips of rough (R)ER predominate. in this fraction, enriched many-fold in RER markers and apo B. Markers for other organelles are low. RER contents, released at pH 11.2, are separated from membranes by ultracentrifugation. Most apo B remains membrane-bound, but measurable amounts a~ released. Density gradient ultracentrifugation isolates spherical HDULDL-like particles, -125-250 A in diameter, that contain mom triglycerides (TG) than cholesteryl esters (CE) together with apo B48 and BlOO. Another particle, the size of nascent Golgi VLDL (-450 A), has a similar ratio of TG to CE. Remarkably, these TGrich particles lack detectable apo B, which is consistent with the observation of VLDL-size particles in rat hepatocytes that do not immunostain for apo B (Alexander et al. J Cell Rio1 1976; 69: 241-263). These results are consistent with the two-step model of nascent VLDL assembly: the smaller particles are produced as apo B is sequestered in the RER lumen by a process requiring neutral lipid transfer protein; subsequently this particle coalesces with a larger TG-rich particle sequestered in the absence of ape B in the lumen of the SER. Small particles produced in the RER are sometimes secreted; apo B-deficient TG-rich particles probably are not.
Multivesicular bodies (MVB), the compartment of uncoupling of receptors and ligands (CURL) and the receptor-recycling compartment (RRC) were isolated from rat livers to investigate the intracellular trafficking of different lipoprotein components, These three endosomal fractions are characterized by high concentrations of annexin VI, which has recently been identified as the main constituent of hepatocytic endosomes. Annexin VI is virtually absent from lysosomes and plasma membranes. It is localized exclusively on the cytoplasmatic leaflet of the endosoma1 membrane and may participate in interactions of endosomes with the cytoskeleton. LDL, j3-VLDL, small chylomicron remnants and HDLparticles are taken up first into CURL and am subsequently transferred to the prelysosomal compartment of MVB. Finally these lipoproteins are degraded in lysosomes. Although large chylomicrons, prepared from lymph of lipidfed rats, and their chylomicron remants were rapidly taken up by the liver, large chylomicrons and large chylomicron remnants accumulate in endosomes at a lower rate than small chylomicrons and small chylomicron remnants. These findings suggest that large chylomicron remnants interact with binding sites that do not primarily lead to endocytosis or that extracellular processing precedes endocytosis of large chylomicron remnants. HDL-associated cholesteryl esters in the whole liver exceeded HDL-associated apolipoprotein A-I two- to three-fold, which is compatible with selective uptake of HDL-cholesteryl esters in addition to uptake of intact HDL-particles. The excess cholesteryl esters accumulated in a non-endosomal fraction, whose major proteins differed from the integral proteins of endosomes. These data suggest two distinct intracellular routes of hepatocytic HDL trafficking in vivo: HDL-particles are predominantly transported to endosomes and are finally subjected to lysosomal degradation, while HDL-cholesteryl esters accumulate in a non-endosomal, non-lysosomal compartment. Mechanisms regulating hepatic chylomicron remnant uptake and metabolism Windle& Greeve J, Jlckle S, Rinninger F, Greten H, Medizinische Kernklinik und Poliklinik, Universitiits-Krankenhaus Eppendot$ Martinistr. 52, D-20246, Hamburg, Germany
Exogenous lipids am transported in chylomicron remnants to the liver. Several aspects of the regulation of their hepatic uptake and metabolism have been investigated. A quantitative reverse transcription PCR using an external standard has been established to determine LDL-receptor mRNA. In the rat, hepatic LDL-receptor mRNA is suppressed by exogenous cholesterol, saturated fatty acids and bile acids and by uptake of cholesterol-rich /I-VLDL. Removal of small chylomicron remnants by perfused rat livers closely correlates with the degree of suppression of LDL-receptor mRNA. In contrast, removal of remnants of large chylomicrons is not appreciably influenced by the level of LDL-receptor mRNA. The asialoglycoprotein receptor may serve as an alternative mechanism of remnant removal, Transient and stable transfection of the cDNAs of the two subunits of the human asialoglycoprotein receptor markedly increased the binding capacity for chylomicron remnants. ln rats with permanent venous catheters and biliodigestive anastomosis the catabolism of cholesterol from chylomicron rem-
Atherosclerosis X, Montreal, October 1994