Glycosylation of apoproteins of rat very low density lipoproteins during transit through the hepatic Golgi apparatus

1977 ◽  
Vol 55 (1) ◽  
pp. 83-90 ◽  
Author(s):  
Peter J. Dolphin ◽  
David Rubinstein
Keyword(s):  
Golgi Apparatus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Density Lipoprotein ◽  
Low Density Lipoproteins ◽  
Secretory Vesicles ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Experimental Conditions ◽  
Apo B

The glycosylation of apo very low density lipoproteins (apo-VLDL) in vivo was studied by following the incorporation of [14C]glucosamine into several groups of apoproteins of VLDL isolated from hepatic Golgi fractions and from serum of sucrose-fed, colchicine-treated rats. Simultaneous incorporation of [3H]leucine was used to quantitate the apoproteins following separation by polyacrylamide gel electrophoresis. Experimental conditions were selected so that the 14C:3H ratio in the apoproteins permitted estimations of the extent of glycosylation by glucosamine and its metabolites. A rapidly decreasing 14C:3H ratio was noted in serum apo-VLDL for the first 30 min after administration of the isotopically labelled precursors, followed by stabilization of the ratio. These data are consistent with the glycosylation of a preformed pool of apo-VLDL, probably apo-B. Glucosamine was progressively incorporated into apo-VLDL during transition from the forming face of the Golgi apparatus to the secretory vesicles, as indicated by an increasing 14C:3H ratio. On the other hand, the ratio of the rapidly migrating apoproteins of VLDL, corresponding to the apo-C-II and apo-C-III, showed the opposite trend, as did total apo high density lipoprotein (apo-HDL) and the rapidly migrating bands of apo-HDL. Division of the rapidly migrating apoproteins of VLDL into upper bands (probably apo-C-II and apo-C-III-0) and lower bands (probably apo-C-III-3) resulted in a 14C:3H ratio near zero in the upper band apoproteins, consistent with the absence of carbohydrates. The lower band showed a rising 14C:3H ratio during transition through the Golgi apparatus, suggesting increased glycosylation. The decreasing 14C:3H ratio in the rapidly migrating proteins is therefore due to the acquisition of apo-C-II and apo-C-III-0 by VLDL during passage from the forming face to the secretory vesicles of the Golgi apparatus.

The synthesis of apoproteins of very low density lipoproteins isolated from the Golgi apparatus of rat liver

1976 ◽  
Vol 54 (7) ◽  
pp. 617-628 ◽  
Author(s):  
A. Christine Nestruck ◽  
David Rubinstein
Keyword(s):  
Golgi Apparatus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Low Density Lipoproteins ◽  
Polyacrylamide Gel ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Apo B ◽  
Relationship Analysis ◽  
Discontinuous Sucrose Gradient

The incorporation of [3H]leucine in vivo into very low density lipoproteins (VLDL) from the rat hepatic Golgi apparatus and serum was studied. A Golgi-rich fraction isolated on a discontinuous sucrose gradient between 0.5 and 1.1 M was found to contain VLDL having common antigenic determinants with serum VLDL. The incorporation of the [3H]leucine into the Golgi VLDL and serum VLDL suggested a precursor–product relationship. Analysis of the apoproteins of the Golgi VLDL by polyacrylamide gel electrophoresis revealed protein bands with similar mobility to those of serum VLDL, except that the former contained virtually no rapidly migrating peptides with the mobility of serum apo-C-II and apo-C-III. The pattern of incorporation of the [3H]leucine into the apoproteins was similar in VLDL from Golgi apparatus and serum, except for the absence of radioactivity in the area of the gel of Golgi apo-VLDL corresponding to apo-C-II and apo-C-III. The radioactive amino acid was incorporated predominantly into the Golgi apo-VLDL bands with similar mobility to apo-B and an apoprotein or group of apoproteins containing the arginine-rich peptide of serum VLDL. In vitro incubation of the Golgi VLDL with [3H]leucine-labeled HDL resulted in the acquisition of a number of proteins, including the rapidly migrating proteins. Administration of colchicine prior to the injection of [3H]leucine resulted in the appearance of gel bands and radioactivity in the apo-C-II and apo-C-III areas of Golgi apo-VLDL, suggesting that these can be acquired if secretion of VLDL is slowed or inhibited. The hepatic Golgi apparatus was then divided into fractions of predominantly forming face (GF3) or secretory granules (GF1). After polyacrylamide gel electrophoresis of the apo-VLDL from GF3, no visible bands or incorporation of [3H]leucine was found in the region of apo-C-II or apo-C-III. However VLDL from GF1 showed visible and radioactive bands in the apo-C-II and apo-C-III area although they represented a much smaller proportion of the total apoprotein than was found in the corresponding serum apo-VLDL. In the isolated perfused liver the percentage incorporation of [3H]leucine into the rapidly migrating apoproteins of Golgi VLDL was considerably less than that found in the corresponding apoproteins of perfusate VLDL, where circulating C lipoproteins are virtually absent.The data indicate that nascent VLDL begins to acquire the C-II and C-III apoproteins during its passage through the Golgi apparatus but that the main acquisition occurs during or after secretion into the space of Disse.

A comparison of some immunological characteristics of very low density lipoproteins of normal and hypercholesterolemic rat sera

1978 ◽  
Vol 56 (6) ◽  
pp. 673-683 ◽  
Author(s):  
Peter J. Dolphin ◽  
Laurence Wong ◽  
David Rubinstein
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Normal Serum ◽  
Low Density Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Rat Serum ◽  
Apo B ◽  
Apo E

The immunological characteristics of very low density lipoproteins (VLDL) from normal and hypercholesterolemic rat sera were compared using polyspecific antisera to VLDL and high density lipoproteins (HDL) and monospecific antisera to apo-B, apo-C, apo-A-I, and apo-E. Ultracentrifugally isolated VLDL from normal serum were studied by immunodiffusion and found to contain both discrete and associated (with apo-B) apo-C and apo-E, probably in the form of lipid-containing lipoproteins. However, immunoelectrophoresis of whole serum revealed only an associated form of the lipoprotein having pre-β mobility (i.e., VLDL), suggesting that the presence of discrete lipoproteins in isolated VLDL, each containing a single apoprotein family, may represent ultracentrifugal artifacts. Ultracentrifugally isolated VLDL from diet-induced hypercholesterolemic rat serum contained only trace amounts of apo-C and large quantities of apo-E, both of which were totally associated with apo-B. VLDL isolated by ultracentrifugation from perfusate of normal and hypercholesterolemic livers contained only associated lipoprotein complexes made up of apo-B, apo-C, and apo-E in the former but only apo-B and apo-E in the latter. These data suggest that normal VLDL are secreted as lipoprotein complexes containing apo-B, apo-C, and apo-E which may become destabilized in the circulation. However, VLDL from hypercholesterolemic serum show a marked diminution in the quantity of apo-C as indicated by the relative incorporation of [3H]leucine in vivo and by polyacrylamide gel electrophoresis of apo-VLDL.

scholarly journals Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus.

1985 ◽  
Vol 100 (5) ◽  
pp. 1558-1569 ◽  
Author(s):  
C A Hornick ◽  
R L Hamilton ◽  
E Spaziani ◽  
G H Enders ◽  
R J Havel
Keyword(s):  
Golgi Apparatus ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density Lipoproteins ◽  
Endocytic Pathway ◽  
Multivesicular Bodies ◽  
Secretory Vesicles ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Isolation And Characterization

Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor-mediated endocytosis. MVBs also contained numerous small vesicles, 0.05-0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized triglycerides. Membranes of MVBs contained much more cholesterol and less protein than did Golgi membranes. We conclude that two distinct lipoprotein-filled organelles are located in the bile canalicular pole of hepatocytes. MVBs, a major prelysosomal organelle of low density in the endocytic pathway, contain remnants of triglyceride-rich lipoproteins, whereas secretory vesicles of the Golgi apparatus contain nascent very low density lipoproteins.

scholarly journals The metabolic conversion of very-low-density lipoprotein into low-density lipoprotein by the extrahepatic tissues of the rat

1979 ◽  
Vol 178 (2) ◽  
pp. 455-466 ◽  
Author(s):  
B S Suri ◽  
M E Targ ◽  
D S Robinson
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Quantitative Information ◽  
Low Density Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Apo B ◽  
Extrahepatic Tissues

1. The work reported was designed to provide quantitative information about the capacity of the extrahepatic tissues of the rat to degrade injected VLD lipoproteins (very-low-density lipoproteins, d less than 1.006) to LD lipoproteins (low-density lipoproteins, d 1.006–1.063) and to study the fate of the different VLD-lipoprotein apoproteins during the degradative process. 2. Rat liver VLD lipoproteins, radioactively labelled in their protein moieties, were produced by the perfusion of the organ and were either injected into the circulation of the supradiaphragmatic rats or incubated in rat plasma at 37 degrees C. At a time (75 min) when approx. 90% of the triacylglycerol of the VLD lipoproteins had been hydrolysed the supradiaphragmatic rats were bled and VLD lipoproteins, LD lipoproteins and HD lipoproteins (high-density lipoproteins, d 1.063–1.21) were separated from their plasma and from the plasma incubated in vitro. The apoproteins of each of the lipoprotein classes were resolved by gel-filtration chromatography into three main fractions, designated peaks I, II and III. 3. Incubation of the liver VLD lipoproteins in plasma in vitro led to the transfer of about 30% of the total protein radioactivity to the HD lipoproteins. The transfer mainly involved the peak-II (arginine-rich and/or apo A-I) and peak-III (apo C) proteins. There was also a small transfer of radioactivity (about 5% of the total) to the LD lipoproteins. 4. Injection of the liver VLD lipoproteins into the circulation of the supradiaphragmatic rat resulted in the transfer of about 15% of the total VLD-lipoprotein radioactivity to the LD lipoproteins. The transfer involved mainly the peak-I (apo B) proteins and accounted for about 20% of the total apo B protein radioactivity of the injected VLD lipoproteins. When the endogenous plasma VLD lipoprotein was taken into account the transfer of apo B protein was about 35%. 5. The transfer of peak-II protein radioactivity from the VLD to the HD lipoproteins was greater in the plasma of the supradiaphragmatic rat than in the incubated plasma suggesting that there was a net transfer of peak-II apoproteins during the VLD lipoprotein degradation. The transfer of peak-III protein radioactivity was not greater in the plasma of the supradiaphragmatic rat, but there was a loss of this radioactivity from the circulation.

scholarly journals Comparison of apoprotein B of low density lipoproteins of human interstitial fluid and plasma

1984 ◽  
Vol 222 (1) ◽  
pp. 49-55 ◽  
Author(s):  
J L Hong ◽  
J Pflug ◽  
D Reichl
Keyword(s):  
Interstitial Fluid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Low Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Apo B ◽  
Apoprotein B ◽  
Human Interstitial Fluid ◽  
Nonionic Detergent

Virtually all apoprotein B (apoB)-containing lipoproteins of the peripheral interstitial fluid of subjects with primary lymphoedema float in the ultracentrifugal field in the density interval 1.019-1.063 g/ml; in this respect they are similar to plasma low-density lipoproteins (LDL). 2. Virtually all apo-B-containing lipoproteins of interstitial fluid migrate in the electrophoretic field with pre-beta mobility; in this respect they are similar to plasma very-low-density lipoproteins. 3. The apoB of lipoproteins of interstitial fluid does not differ in terms of Mr from apoB-100 of human plasma [Kane, Hardman & Paulus (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2465-2469] as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. Both apoB of interstitial fluid and plasma are heterogenous in terms of their charge as determined by isoelectric focusing of their complexes with the nonionic detergent Nonidet P40. ApoB of plasma LDL focuses between pH5.9 and 6.65, and that of interstitial fluid LDL between pH 5.9 and 6.1. Thus the overall charge of apoB of interstitial fluid is more negative than that of its plasma LDL counterpart.

Transfer of newly made triglyceride from hepatocytes to preexisting extracellular very low density lipoproteins

1987 ◽  
Vol 65 (3) ◽  
pp. 337-343
Author(s):  
Gen Yoshino ◽  
George Steiner
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Liver Cells ◽  
Low Density Lipoproteins ◽  
In Vivo Studies ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Apoprotein B

Previous in vivo studies suggested a new model to describe the metabolism of very low density lipoproteins (VLDL). It was hypothesized that some of the lipoprotein triglyceride was transferred directly from hepatocytes and intestinal mucosal cells into preexisting extracellular VLDL particles. These studies employ an in vitro system to test this hypothesis. Isolated rat liver cells containing newly made radioactive triglyceride were prepared. These cells were incubated in medium to which exogenous VLDL had or had not been added. The presence of extracellular VLDL (rat or human) stimulated the transfer of labeled triglyceride out of the liver cells. This triglyceride was recovered in the medium's VLDL (as determined by its density and its precipitability by MnCl2–heparin or by anti-apoprotein B). Although these studies focussed on VLDL, preliminary data showed that similar triglyceride transfer occurred in the presence of the other apoprotein B containing lipoprotein, low density lipoprotein (LDL). However, in the presence of equivalent amounts of LDL, this triglyceride transfer was less than that seen in the presence of exogenous VLDL. Furthermore, the increased triglyceride released in the presence of LDL occurred entirely in the d < 1.006 fraction of the medium. That released in the presence of VLDL was recovered in the d > 1.006 fraction. Hence, we conclude that the transfer of the newly made triglyceride was from the cell to the extracellular lipoprotein that had been added to the medium. The transfer of triglyceride to VLDL did not depend on the synthesis and release of new VLDL particles because it was not accompanied by a change in the production of [14C]leucine VLDL protein, it was not blocked by chloroquine, and the LDL induced triglyceride release occurred into the d > 1.006 fraction. This transfer did not depend on the previously described triglyceride-transfer factor. The present in vitro studies support the model suggested by our earlier in vivo studies. The VLDL particle does not appear to be metabolized as a complete intact unit. Rather, some of its major lipid component, triglyceride, can move directly into and out of already existing extracellular lipoproteins.

scholarly journals Subcellular localization of B apoprotein of plasma lipoproteins in rat liver.

1976 ◽  
Vol 69 (2) ◽  
pp. 241-263 ◽  
Author(s):  
C A Alexander ◽  
R L Hamilton ◽  
R J Havel
Keyword(s):  
Golgi Apparatus ◽  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
Antigenic Determinants ◽  
Secretory Vesicles ◽  
Low Density ◽  
Bile Canaliculi ◽  
Very Low Density Lipoproteins ◽  
Rough Er ◽  
Space Of Disse

Multispecific antigen-binding fragments (Fab) from rabbit antisera against rat very low density lipoproteins (VLDL) and Fab against rat low density lipoproteins that were monospecific for the B apoprotein were conjugated to horseradish peroxidase. Conjugates were incubated with 6-mum frozen sections from fresh and perfusion-fixed livers and with tissue chopper sections (40 mum thick) from perfusion-fixed livers. In the light microscope, specific reaction product was present in all hepatocytes of experimental sections as intense brown to black spots whose locations corresponded to the distribution of the Golgi apparatus: along the bile canaliculi, near the nuclei, and between the nuclei and bile canaliculi. Perfusion fixation with formaldehyde produced satisfactory ultrastructural preservation with retention of lipoprotein antigenic determinants. In the electron microscope, patches of cisternae and ribosomes of the rough endoplasmic reticulum (ER) and particularly its smooth-surfaced ends, vesicles located between the rough ER and the Golgi apparatus, the Golgi apparatus and its secretory vesicles and VLDL particles in the space of Disse all bore reaction product. The tubules and vesicles of typical hepatocyte smooth ER did not contain reaction product, nor did the osmiophilic particles contained therin. The localization obtained in this study together with other evidence suggests a sequence for the biosynthesis of VLDL that differs in some respects from that proposed by others: (a) the triglyceride-rich particle originates in smooth ER where triglycerides are synthesized; (b) at the junction of the smooth and rough ER the particle receives apoproteins synthesized in the rough ER; (c) specialized tubules transport the particle, now a nascent lipoprotein, to the Golgi apparatus where concentration occurs in secretory vesicles; (d) secretory vesicles move to the sinusoidal surface where the particles are secreted into the space of Disse by fusion of the vesicular membrane with the plasma membrane of the hepatocyte.

Effects of oxidized low-density lipoproteins on the hepatic microvasculature

2011 ◽  
Vol 301 (4) ◽  
pp. G684-G693 ◽  
Author(s):  
Ana Oteiza ◽  
Ruomei Li ◽  
Robert S. McCuskey ◽  
Bård Smedsrød ◽  
Karen Kristine Sørensen
Keyword(s):  
Electron Microscopy ◽  
Linear Trend ◽  
Density Lipoprotein ◽  
Low Density Lipoproteins ◽  
Intercellular Adhesion Molecule ◽  
Organ Distribution ◽  
Low Density ◽  
Hepatic Microcirculation ◽  
Oxidized Low Density Lipoproteins

Oxidized low-density lipoproteins (oxLDLs) are involved in proinflammatory and cytotoxic events in different microcirculatory systems. The liver is an important scavenger organ for circulating oxLDLs. However, the interaction of oxLDL with the hepatic microcirculation has been poorly investigated. The present study was conducted to examine the effects of differently modified oxLDLs on the hepatic microvasculature. C57Bl/6J mice were injected intravenously with low-density lipoprotein (LDL), or LDL oxidized for 3 h (oxLDL3) or 24 h (oxLDL24), at doses resembling oxLDL plasma levels in cardiovascular disease patients. Radioiodinated ligands were used to measure blood decay and organ distribution, and nonlabeled ligands to evaluate microcirculatory responses, examined by in vivo microscopy 30–60 min after ligand injection, immunohistochemistry, and scanning and transmission electron microscopy. Mildly oxLDL (oxLDL3) was cleared from blood at a markedly slower rate than heavily oxLDL (oxLDL24), but significantly faster than LDL ( P < 0.01). Injected oxLDLs distributed to liver. OxLDL effects were most pronounced in central areas of the liver lobules where oxLDL3elicited a significant ( P < 0.05) reduction in perfused sinusoids, and both oxLDL3and oxLDL24significantly increased the numbers of swollen endothelial cells and adherent leukocytes compared with LDL ( P < 0.05). OxLDL-treated livers also exhibited increased intercellular adhesion molecule (ICAM)-1 centrilobular staining. Electron microscopy showed a 30% increased thickness of the liver sinusoidal endothelium in the oxLDL3group ( P < 0.05) and a reduced sinusoidal fenestration in centrilobular areas with increased oxidation of LDL ( P for linear trend <0.05). In conclusion, OxLDL induced several acute changes in the liver microvasculature, which may lead to sinusoidal endothelial dysfunction.

scholarly journals Macrophages Create an Acidic Extracellular Hydrolytic Compartment to Digest Aggregated Lipoproteins

2009 ◽  
Vol 20 (23) ◽  
pp. 4932-4940 ◽  
Author(s):  
Abigail S. Haka ◽  
Inna Grosheva ◽  
Ethan Chiang ◽  
Adina R. Buxbaum ◽  
Barbara A. Baird ◽  
...  
Keyword(s):  
Free Cholesterol ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density Lipoproteins ◽  
Critical Event ◽  
Low Density ◽  
Acid Hydrolases ◽  
Detailed Understanding ◽  
Physical Features

A critical event in atherogenesis is the interaction of macrophages with subendothelial lipoproteins. Although most studies model this interaction by incubating macrophages with monomeric lipoproteins, macrophages in vivo encounter lipoproteins that are aggregated. The physical features of the lipoproteins require distinctive mechanisms for their uptake. We show that macrophages create an extracellular, acidic, hydrolytic compartment to carry out digestion of aggregated low-density lipoproteins. We demonstrate delivery of lysosomal contents to these specialized compartments and their acidification by vacuolar ATPase, enabling aggregate catabolism by lysosomal acid hydrolases. We observe transient sealing of portions of the compartments, allowing formation of an “extracellular” proton gradient. An increase in free cholesterol is observed in aggregates contained in these compartments. Thus, cholesteryl ester hydrolysis can occur extracellularly in a specialized compartment, a lysosomal synapse, during the interaction of macrophages with aggregated low-density lipoprotein. A detailed understanding of these processes is essential for developing strategies to prevent atherosclerosis.

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