Isolation and characterization of hepatic Golgi lipoproteins from hypercholesterolemic rats.

Swift, Larry L.; Manowitz, Neil R.; Dunn, G. Dewey; LeQuire, Virgil S.

doi:10.1172/jci109871

Cited by 66 publications

(15 citation statements)

References 34 publications

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“…Biochemically, the purity of Golgi-rich fractions was assessed by the activity of galactosyltransferase, a Golgi-specific enzyme. Golgi-rich fractions were enriched in galactosyltransferase activity from 43-to 96-fold compared with the initial liver homogenates, not unlike values obtained using rat liver (19).…”

Section: Resultsmentioning

confidence: 55%

“…The mice were killed by cervical dislocation, and the liver was quickly removed, trimmed of excess fat and connective tissue, rinsed in distilled water, blotted, and weighed. Golgi apparatus-rich fractions were isolated from the liver according to a modification of the method of Swift et al (19,20). The livers were minced finely with scalpel blades and placed in homogenization buffer (0.1 M phosphate-buffered saline, pH 7.3, 0.25 M sucrose, 1% dextran, and 0.01 M MgCl 2 ).…”

Section: Preparation Of Iodinated D ͻ 1019mentioning

confidence: 99%

“…Elimination of this contaminant is crucial in assessing the true secretory products within the Golgi fraction. Our methodology for preparation of Golgi fractions from mouse liver was based on procedures developed in our laboratory (29) and subsequently modified specifically to eliminate endosomal contamination (23,30). Enzymatic analysis of our preparations demonstrated enrichment in galactosyltransferase, a recognized marker of Golgi purity.…”

Section: Fig 5 Evidence That Apoe Recycling Through the Golgi Is Dementioning

confidence: 99%

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Recycling of Apolipoprotein E in Mouse Liver

Fazio¹,

Linton

Hasty³

et al. 1999

Journal of Biological Chemistry

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Following the internalization of low density lipoprotein (LDL) by the LDL receptor within cells, both the lipid and the protein components of LDL are completely degraded within the lysosomes. Remnant lipoproteins are also internalized by cells via the LDL receptor as well as other receptors, but the events following the internalization of these complexes, which use apolipoprotein E (apoE) as their ligand for receptor capture, have not been defined. There is evidence that apoEcontaining ␤-very low density lipoproteins follow differential intracellular routing depending on their size and apoE content and that apoE internalized with lipoproteins can be resecreted by cultured hepatocytes and fibroblasts. In the present studies, we addressed the question of apoE sparing or recycling as a physiologic phenomenon. Remnant lipoproteins (d < 1.019 g/ml) from normal mouse plasma were iodinated and injected into normal C57BL/6 mice. Livers were collected at 10, 30, 60, and 120 min after injection, and hepatic Golgi fractions were prepared for gel electrophoresis analysis. Golgi preparations were analyzed for galactosyltransferase enrichment (>40-fold above cell homogenate) and by appearance of the Golgi stacks and vesicles on electron microscopy. Iodinated apoE was consistently found in the Golgi fractions peaking at 10 min and disappearing by 2 h after injection. Although traces of apoB48 were present in the Golgi fractions, the apoE/ apoB ratio in the Golgi was 50-fold higher compared with serum. Quantitatively similar results were obtained when the very low density lipoprotein remnants were injected into mice deficient in either apoE or the LDL receptor, indicating that the phenomenon of apoE recycling is not influenced by the production of endogenous apoE and is not dependent on the presence of LDL receptors. In addition, radioactive apoE in the Golgi fractions was part of d ‫؍‬ 1.019 -1.21 g/ml complexes, indicating an association of recycled apoE with either newly formed lipoproteins or the internalized complexes. These studies show that apoE recycling is a physiologic phenomenon in vivo and establish the presence of a unique pathway of intracellular processing of apoEcontaining remnant lipoproteins.The series of events following internalization of plasma low density lipoprotein (LDL) 1 has been dissected in detail by the classic studies of Goldstein and Brown (1, 2). The LDL particle, which contains apolipoprotein B100 (apoB100) as the sole protein component, is internalized as a lipoprotein-receptor complex within an endosomal compartment that subsequently fuses with lysosomes. Whereas the LDL receptor recycles back to the cell surface, the lipoprotein particle continues on its routing toward the lysosomal compartment where complete hydrolysis of apoB and the lipid components of the particle occurs (3). It is not known whether other lipoproteins follow the same intracellular pathways of LDL after internalization. Chylomicron and very low density lipoprotein (VLDL) remnants, for example, despite containing apoB utili...

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Section: Resultsmentioning

confidence: 55%

Section: Preparation Of Iodinated D ͻ 1019mentioning

confidence: 99%

Section: Fig 5 Evidence That Apoe Recycling Through the Golgi Is Dementioning

confidence: 99%

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Recycling of Apolipoprotein E in Mouse Liver

Fazio¹,

Linton

Hasty³

et al. 1999

Journal of Biological Chemistry

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“…Two groups of emulsions with lipid compositions similar to those of nascent (Green et al, 1979;Swift et al, 1980), group " Calculated from the data presented in Tables I and 11. Values for the mean * 1 SD (n = 3) lipid ratios.…”

Section: Resultsmentioning

confidence: 99%

Triolein-cholesteryl oleate-cholesterol-lecithin emulsions: structural models of triglyceride-rich lipoproteins

Miller¹,

Small²

1983

Biochemistry

100

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The organization of lipids within emulsions composed of triolein (TO), cholesteryl oleate (CO), cholesterol (C), and egg yolk phosphatidylcholine (L) was examined. CO was substituted for TO in a series of emulsions to obtain TO:CO ratios comparable to the triglyceride:cholesterol ester ratios observed in subfractions of triglyceride-rich lipoproteins. The weight fraction of TO in the surface phase (0.02-0.05) was independent of the TO content of the emulsions. However, the weight fraction of CO in the surface phase depended upon the percentage of CO in the emulsions and was less than 0.004 even when 13.7% CO was present in the emulsion. When CO was substituted for TO, the percent of the total particle C which was carried in the droplet oil phase was increased. The interparticle equilibration of lipids was studied in subfractions of sonicated emulsions with particle sizes comparable to triglyceride-rich lipoproteins. The TO:CO ratios of the subfractions of a given emulsion were constant and independent of size, but the C:L ratio decreased in particles of smaller diameter. However, the surface C:L ratio was the same in all particles from a given emulsion. The size dependence of the C:L ratios was attributed to the partitioning of C into the oil cores of the emulsions. Because large droplets have the greatest core:surface mass ratios, more of their total particle C is carried in the core.

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“…Nakaya et al (11,12) demonstrated de novo synthesis and secretion of lipoproteins with LDL density by the isolated perfused pig liver. Swift et al (13,14) isolated a cholesteryl ester-rich LDL from the hepatic Golgi apparatus of cholesterol-fed rats, and Noel et al (15) demonstrated production of a cholesteryl ester-rich LDL by the isolated perfused liver of cholesterol-fed rats. Guo et al (16) have recently identified cholesteryl ester-rich LDL in perfusates of livers from normal and cholesterol-fed guinea pigs, but suggest that many of these particles represent washout from liver of adsorbed plasma LDL.…”

Section: Introductionmentioning

confidence: 99%

Studies on the production of low density lipoproteins by perfused livers from nonhuman primates. Effect of dietary cholesterol.

Johnson¹,

Clair²,

Rudel³

1983

J. Clin. Invest.

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A B S T R A C T Nonhuman primates consuming diets containing cholesterol develop coronary artery atherosclerosis that we have found to be highly correlated with an increase in the size and cholesteryl ester content of plasma low density lipoproteins (LDL). The present studies were designed to determine whether the enlarged plasma LDL are produced directly by the liver of cholesterol-fed monkeys. African green monkeys were fed a diet containing 40% of calories as butter fat and either 0.16 mg cholesterol/kcal (control diet) or 0.78 mg cholesterol/kcal (test diet). The livers of these monkeys were perfused by recirculation with a lipoprotein-free medium for 4 h. The rate of accumulation of perfusate cholesterol was linear and greater in liver perfusates from test diet-fed vs. control diet-fed monkeys and was positively correlated with both the plasma cholesterol concentration and LDL size in the donor animal. All perfusate d < 1.063 g/ ml lipoprotein subfractions from livers of test diet-fed monkeys were enriched in cholesteryl ester severalfold over the corresponding subfractions from control dietfed monkeys and contained only the larger form of apolipoprotein B typical of plasma LDL. However, the perfusate lipoproteins in the LDL density range did not have an average size or composition typical of LDL from plasma. Rather, they were relatively enriched in phospholipid and unesterified cholesterol and were deficient in cholesteryl esters. In addition, perfusate high density lipoproteins were discoidal particles. These data show that the enzyme lecithin:cholesterol acyltransferase (LCAT) was essentially inactive in Received for publication 20 December 1982 and in revised form 9 March 1983. these perfusates and, as a result, the dietary cholesterol-induced enrichment of perfusate d < 1.063 g/ml lipoproteins with cholesteryl esters probably resulted from increased hepatic secretion of cholesteryl esters and not from modification of lipoproteins by LCAT during recirculating perfusion. In spite of this increase, enlarged cholesteryl ester-rich LDL were not found in the perfusate, suggesting that large molecular weight plasma LDL are not directly secreted by the liver but instead probably result from further intravascular metabolism of cholesteryl ester-enriched hepatic precursor lipoproteins.

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Isolation and characterization of hepatic Golgi lipoproteins from hypercholesterolemic rats.

Cited by 66 publications

References 34 publications

Recycling of Apolipoprotein E in Mouse Liver

Recycling of Apolipoprotein E in Mouse Liver

Triolein-cholesteryl oleate-cholesterol-lecithin emulsions: structural models of triglyceride-rich lipoproteins

Studies on the production of low density lipoproteins by perfused livers from nonhuman primates. Effect of dietary cholesterol.

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