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The metabolic fate of homologous high density lipoprotein (HDL) was studied in the rat, tracing the apoprotein A-I (apo A-I) and cholesterol ester moieties simultaneously. The apo A-I was labeled with covalently linked 125I-labeled tyramine cellobiose, which accumulates in the cells degrading the apoprotein; [3H]cholesterol ethers, which cannot be hydrolyzed or mobilized after uptake, were incorporated into the lipid core of reconstituted HDL to reflect the fate of the cholesterol esters. Several lines of evidence, including direct comparison with biologically labeled HDL, are presented to support the validity of this approach. The liver was the major organ of cholesterol ether uptake, accounting for 65% of the total; the adrenal gland and ovary were the most active organs per gram (wet) of weight. Uptake of cholesterol ether was 7-fold greater than that of apo A-I in adrenal, 4-fold greater in the ovary, and greater than 2-fold greater in the liver. The remaining tissues took up apo A-I and cholesterol ethers at more nearly equal rates. Transfer of HDL-associated cholesterol ethers and 125I-labeled apo A-I to other lipoprotein fractions was not observed; thus, the results reflect direct uptake from HDL itself. Whereas uptake of low density lipoprotein appears to involve endocytosis of intact particles, uptake of HDL in at least some rat tissues involves additional, more complex, transfer mechanisms.

Low density lipoprotein (LDL) catabolism was studied using WHHL rabbits, an inbred strain deficient in LDL receptor activity and, thus, an animal model for homozygous familial hypercholesterolemia. WHHL and normal rabbits were injected with [14C]sucrose-LDL and the tissue sites of LDL degradation were determined 24 h later. On degradation of [14C]sucrose-LDL, the [14C]sucrose ligand remains trapped within tissues as a cumulative measure of degradation. The fractional catabolic rate of [14C]sucrose-LDL in Watanabe heritable hyperlipidemic (WHHL) rabbits was reduced (0.024 +/- 0.010 versus 0.063 +/- 0.026 h-1) but, by virtue of the increased plasma pool, total LDL flux was increased (33.5 +/- 9.6 versus 10.6 +/- 4.4 mg of LDL protein/kg/day). Liver was the predominant site of catabolism in both WHHL and normal rabbits (52.7 +/- 6.9 and 56.6 +/- 6.2% of total degradation). About 90% of hepatic catabolism was attributable to parenchymal cells in both cases. Thus, Kupffer cells, a major component of the reticuloendothelial system, do not play a major role in LDL catabolism in WHHL rabbits. Despite receptor deficiency, the relative contribution of various tissues to overall LDL degradation was not greatly altered and the absolute rate of delivery of LDL to all tissues was increased with the exception of the adrenal. Thus, there was no evidence that the increased degradation occurred in any special subset of "scavenger" cells. Nevertheless, local scavenger cell uptake may be critically important, especially in atherogenesis. If it is assumed that receptor-independent degradation occurs at the same rate in the tissues of WHHL and normal rabbits and that catabolism in the absence of receptors is a linear function of concentration, then one can estimate the fraction of uptake in normal tissues mediated by receptors. The difference in the fraction of the plasma LDL pool cleared per unit of time in normal and WHHL rabbits would reflect the contribution of receptors to fractional clearance. By this calculation, receptor-mediated degradation in normal rabbits was 62% overall, 63% in liver, 92% in adrenal, and 83% in gut.

A procedure has been developed for the small-scale isolation and characterization of lipoproteins secreted by cultured rat liver hepatocytes. The lipoproteins in the culture medium were separated into VLDL, LDL, HDL and a fraction with d greater than 1.21 on single-spin density-gradients. The lipoproteins were removed from the gradients by adsorption onto Cab-O-Sil, a hydrated colloidal silica. The lipid components were extracted from the silica with CHCl3/CH3OH and the apoproteins solubilized in a buffer that contained 2% sodium dodecyl sulfate and 6 M urea. The proteins were analyzed on 3-20% acrylamide electrophoresis gels that contained 1% sodium dodecyl sulfate. The two major rat-plasma lipoproteins, VLDL and HDL, were well separated by the gradients. The Cab-O-Sil was shown to bind 90-95% of the HDL and VLDL in the fractions from the gradient. The recovery of the lipid components was essentially quantitative. The recovery of the apolipoproteins was only about 60% but with very good precision. Over a 20 h period, the lipid phosphorus associated with secreted lipoproteins increased linearly. The secretion of apolipoprotein A1 and apolipoprotein E associated with HDL and apolipoprotein B associated with VLDL also increased as a nearly linear function with time. The secretion of apolipoprotein E associated with VLDL was linear only up to approx. 6 h. The availability of this procedure should greatly facilitate further studies on the characterization of lipoproteins secreted by hepatocytes and mechanisms that regulate lipoprotein synthesis and secretion.

Synthesis and intracellular transport of two secretory proteins, serum albumin (SA) and apolipoprotein B (apo B) have been synchronized in primary cultures of normal rat hepatocytes to make possible immunocytochemical study of the transport pathway. Under appropriate conditions of cycloheximide treatment, synthesis of new protein was inhibited and, by double immunofluorescent labeling, the cells were found to be largely depleted of the SA and apo B previously synthesized. Re-initiation of protein synthesis led to sequential appearance of SA and apo B, first in the endoplasmic reticulum, then in the Golgi complex, and finally at the cell surface. These results indicate that it should be feasible to use this cell system for high-resolution investigation of the sequence of structures involved in intracellular transport of SA and apo B by corresponding immunolabeling experiments as observed by electron microscopy.

It has been postulated that hyperlipidemia in the nephrotic syndrome is due to overproduction of lipoproteins and that low colloid osmotic pressure (due to hypoalbuminemia) triggers this. Secretion of very low density lipoproteins (VLDL) by cultured rat hepatocytes has been shown to be inhibited by albumin, globulins, and dextrans, but the effect did not correlate with osmolarity. In the present studies we tested the hypothesis that viscosity rather than osmolarity might be the parameter determining the effectiveness of macromolecules in inhibiting VLDL synthesis and secretion by cultured rat hepatocytes. Synthesis and secretion of VLDL was measured in terms of incorporation of [3H] glycerol into medium triglycerides and also from changes in the mass of secreted VLDL triglycerides and apoproteins. The viscosity of the culture medium was increased by addition of dextran-500, gelatin or methylcellulose MX 880. Synthesis and secretion of VLDL was inhibited in direct proportion to increasing viscosity. At a viscosity of 2, which is about that of normal plasma, VLDL secretion was reduced by 20%. An inhibition of 60-70% in secretion and 30-40% in synthesis of VLDL lipid and protein components was observed at a relative viscosity of approximately 3.7. This viscosity was obtained by addition of any of the following: 3% dextran, 3% gelatin, 0.2% methylcellulose, or a combination of 0.1% methylcellulose plus 2% gelatin. Thus, similar viscosities resulted in similar degrees of inhibition despite differences of up to 16-fold in mass concentration and up to 20-fold in osmolarity.

We have studied hepatocytes cultured from Watanabe-heritable hyperlipidemic (WHHL) rabbits, animals that display striking elevation of plasma low density lipoprotein (LDL), spontaneous atherosclerosis, and an absence of LDL receptor activity in cultured fibroblasts. Degradation of LDL by WHHL hepatocytes differed from degradation by normal hepatocytes in several ways: 1) degradation by normal hepatocytes as a function of LDL concentration was curvilinear with a saturable component, while degradation by WHHL hepatocytes was a linear function of concentration; 2) degradation of 125I-labeled LDL by mutant cells was not decreased by excess unlabeled LDL, while degradation by normal cells was; 3) degradation of LDL by normal cells was inhibited by colchicine and chloroquine while degradation by the mutant cells was not; 4) both cell types catabolized LDL at nearly equal rates, but activity of 3-hydroxy-3-methylglutaryl-CoA reductase was suppressed only in the normal cells. These differences are analogous to those previously reported in describing the qualitatively different pathways for receptor-dependent and receptor-independent catabolism of lactosylated and native human LDL in rat hepatocytes. Thus, hepatocytes from WHHL rabbits lack LDL receptor activity. The peripheral and hepatic LDL receptors most likely are products of the same gene or depend for their activity on a single gene product.

The uptake of high-density lipoprotein (HDL)-associated apolipoprotein A-I and cholesterol esters was estimated in 16 tissues of the rat using rat HDL doubly labeled with nondegradable tracers; covalently attached 125I-tyramine-cellobiose traced apo-A-I, and [3H]cholesteryl linoleyl ether traced cholesterol esters. Both labels remained associated with the HDL fraction in the plasma, adequately traced their unlabeled counterparts, and were well trapped at their sites of uptake. Cholesteryl ether was taken up at a greater fractional rate than apo-A-I by adrenal, ovary, and liver: 7-fold, 4-fold, and 2-fold greater, respectively. The rates of uptake of cholesteryl ether and apo-A-I were about equal in the other tissues (except kidney). The disproportionate uptake of HDL cholesteryl ether relative to HDL apo-A-I was also observed in primary cultures of rat adrenal cells and hepatocytes. Uptake of both moieties in both cell types showed saturability. Both the absolute rate of uptake of [3H]cholesteryl ether and the ratio of ether uptake to apo-A-I uptake were greater in adrenal cells than in hepatocytes, consonant with the in vivo observations. Very similar results were obtained using HDL biologically labeled with [3H]cholesterol esters. The disproportionate uptake of [3H]cholesteryl ether was not significantly decreased by depletion of apo-E from the HDL nor by reductive methylation of the apo-E to block its recognition by receptors. However, apo-A-I uptake was decreased, suggesting that apo-E mediates the uptake of particles containing apo-A-I but does not contribute to the disproportionate uptake of [3H]cholesteryl ether.

Very low density lipoproteins, newly secreted by cultured rat hepatocytes into a serum-free medium, contain some cholesteryl esters although the percentage of total cholesterol in ester form is less than that in plasma very low density lipoproteins. When acyl CoA:cholesterol acyltransferase activity in hepatocytes was stimulated by the addition of 25-hydroxycholesterol (10 microgram/ml) or mevalonolactone (1 mM), the absolute amount of esterified cholesterol secreted in very low density lipoproteins increased significantly, but the amount of free cholesterol decreased or showed no change. Thus the percentage of very low density lipoprotein cholesterol in ester form increased, in some experiments to as much as 50% of the total. These results provide additional evidence that hepatic acyl CoA:cholesterol acyltransferase plays a role in the generation of some of the cholesteryl ester in newly-secreted lipoproteins. They further suggest that changes in the activity of the enzyme can potentially regulate the fraction of cholesterol secreted in esterified form.

The biosynthesis and secretion of very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) by cultured normal rat hepatocytes was investigated with particular emphasis on its modification by monensin. This acidic ionophore coordinately inhibited the rates of secretion of the several VLDL apolipoproteins and the VLDL lipids, suggesting an effect late in the process of biosynthesis and secretion, probably at the stage of exiting from the Golgi apparatus. The secretion of immunoreactive albumin into the medium was comparably inhibited, implying that the pathway and mechanisms involved in albumin secretion may be closely similar to those for VLDL synthesis and secretion. Secretion of phospholipids and of apolipoproteins E and A-I in the HDL fraction increased progressively with time over 18 h in control incubations but was strongly inhibited by monensin. During extended incubation with monensin at high concentrations (10 microM), there was a net release to the medium of a number of hepatocyte proteins, including some that comigrated with apolipoprotein A-I and apolipoprotein C, making it appear that monensin increased the secretion of these apolipoproteins. However, using labeled amino acids, it was shown by autoradiography and by immunoprecipitation that secretion of newly-synthesized, radioactive apolipoprotein A-I and apolipoprotein C was actually inhibited by monensin. These results are compatible with the conclusion that HDL synthesis and secretion may occur by mechanisms closely related to those for synthesis and secretion of albumin and VLDL.

Adult rat parenchymal liver cells were isolated and cultured in monolayers. Cholesterol esterification in the intact cultured cells was determined by measuring incorporation of tritiated oleic acid into cell cholesterol ester. Addition of 10 microgram/ml of 25-hydroxycholesterol to the medium gave a 3- to 6-fold increase in cholesterol esterification, while the incorporation of oleic acid into phospholipids and triglycerides remained unaltered. Similar stimulation of cholesterol esterification by 25-hydroxycholesterol was also found if [14C]mevalonolactone or [3H]cholesterol (the latter presented to the cells in high density lipoproteins) were used as precursors. The stimulatory effect of 25-hydroxycholesterol was maximal after only 15-min incubation and was independent of protein synthesis. After 4 to 6 h of incubation with 25-hydroxycholesterol, its stimulatory effect was reduced significantly, and after 18 h of incubation no stimulation was observed. Thus, the liver cells in some fashion adapt to the continuing presence of 25-hydroxycholesterol. Isolated microsomes prepared from cells previously incubated with 25-hydroxycholesterol showed acyl-CoA:cholesterol acyltransferase activity 3 times that of microsomes from control cells. Incubation of isolated microsomes with 25-hydroxycholesterol increased acyl-CoA:cholesterol acyltransferase activity 2-fold. The net cellular content of ester cholesterol increased after 2 to 6 h incubation of hepatocytes with 25-hydroxycholesterol; there was a net decrease in cellular free cholesterol. Mevalonolactone (10 mM) also stimulated cholesterol esterification and inreased the cellular content of ester cholesterol (3- to 4-fold). The effectiveness of mevalonolactone did not diminish with longer periods of preincubation. Furthermore, the stimulatory effects of 25-hydroxycholesterol and mevalonolactone added together were at least 50 to 100% greater than the effects of either agent alone, suggesting that the mechanisms by which they increase cellular cholesterol esterification are different. Both 25-hydroxycholesterol and mevalonolactone rapidly inhibited microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity. Pure cholesterol had no effect on cellular cholesterol esterification or on 3-hydroxy-3-methylglutaryl-CoA reductase activity at concentrations up to 10 microgram/ml.

It has been suggested that the liver may be a major site for irreversible degradation of low-density lipoprotein (LDL). The disappearance of autologous (125)I-labeled LDL from plasma was compared in intact and in hepatectomized swine. Contrary to expectations, the rate of irreversible removal of LDL from plasma was increased rather then decreased by hepatectomy. These studies suggest that the liver is not a major site for LDL removal. We propose further that the liver (or some function requiring an intact liver) may affect the metabolism of LDL in a manner that prolongs its lifetime in the plasma compartment.

Removal of the terminal sialic acid residues from many serum glycoproteins results in exposure of their penultimate galactose residues and rapid clearance from circulation by the liver. Low-density lipoprotein is a glycoprotein containing 21 galactose and 9 sialic acid residues per particle. Studies in this laboratory and others have shown that both the liver and extrahepatic tissues contribute to the degradation of low-density lipoprotein. This study was undertaken to determine whether desialylation of pig low-density lipoprotein alters its removal from circulation. Low-density lipoprotein was incubated at 37 degrees C with an agarose-bound neuraminidase, proteinase-free, from Clostridium perfringens. After 18 h at pH 5.0, 70% of the sialic acid residues were removed. The desialylated 131I-labelled and native 125I-labelled low-density lipoproteins were simultaneously injected into a pig, and their disappearance from plasma was followed for 96 h. The turnovers of the two were identical. In contrast, neuraminidase-treated fetuin was cleared about 200-fold faster than native fetuin. Studies were also performed in cultured rat hepatocytes. Rates of degradation of native and neuraminidase-treated low-density lipoprotein were similar, whereas asialo-fetuin was degraded at six to ten times the rate of native fetuin. Thus desialylation does not appear to alter low-density-lipoprotein catabolism by hepatic or extrahepatic cells.

Human low density lipoprotein (LDL) covalently conjugated with 200-250 residues of lactose per LDL particle (Lac-LDL) was bound and rapidly taken up by the galactose-specific receptor of rat hepatocytes. Uptake of Lac-LDL was associated with inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase and stimulation of cholesterol esterification. Uptake of native human LDL had no significant effects on these enzyme activities even when the rates of LDL uptake equaled those of Lac-LDL. When injected into rats, Lac-LDL was selectively removed by the liver (98% of injected dose). The hepatic subcellular distribution of simultaneously injected native 125I-labeled LDL and 131I-labeled Lac-LDL differed significantly, Lac-LDL was associated with fractions enriched in lysosomal hydrolases whereas native LDL was found predominantly in the supernatant fraction enriched in lactate dehydrogenase. Chloroquine (0.1 mM) markedly suppressed uptake of Lac-LDL by cultured rat hepatocytes (> 80%) but had only a small effect on uptake of native LDL. Leupeptin (0.625 mM) inhibited degradation of Lac-LDL more than it did degradation of native LDL. Colchicine (0.25 microM) dramatically suppressed uptake of Lac-LDL (> 70%) but did not affect native LDL uptake even at concentrations as high as 10 microM. Uptake of human LDL by rat hepatocytes occurs largely by nonspecific mechanisms, including fluid endocytosis, whereas Lac-LDL, as shown here, is taken up by a specific receptor-mediated mechanism. The results show further that native human LDL, representing an example of a protein taken up nonspecifically, is processed intracellularly by a pathway qualitatively distinct from that for Lac-LDL, an example of a protein taken up by a specific mechanism. Lac-LDL may serve as a vehicle for specifically delivering drugs, hormones, or radioactive compounds to hepatocytes for therapeutic or diagnostic purposes.