High-density Lipoprotein Degradation Research Articles

High insulin level promotes the degradation of high density lipoprotein generation-related functional protein ABCA1 through calpain and proteasome pathway in 3T3-L1 adipocytes

To explore effects and potential mechanisms of high insulin environment on high density lipoprotein (HDL) generation-related functional protein ABCA1. [(3)H] labeled cholesterol efflux from mature 3T3-L1 adipocytes was detected by liquid scintillation counting. ABCA1 mRNA and protein expression in mature 3T3-L1 adipocytes post stimulation with various concentrations of insulin was detected by real-time fluorescence-based quantitative techniques and Western blot, respectively, in the absence and presence of CHX (cycloheximide, CHX), calpeptin (calpain pathway inhibitor) or MG-132 (proteasome pathway inhibitor). Cholesterol efflux rates were reduced post insulin stimulation in a dose-dependent manner ((7.06 ± 0.27)%, (6.59 ± 0.30)%, (6.34 ± 0.24)%, (5.07 ± 0.40)%, and (4.71 ± 0.40)% at 0, 1, 10, 10², and 10³ nmol/L of insulin, P < 0.05). Cholesterol efflux rates decreased in a time-dependent manner post 10³ nmol/L insulin stimulation (6.52 ± 0.30)%, (5.59 ± 0.71)%, (5.44 ± 0.37)%, (4.52 ± 0.32)%, and (4.38 ± 0.33)% at 0, 2, 4, 6, 12 h, respectively). ABCA1mRNA levels were not affected by insulin (P > 0.05). ABCA1 protein level was significantly downregulated in 10³ nmol/L insulin group compared to 0 nmol/L insulin group (P < 0.01). Compared with the 0 h group, ABCA1 protein level was significantly reduced in 6 h group (P < 0.05) and further reduced in 12 h group (P < 0.01). Both calpeptin and MG-132 could partly reduce insulin-induced degradation of ABCA1. Compared with the negative control group, ABCA1 protein levels were significantly upregulated by cotreatment with calpeptin and MG-132, respectively (both P < 0.01). Our data suggest that high insulin level could promote the ABCA1 protein degradation and reduce cholesterol efflux from mature 3T3-L1 adipocytes through calpain and proteasome pathway, thus, produce a circumference not suitable for nascent HDL formation in 3T3-L1 adipocytes.

High-density lipoprotein endocytosis in endothelial cells

To describe the way stations of high-density lipoprotein (HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescence microscopy. HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type I mediated selective uptake without concomitant HDL endocytosis. HDL endocytosis occurs via clathrin-coated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.

A Chinese Hamster Ovarian Cell Line Imports Cholesterol by High Density Lipoprotein Degradation

Plasma high density lipoprotein (HDL) is inversely associated with the development of atherosclerosis. HDL exerts its atheroprotective role through involvement in reverse cholesterol transport in which HDL is loaded with cholesterol at the periphery and transports its lipid load back to the liver for disposal. In this pathway, HDL is not completely dismantled but only transfers its lipids to the cell. Here we present evidence that a Chinese hamster ovarian cell line (CHO7) adapted to grow in lipoprotein-deficient media degrades HDL and concomitantly internalizes HDL-derived cholesterol. Delivery of HDL cholesterol to the cell was demonstrated by a down-regulation of cholesterol biosynthesis, an increase in total cellular cholesterol content and by stimulation of cholesterol esterification after HDL treatment. This HDL degradation pathway is distinct from the low density lipoprotein (LDL) receptor pathway but also degrades LDL. 25-Hydroxycholesterol, a potent inhibitor of the LDL receptor pathway, down-regulated LDL degradation in CHO7 cells only in part and did not down-regulate HDL degradation. Dextran sulfate released HDL bound to the cell surface of CHO7 cells, and heparin treatment released protein(s) contributing to HDL degradation. The involvement of heparan sulfate proteoglycans and lipases in this HDL degradation was further tested by two inhibitors genistein and tetrahydrolipstatin. Both blocked HDL degradation significantly. Thus, we demonstrate that CHO7 cells degrade HDL and LDL to supply themselves with cholesterol via a novel degradation pathway. Interestingly, HDL degradation with similar properties was also observed in a human placental cell line.

SR-BI-mediated High Density Lipoprotein (HDL) Endocytosis Leads to HDL Resecretion Facilitating Cholesterol Efflux

The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ultrasensitive microscopy. In this study we show that this uptake of entire HDL particles is followed by resecretion. After uptake, HDL is localized in endocytic vesicles and organelles en route to the perinuclear area; many HDL-positive compartments were classified as multivesiculated and multilamellated organelles by electron microscopy. By using 125I-labeled HDL, we found that approximately 0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted. 125I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux.

High Density Lipoprotein Catabolism in Primary Cultured Hepatocytes from Daunomycin-Induced Nephrotic Rats

We investigated into HDL (high density lipoprotein) catabolism with primary cultured hepatocytes to elucidate the causes of increased HDL apolipoproteins in the plasma of daunomycin-induced nephrotic rats (D rats). The phospholipid, triglyceride, cholesterol, cholesteryl ester and apolipoprotein contents in HDL increased in D rats compared with control rats (C rats). The uptake (binding plus internalization) of ¹²⁵I-HDL from D rats to two groups of hepatocytes was significantly greater than that of ¹²⁵I-HDL from C rats. Uptake of ¹²⁵I-HDL from D rats to D rats’ hepatocytes was significantly greater than that of ¹²⁵I-HDL from C rats to C rats’ hepatocytes. The degradation of ¹²⁵I-HDL from D rats was greater than that of ¹²⁵I-HDL from C rats using two groups of hepatocytes. These results demonstrated that the uptake and degradation of HDL to D rats’ hepatocytes were greater than those of HDL to C rats’ hepatocytes. The increased HDL apolipoprotein content in the plasma of D rats may not be due to decreased uptake and degradation of HDL in hepatocytes compared with C rats.

Uptake of high-density lipoprotein by Y-organs of the crab Cancer antennarius: III. Evidence for adsorptive endocytosis and the absence of lysosomal processing.

Ecdysteroid hormones in crustaceans are synthesized from cholesterol in the Y-organs. Circulating cholesterol is bound to high-density lipoprotein (HDL). Experiments were conducted to find the mode of cholesterol uptake by Y-organ cells. The working hypothesis is as follows: Cholesterol is taken up by endocytosis of the entire HDL-cholesterol complex. HDL was doubly labeled in the apolipoprotein and cholesterol components with 125I and 3H, respectively. The time courses of uptake of the two labels by Y-organ segments in vitro were parallel throughout 24 hr of incubation. Chloroquine, an inhibitor of lysosomal function, had no effect on the uptake and degradation of HDL. Cycloheximide, an inhibitor of protein synthesis, depressed HDL uptake. Transmission electron microscopy of Y-organ tissue showed features characteristic of adsorptive endocytosis, including coated pits in the plasma membranes and their invagination and vesiculation. Visualization of HDL with immunogold binding showed profiles consistent with endocytosis. Quantitation of the distribution of HDL-gold particles indicated that about 75% were associated with cellular formed elements, but not with lysosomes, and significantly more particles overall were present in cells from de-eyestalked crabs than those from intact crabs. These data demonstrate that (a) cholesterol-carrying HDL is taken into Y-organ cells by adsorptive endocytosis, (b) its uptake is dependent on de novo protein synthesis, (c) its intracellular processing is independent of lysosomal enzymes, and (d) its uptake is depressed by the molt-inhibiting hormone secreted by the eyestalks.

Cellular uptake and catabolism of high-density-lipoprotein triacylglycerols in human cultured fibroblasts: degradation block in neutral lipid storage disease

High-density lipoprotein (HDL)-[3H]triolein (i.e. [3H]triolein incorporated into reconstituted HDL) was taken up by cultured fibroblasts through an apparently saturable process, competitively inhibited by non-labelled HDL and independent of the LDL receptor. Using 125I-HDL and HDL-[3H]triolein, binding experiments (at 0 degrees C) followed by a short-time 'chase' at 37 degrees C showed that 125I radioactivity was rapidly released in the culture medium (as trichloroacetic acid-precipitable material), whereas 3H radioactivity remained associated with the cell. The cell-associated HDL-[3H]triolein was rapidly degraded in normal fibroblasts, and the liberated [3H]oleic acid was incorporated into newly biosynthesized phospholipids. In Wolman-disease fibroblasts HDL-[3H]triolein was degraded at a normal rate, and thus independently of the lysosomal compartment. In contrast, the degradation of HDL-[3H]triolein was blocked in fibroblasts from Neutral Lipid Storage Disease (NLSD), similarly to that of endogenously biosynthesized triacylglycerols [Radom, Salvayre, Nègre, Maret and Douste-Blazy (1987) Eur. J. Biochem. 164, 703-708]. Trypsin-treated HDL-[3H]triolein was also taken up by cells and degraded quite similarly to HDL-[3H]triolein. In conclusion, all these data taken together suggest that HDL-[3H]triolein is: (i) associated with the cell through a process independent of intact apolipoprotein (apo) As, thus probably independent of an apoA-receptor-mediated uptake; (ii) internalized by cells, whereas 125I-apoAs are released in the culture medium; (iii) directed to the same non-lysosomal catabolic pool (blocked in NLSD) as for endogenously biosynthesized triacylglycerols.

Cytoplasmic triacylglycerols and cholesteryl esters are degraded in two separate catabolic pools in cultured human fibroblasts

The sources and the catabolic pathways of cytoplasmic pools of triacylglycerols and cholesteryl esters have been comparatively investigated in cultured fibroblasts from normal subjects and from patients affected with neutral lipid storage disease (NLSD) and Wolman disease (WD). (i) Endogenously biosynthesized triacylglycerols and cholesteryl esters were degraded extra-lysosomaily since they were catabolized at similar rates in normal and in WD fibroblasts. In NLSD fibroblasts, the degradation of endogenous triacylglycerols was severely deficient, whereas that of endogenous cholesteryl esters was in the normal range, (ii) Reconstituted high density lipoproteins (HDL) containing radiolabelled [ 3H]triolein and cholesteryl [ 14C]oleate were taken up by cultured fibroblasts and rapidly degraded in a non-lysosomal compartment. In NLSD fibroblasts the degradation of HDL-[ 3H]triolein was blocked whereas that of HDL-[ 14C]cholesteryl oleate was in the normal range. These data suggest that: (i) the cytoplasmic pools of triacylglycerols and cholesteryl esters originate from HDL uptake and from endogenous biosynthesis as well; (ii) cytoplasmic (non-lysosomal) triacylglycerols and cholesteryl esters are degraded by two separate catabolic pathways.

Extralysosomal degradation of high-density lipoproteins in a human hepatoma cell line, Mahlavu

Lipoprotein metabolism has been investigated in a novel human hepatoma cell line, Mahlavu, which has been reported to possess the characteristics of hepatocytes. Low-density lipoprotein (LDL) was degraded by Mahlavu cells. LDL taken up by the cells suppressed intracellular cholesterol biosynthesis and promoted cholesterol esterification in a manner similar to that of HepG2 cells. High-density lipoprotein (HDL) was also degraded by Mahlavu cells, whereas it was not degraded by fibroblasts. We compared the mode of intracellular metabolism of HDL to that of LDL. In contrast to the LDL receptor pathway, the degradation of HDL was not inhibited by 100 μM chloroquine added to the medium, indicating that the degradation may not occur in lysosomes. Cholesterol taken up as HDL-cholesterol by the Mahlavu cells had no effect on the intracellular biosynthesis of cholesterol nor on cholesterol esterification. The conditioned media in which Mahlavu cells had been cultured did not promote the degradation of HDL, suggesting that HDL is degraded intracellularly. These data suggest that HDL is taken up and degraded by the liver cells in contrast to extrahepatic peripheral cells such as fibroblasts and macrophages in which the degradation of HDL does not occur. The results indicate that HDL-associated cholesterol may be processed via a pathway different from that of LDL metabolism and that the degradation of HDL occurs extralysosomally.

Characterization in vitro of interaction of human apolipoprotein E-free high density lipoprotein with human hepatocytes.

Characterization of the interaction of iodinated apolipoprotein (apo) E-free high density lipoprotein (HDL) with cultured human hepatocytes provides evidence for a saturable, Ca2(+)-independent, high affinity binding site with an apparent km value of 20 micrograms/ml of apolipoprotein. Nitrated HDL and low density lipoprotein (LDL) did not compete for the binding of HDL, in contrast to very low density lipoprotein (VLDL). It is suggested that VLDL competition is exerted by the presence of apo Cs. Degradation of HDL was relatively low and in some cases not detectable. In cases where degradation was found, inhibitors of the lysosomal pathway of protein degradation had no effect, while LDL degradation was inhibited more than 80%. In the presence of 10 microM of monensin, the cell-association of HDL was unaffected, but the degradation was inhibited by 30%. Under similar conditions, LDL association was inhibited by 40% and LDL degradation, by 90%. Incubation of human hepatocytes with fluorescently labeled HDL (Dil-HDL) revealed (in contrast to Dil-LDL) mainly strong membrane-bound fluorescence and hardly any labeling of small intracellular vesicles. It is concluded that human hepatocytes possess a specific high affinity site for human HDL with recognition properties similar to those described earlier on rat hepatocytes. No evidence that the binding of HDL is actively coupled to uptake and lysosomal degradation could be obtained, indicating that binding of LDL and HDL to human hepatocytes is coupled differently to intracellular pathways.

Ecdysteroid biosynthesis in the crustacean Y‐organ and control by an eyestalk neuropeptide

AbstractThe endocrine axis is described that controls growth, molting, and regeneration in crustaceans: The ecdysial glands (Y‐organs), sources of ecdysteroid hormones, and eyestalk neurosecretory centers, sources of a putative peptide (molt‐inhibiting hormone, MIH) that exerts negative control of Y‐organ function. Current knowledge of the physiology of these components is briefly reviewed, and in this context Y‐organs are discussed as useful models for study of steroidogenesis generally in animals at the cellular and molecular levels. Data are emphasized that demonstrate the utilization of cholesterol by Y‐organs for biosynthesis of at least two ecdysteroid secretion products, and suppression of the process by MIH. Evidence from radioactive tracer experiments is presented to show that (a) cholesterol in circulating hemolymph and in vitro in the presence of hemolymph serum is quantitatively bound to high‐density lipoprotein (HDL), (b) Y‐organs take up cholesterol by an active process, and (c) uptake is greatly accelerated in the absence of MIH (eyestalks absent) or inhibited by eyestalk extract. Results of in vitro experiments with 125I‐labeled HDL suggest that the HDL‐cholesterol complex is taken into Y‐organ cells mediated by receptors for the HDL apoprotein. Measured as a function of HDL concentration in the medium, cellular acid‐precipitable radioactivity (HDL uptake), acid‐soluble radioactivity (HDL degradation), and ecdysone secretion each exhibited saturation kinetics. Also, at saturation levels of external HDL, uptake, degradation, and secretion were significantly higher in Y‐organs from de‐eyestalked crabs than in glands from intact crabs.

The effects of phthalimide and saccharin derivatives on low-density lipoprotein (LDL) and high-density lipoprotein (HDL) receptor activity and related enzyme activities.

The hypolipidemic agents, phthalimide, saccharin, o-(N-phthalimido) acetophenone, N-(p-chlorobenzoyl) sulfamate, and o-chlorobenzylsulfonamide affected low-density lipoprotein (LDL) and high-density lipoprotein (HDL) receptor activity and lipoprotein degradation. In isolated rat hepatocytes, rat aorta foam cells, and human fibroblasts, LDL receptor activity, which is dependent on apo-B and -E, was inhibited by the drugs in a dose-dependent manner. LDL degradation was accelerated in the hepatocytes, while it was inhibited in aorta cells and fibroblasts. The drugs enhanced HDL receptor activity, dependent on apo-E and -A1, and HDL degradation in the hepatocytes, whereas in fibroblasts and aorta cells HDL receptor binding and degradation were suppressed. In parallel, activities of acyl CoA acyl transferase, sn-glycerol-3-phosphate acyl transferase, and heparin-induced lipoprotein lipase decreased and activities of HMG-CoA reductase and cholesterol oleate-ester hydrolase increased. In fibroblasts the presence of drugs enhanced HDL binding of intracellular cholesterol. In vivo studies demonstrated that phthalimide and saccharin treatment enhanced the clearance of HDL and decreased the clearance of LDL from the serum of rats. The results suggest that the mode of action of the agents is to modulate the lipoprotein receptor and, thereby, the clearance of lipids from peripheral tissue as part of the hypolipidemic activity.

Significance of hepatic triglyceride lipase activity in the regulation of serum high density lipoproteins in type II diabetes mellitus.

We investigated the regulation of serum high density lipoprotein (HDL) cholesterol metabolism in patients with type II diabetes mellitus by determining the activities of the two lipolytic enzymes that play major roles in the production and degradation of HDL. The activity of lipoprotein lipase (LPL), the enzyme responsible for HDL cholesterol production, and the activity of hepatic triglyceride lipase (HTGL), the enzyme that facilitates the catabolism of HDL, were measured in plasma obtained after iv injection of heparin. Thirty patients were selected to represent a wide range of serum HDL cholesterol concentrations (low, normal, and high HDL cholesterol groups). Mean lipoprotein lipase activity was similar in all three groups [122 +/- 10 (SEM) U/mL in the low HDL, 141 +/- 11 U/mL in the normal HDL, and 148 +/- 30 U/mL in the high HDL group]. Mean HTGL activity was markedly decreased in the high HDL group; the mean values were 346 +/- 28 U/mL in the low HDL, 320 +/- 25 U/mL in the normal HDL, and 191 +/- 23 U/mL in the high HDL groups, respectively. Body weight and insulin requirement correlated directly with HTGL activity and inversely with serum HDL cholesterol levels. These findings suggest that in type II diabetes mellitus low serum HDL cholesterol levels may be due to an increased rate of clearance by HTGL.

Cholesterol regulates high-density lipoprotein interaction with isolated epithelial cells of human small intestine

The effect of cholesterol on the interaction of high-density lipoprotein (HDL) with isolated human small intestine epithelial cells (enterocytes) was studied. 125I-labeled HDL 3 binding by these cells was enhanced in response to cholesterol loading of the cells in a time- and dose- dependent manner. Preincubation of the cells with cholesterol led to the enhancement both of the number of binding sites and the binding affinity. The enhancement of binding correlated with the cellular cholesterol content. Cycloheximide (0.5 mM) inhibited uptake of cholesterol by enterocytes and blocked its effect on 125I-labeled HDL 3 binding. The effect of cholesterol on 125I-labeled HDL 3 degradation had a double-phase character. At concentrations 10–20 μg/ml, the degradation rate was rapidly elevated, but further increase in cholesterol concentration led to a fall in the degradation rate. Incubation of enterocytes with HDL 3 resulted in the efflux of cholesterol from cells and its incorporation into HDL 3. The results obtained make it possible to assume that binding and degradation of 125I-labeled HDL 3 by human enterocytes are independently regulated by the cell total cholesterol content. Binding of HDL by enterocytes may result both in the degradation of HDL and cholesterol efflux from cells.

Specific high affinity binding and degradation of high density lipoproteins by isolated epithelial cells of human small intestine

The interaction of high density lipoproteins (HDL) with isolated epithelial cells of human small intestine (enterocytes) was studied. 125I-HDL 3 (density = 1,125 to 1,126 g/cm 3) exhibit a high-affinity (Kd = 8.3 × 10 −8. Bmax = 886 ng/mg cell protein), saturable and reversible binding to isolated enterocytes. In the presence of excess unlabeled HDL 3, the cell surface-bound 125I-HDL 3 are released into the medium nondegraded. Treatment of cells with pronase does not affect 125I-HDL 3 binding. The binding is accompanied with internalization and degradation of 125I-HDL 3. Chloroquine inhibits the degradation and increases 125I-HDL 3 uptake. A threefold excess of HDL 3 and HDL 2 inhibits the binding and degradation of 125I-HDL 3 by 60%, whereas a 20-fold excess of low density lipoproteins (LDL), only by 20%. HDL 3 (20 to 1,000 μg/mL) stimulates the synthesis of sterols and inhibits sterol ester synthesis in enterocytes. The obtained results make it possible to assume that epithelial cells of the small intestine may participate in the catabolism of HDL in human organism.

Modifications and degradation of high density lipoproteins.

It is evident that lipoprotein modifications, degradation and clearance from plasma and interstitial compartments involves both cellular and extracellular processing. Cellular uptake of the intact particle as a whole and/or selective removal of constituent apoproteins and lipids by various parenchymal cells goes on continuously. Regulation of these processes undoubtedly varies tissue to tissue and much remains to be clarified in human tissues in vivo. The metabolic effects of chemical, proteolytic, and lipolytic modification of lipoproteins secondary to transient cellular encounters (e.g. during transit through endothelial barriers, or reversible binding to cells) on apolipoprotein clearance remains to be defined. It is likely that multiple post-secretory modifications occur and together represent subtle regulatory events that modulate lipid shuttle functions and cellular targetting properties of HDL particles.

Involvement of microtubules in lipoprotein degradation and utilization for steroidogenesis in cultured rat luteal cells.

Cells isolated from superovulated rat ovaries metabolize low density lipoprotein (LDL) and high density lipoprotein (HDL) of human or rat origin and use the lipoprotein-derived cholesterol as a precursor for progesterone production. Under in vitro conditions, both lipoproteins are internalized and degraded in the lysosomes, although degradation of HDL is of lower magnitude than that of LDL. In this report we have examined the role of cellular microtubules in the internalization and degradation of human LDL and HDL in cultured rat luteal cells. The microtubule depolymerizing agents colchicine, podophyllotoxin, vinblastine, and nocodazole as well as taxol, deuterium oxide, and dimethyl sulfoxide, which are known to rapidly polymerize cellular tubulin into microtubules, were used to block the function of microtubules. When these antimicrotubule agents were included in the incubations, degradation of the apolipoproteins of [125I]iodo-LDL and [125I]iodo-HDL by the luteal cells was inhibited by 50-85% compared to untreated control values. Maximum inhibitory effects were observed when the cells were preincubated with the inhibitor for at least 4 h at 37 C before treatment with the labeled lipoprotein. Lipoprotein-stimulated progesterone production by luteal cells was also inhibited by 50% or more in the presence of antimicrotubule agents. However, basal and hCG-stimulated progesterone production were unaffected by these inhibitors. The binding of [125I]iodo-LDL and [125I]iodo-HDL to luteal cell plasma membrane receptors was not affected by the microtubule inhibitors. Although binding was unaffected and degradation was impaired in the presence of the inhibitors, there was no detectable accumulation of undegraded lipoprotein within the cells during the 24 h of study. From this study we conclude that the uptake and utilization of LDL and HDL by cultured rat luteal cells are mediated by cellular microtubules.

Comparison of binding and degradation of high density lipoprotein by intestinal mucosal cells, fibroblasts and adrenal cortical cells in culture

In this study we have compared the binding and degradation of human high density lipoprotein (HDL 3), devoid of apolipoprotein E, by rat intestinal (mucosal) and adrenal cells and by human fibroblasts in culture. Binding of HDL 3 to adrenal and intestinal cells was characterised by saturable, specific processes whereas skin fibroblasts from normal humans did not convincingly demonstrate saturability and had a lower affinity and capacity compared with adrenal and intestinal cells. Post-receptor events also appeared to differ. Cells from the adrenal cortex and gut showed similar binding affinities for HDL 3, but the capacity for binding and for degrading HDL 3 was much higher with intestinal cells. The large amounts of HDL degraded by intestinal cells suggest a specific role for the gut in HDL catabolism, and that, in the rat, intestinal cholesterol may be derived from circulating HDL. Finally, it is suggested that rat adrenal cortical and intestinal mucosal cells possess surface receptors for HDL 3 which differ from the LDL receptor.

Cholesterol metabolism in cancer cells in monolayer culture: VI. Metabolism of high-density lipoprotein

The metabolism of high-density lipoprotein (HDL) in cells of five human cancer cell lines maintained in monolayer culture was investigated. In cells of some of the lines there was evidence of high-affinity binding sites for HDL, whereas in others this could not be demonstrated. However, in one cell line, viz., HEC-B-296 (human endometrial carcinoma), degradation of the protein component of HDL was demonstrated. The proteolytic activity was specific for HDL in so far as human serum albumin was not degraded by these cells. However, this degradative process did not involve internalization of the HDL molecule and degradation was not mediated by lysosomal proteolytic enzymes. HDL, when present in the medium, did not affect the degradation of low-density lipoprotein and low-density lipoprotein did not affect the degradation of HDL. HDL did not affect significantly cholesterol biosynthesis or cholesteryl ester biosynthesis as estimated from the activity of the regulatory enzymes, 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl-CoA:cholesterol acyltransferase. The degradation of HDL by HEC-B-296 cells was inhibited, to various degrees, when trypsin inhibitor or a protease inhibitor such as leupeptin, was present in the culture medium. It is concluded that degradation of the protein component of HDL by human neoplastic cells of the HEC-B-296 line was the result of activity of a proteolytic enzyme that is present on the external surface of the cells.

The interaction of the high-density lipoprotein with cultured cells of bovine vascular endothelium.

Previous studies have shown that high‐density lipoprotein (HDL) is mitogenic for low‐density vascular endo thelial cell cultures. In contrast, low‐density lipoprotein(LDL) at physiological concentration is cytotoxic for this cell type [Tauber et al. (1980) J.Clin.Invest. 66, 696‐709]. In order to relate these observations to the physico‐chemical events occurring after the interaction of these lipoproteins with the cells, we have studied the binding, internalization, and degradation of HDL as a function of cell density and organization and have compared the results to those obtained with LDL.Cultured vascular endothelial cells bind HDL to an extent which, on a molar basis, is similar to that of LDL. Since an 80% loss of the LDL receptor sites in cells that were preexposed to LDL was not associated with a detectable decrease in HDL binding, it is likely that HDL and LDL binding sites exist on the cell surface as distinct entities A 5‐10 fold molar excess of LDL over 125 I‐HDL was almost as effective as a similar excess of unlabeled HDL in reducing the binding of 125, indicationg that LDL can comete with HDL for HDL binding sites. In contrast, 125 I‐LDL binding and uptake were reduced by less than 20% in the presence of a 50‐fold molar excess of unlabeled HDL. Both uptake and degradation of HDL were 10‐20 fold lower than those of LDL. HDL degradation was only slightly or not inhibited by chloroquine at a concentration which almost fully inhibited the degradation of LDL. Confluent and highly orgainized endothelial cell cultures bind HDL and LDL particles to an extent which is half of the amount bound to sparse or subconfluent culture However. The formation of a resting endothelial cell monlayer was associated with only a twofold decrease in the uptake of HDL, as compard to a 10‐20‐fold decrease in the uptake and degradation of LDL. In both sparse and confluent endothelial cultures, uptake via fluid pinocytosis non‐specific adscorptive endocytosis could account for most of the HDL uptake. The different pathways of internalization of HDL when added at high concentration to sparse and actively growing cultures.