Regulation of low-density lipoprotein receptors in the human hepatoma cell line Hep G2

The human hepatoma cell line Hep G2 can be maintained in continuous culture and secretes numerous plasma proteins and lipoproteins into the medium. To better characterize cholesterol homeostasis in these cells we have examined the binding, internalization and degradation of [125I]LDL by cultured Hep G2 cells. Hep G2 cells express high-affinity low-density lipoprotein (LDL) receptors which facilitate the binding, internalization and degradation of [125I]LDL; these receptors can be induced by growth in LDL-depleted medium and repressed by further incubation in medium supplemented with LDL. The degradation of [125I]LDL by derepressed Hep G2 cells was inhibited by greater than 90% by monensin. Incubation of Hep G2 cells in the presence of increasing concentrations of LDL also inhibited cholesterol biosynthesis. Our results indicate that Hep G2 cells possess high affinity LDL receptors which are subject to metabolic regulation and suggest that this cell line affords a valuable model to further examine cholesterol and lipoprotein metabolism in human liver cells.     

Effect of low density lipoproteins, high density lipoproteins, and cholesterol on apolipoprotein A-I mRNA in Hep G2 cells

We have utilized the human hepatocellular carcinoma cell line, Hep G2, to study the effects of low density lipoproteins (LDL), high density lipoproteins (HDL), and free cholesterol on apolipoprotein (apo) A-I mRNA levels. Incubation of the Hep G2 cells with LDL and free cholesterol led to a significant increase in the cellular content of cholesterol without any effect on the yield of total RNA or in the cellular protein content. Our studies established that incubation with LDL or free cholesterol increased the relative levels of apoA-I mRNA in the Hep G2 cells. In contrast with cholesterol loading, HDL had the effect of lowering the levels of apoA-I mRNA. These results indicate the LDL and HDL pathways as well as intracellular cholesterol may be important in apoA-I gene expression and regulation.     

Cellular free cholesterol in Hep G2 cells is only partially available for down-regulation of low-density-lipoprotein receptor activity.

We have previously shown that in Hep G2 cells and human hepatocytes, as compared with fibroblasts, the low-density lipoprotein (LDL) receptor activity is only weakly down-regulated after incubation of the cells with LDL, whereas incubation with high-density lipoproteins (HDL) of density 1.16-1.20 g/ml (heavy HDL) strongly increased the LDL-receptor activity. To elucidate this difference between hepatocytes and fibroblasts, we studied the cellular cholesterol homoeostasis in relation to the LDL-receptor activity in Hep G2 cells. (1) Interrupting the cholesteryl ester cycle by inhibiting acyl-CoA: cholesterol acyltransferase (ACAT) activity with compound 58-035 (Sandoz) resulted in an enhanced LDL-mediated down-regulation of the receptor activity. (2) The stimulation of the receptor activity by incubation of the cells with cholesterol acceptors such as heavy HDL was not affected by ACAT inhibition. (3) Incubation of the Hep G2 cells with LDL, heavy HDL or a combination of both grossly affected LDL-receptor activity, but did not significantly change the intracellular content of free cholesterol, suggesting that in Hep G2 cells the regulatory free cholesterol pool is small as compared with the total free cholesterol mass. (4) We used changes in ACAT activity as a sensitive (indirect) measure for changes in the regulatory free cholesterol pool. (5) Incubation of the cells with compactin (2 microM) without lipoproteins resulted in a 4-fold decrease in ACAT activity, indicating that endogenously synthesized cholesterol is directed to the ACAT-substrate pool. (6) Incubation of the cells with LDL or a combination of LDL and heavy HDL stimulated ACAT activity 3-5 fold, whereas incubation with heavy HDL alone decreased ACAT activity more than 20-fold. Our results suggest that in Hep G2 cells exogenously delivered (LDL)-cholesterol and endogenously synthesized cholesterol are primarily directed to the cholesteryl ester (ACAT-substrate) pool or, if present, to extracellular cholesterol acceptors (heavy HDL) rather than to the free cholesterol pool involved in LDL-receptor regulation.     

The metabolism in vitro of human low-density lipoprotein by the human hepatoma cell line Hep G2.

The human hepatoma cell line Hep G2 was studied with respect to metabolism of human low-density lipoprotein (LDL). The Hep G2 cells bind, take up and degrade human LDL with a high-affinity saturable and with a low-affinity non-saturable component. The high-affinity binding possesses a KD of 25 nM-LDL and a maximal amount of binding of about 70 ng of LDL-apoprotein/mg of cell protein. The high-affinity binding, uptake and degradation of LDL by Hep G2 cells is dependent on the extracellular Ca2+ concentration and is down-regulated by the presence of fairly high concentrations of extracellular LDL. Incubation of the Hep G2 cells with LDL results in suppression of the intracellular cholesterol synthesis. It is concluded that the human hepatoma cell line Hep G2 possesses specific LDL receptors similar to the LDL receptors demonstrated on extrahepatic tissue cells.     

Estrogens induce low-density lipoprotein receptor activity and decrease intracellular cholesterol in human hepatoma cell line Hep G2

Administration of estrogens in pharmacologic doses to rats and rabbits induces hepatic low-density lipoprotein (LDL) receptor activity. To determine if estrogens can regulate LDL receptor activity in human cells, 125I-LDL binding and ligand blotting studies were performed with the cell line Hep G2, well-differentiated cells derived from a human hepatoma, and with normal human fibroblasts. Addition of estradiol to Hep G2 cells growing in lipoprotein-deficient medium increased cell surface receptor activity by 141%, whereas fibroblast receptors were slightly reduced. Measurement of LDL internalization and degradation showed that estradiol induced the entire LDL receptor pathway and not simply surface receptors for LDL. Scatchard analysis of specific binding data in Hep G2 cells revealed that increased LDL receptor activity was due to high-affinity binding. When Hep G2 cells were incubated with LDL as well as estradiol, estradiol induction of LDL receptor activity did not occur. Estrogen treatment reduced Hep G2 free cholesterol content by 24% as determined by gas-liquid chromatography but had no significant effect on fibroblast free cholesterol, suggesting that estrogens may induce Hep G2 LDL receptor activity indirectly by lowering intracellular cholesterol. LDL receptor activity in Hep G2 cells grown in the absence of estradiol was resistant to down-regulation by LDL; incubation of cells with LDL for 48 h reduced receptor activity by only 25.8% in Hep G2 cells compared to 80.3% in fibroblasts. The Hep G2 LDL receptor was shown to be biochemically similar to the fibroblast receptor by ligand blotting and immunoblotting with IgG-C7, a monoclonal antibody to the extrahepatic LDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired hepatocyte binding, uptake and degradation of glucosylated low-density lipoproteins

The catabolism of low-density lipoproteins (LDL), the major cholesterol-carrying lipoproteins in plasma, is mediated in part via a high-affinity uptake pathway in the liver. Non-enzymatic glucosylation of lysine residues of apolipoprotein B, the major protein of LDL, blocks receptor-mediated uptake of LDL by fibroblasts and endothelial cells. We investigated the effect of the degree of glucosylation on the binding, uptake and degradation of radioiodinated LDL by the human hepatoma cell line Hep G2. Human LDL was glucosylated with 250 mM glucose and 30 mM cyanoborohydride at 37 degrees C. Incubations ranging from 3 to 48 h in duration resulted in the formation of 6-27% of glucitol-lysine adducts as demonstrated by coincubation with [14C]glucose. The degree of glucose incorporation corresponded to the extent of inhibition of binding, uptake and degradation of LDL (10-90%). The data are consistent with the view that glucosylation of LDL markedly impairs their catabolism. This phenomenon may be related to the pathophysiology of the premature atherosclerosis observed in diabetes mellitus.     

Lipoprotein secretion by the human hepatoma cell line Hep G2: differential rates of accumulation of apolipoprotein B and lipoprotein lipids in tissue culture media in response to albumin, glucose and oleate

Serum low-density lipoprotein (LDL) concentration is a major determinant of susceptibility to the development of atherosclerosis. A major component of the protein moiety of LDL and its precursor very-low-density lipoprotein is apolipoprotein B (apo B). The human hepatoma cell line, Hep G2, was used as a model for the investigation of mechanisms which control hepatic secretion of the apo B and lipid components of lipoproteins. Using a sensitive immunoradiometric assay for apo B developed in this laboratory, we showed that bovine serum albumin inhibited and glucose, and fatty acids enhanced the rate of accumulation of apo B in the culture medium of Hep G2 cells. However, these substances did not necessarily affect LDL lipids in the same way as apo B. This finding appeared to be due to Hep G2 cells expressing lipase activities which led to triacylglycerol and phospholipid hydrolysis and lipid reuptake. Reuptake of apo B also occurred, but its rate of accumulation in the culture medium suggested it was a closer reflection of its true secretory rate.     

Niacin and cholesterol: role in cardiovascular disease (review)

Niacin has been widely used as a pharmacologic agent to regulate abnormalities in plasma lipid and lipoprotein metabolism and in the treatment of atherosclerotic cardiovascular disease. Although the use of niacin in the treatment of dyslipidemia has been reported as early as 1955, only recent studies have yielded an understanding about the cellular and molecular mechanism of action of niacin on lipid and lipoprotein metabolism. In brief, the beneficial effect of niacin to reduce triglycerides and apolipoprotein-B containing lipoproteins (e.g., VLDL and LDL) are mainly through: a) decreasing fatty acid mobilization from adipose tissue triglyceride stores, and b) inhibiting hepatocyte diacylglycerol acyltransferase and triglyceride synthesis leading to increased intracellular apo B degradation and subsequent decreased secretion of VLDL and LDL particles. The mechanism of action of niacin to raise HDL is by decreasing the fractional catabolic rate of HDL-apo AI without affecting the synthetic rates. Additionally, niacin selectively increases the plasma levels of Lp-AI (HDL subfraction without apo AII), a cardioprotective subfraction of HDL in patients with low HDL. Using human hepatocytes (Hep G2 cells) as an in vitro model system, recent studies indicate that niacin selectively inhibits the uptake/removal of HDL-apo AI (but not HDL-cholesterol ester) by hepatocytes, thereby increasing the capacity of retained HDL-apo AI to augment cholesterol efflux through reverse cholesterol transport pathway. The studies discussed in this review provide evidence to extend the role of niacin as a lipid-lowering drug beyond its role as a vitamin.     

Complete down-regulation of low-density-lipoprotein-receptor activity in the human hepatoma cell line Hep G2 by beta-migrating very-low-density lipoprotein and non-lipoprotein cholesterol. Different cellular regulatory pools of cholesterol.

Regulation of low-density-lipoprotein-receptor activity by low-density lipoprotein (LDL), cholesteryl-ester-rich beta-migrating very-low-density lipoprotein (beta-VLDL) and non-lipoprotein cholesterol was investigated in the human hepatoma cell line Hep G2. Competition studies indicate that LDL and beta-VLDL are bound to the same recognition site, tentatively the LDL receptor. The regulatory response of the LDL receptor upon prolonged incubation with LDL or beta-VLDL was, however, markedly different. 22 h preincubation of Hep G2 cells with excess LDL caused a partial down regulation to 31% of the initial level of the high-affinity association of LDL and 26% of the high-affinity degradation of LDL, while with beta-VLDL a complete down regulation of the LDL-receptor activity is observed. Preincubation of Hep G2 cells with beta-VLDL for 22 h led to a fourfold increase in intracellular cholesterol esters and a twofold increase in acyl-coA:cholesterol acyltransferase activity. With LDL, the amount of intracellular cholesterol esters is increased 1.6-fold. The more effective down regulation of LDL receptors by beta-VLDL as compared to LDL can be explained by the more effective intracellular cholesterol delivery with beta-VLDL than with LDL. Preincubation of Hep G2 cells for 22 h with acetylated LDL hardly influenced the LDL-receptor activity. Non-lipoprotein cholesterol, however, caused a complete down regulation of LDL-receptor activity at even lower extracellular cholesterol concentrations than with beta-VLDL. The complete down regulation of LDL receptors by non-lipoprotein cholesterol is not accompanied by a significant increase in acyl-coA:cholesterol acyltransferase activity, while the intracellular cholesterol ester concentration is only increased 1.6-fold. It is suggested that the effectiveness of non-lipoprotein cholesterol to regulate LDL receptors is caused by its efficiency to reach the sterol regulatory site. The inability of LDL to down regulate its receptor completely can thus be explained by the inability of LDL to deliver cholesterol adequately at the intracellular regulatory site of the LDL receptor. The observed complete down regulation of the LDL receptor by beta-VLDL may be responsible for the cholesterol-rich-diet induced, complete down regulation of LDL-receptor-mediated clearance of LDL in vivo.     

Characterization of lipoproteins produced by the human liver cell line, Hep G2, under defined conditions

Confluent monolayers of the human hepatoblastoma-derived cell line, Hep G2, were incubated in serum-free medium. Conditioned medium was ultracentrifugally separated into d less than 1.063 g/ml and d 1.063-1.20 g/ml fractions since very little VLDL was observed. The d less than 1.063 g/ml fraction was examined by electron microscopy; it contained particles of 24.5 +/- 2.3 nm diameter, similar in size to plasma LDL; a similar size was demonstrated by nondenaturing gradient gel electrophoresis. These particles possessed apoB-100 only. The d less than 1.063 g/ml fraction had a lipid composition unlike that of plasma LDL; unesterified cholesterol was elevated, there was relatively little cholesteryl ester, and triglyceride was the major core lipid. The d 1.063-1.20 g/ml fraction was heterogeneous in size and morphology. Electron microscopy revealed discoidal particles (14.9 +/- 3.2 nm long axis and 4.5 +/- 0.2 nm short axis) as well as small spherical ones (7.6 +/- 1.4 nm diameter). Nondenaturing gradient gel electrophoresis consistently showed the presence of peaks at 13.4 11.9, 9.7, and 7.4 nm. The latter peak was conspicuous and probably corresponded to the small spherical structures seen by electron microscopy. Unlike plasma HDL, Hep G2 d 1.063-1.20 g/ml lipoproteins contained little or no stainable material in the (HDL3a)gge region by gradient gel electrophoresis. Hep G2 d 1.063-1.20 g/ml lipoproteins differed significantly in composition from their plasma counterparts; unesterified cholesterol and phospholipid were elevated and the mole ratio of unesterified cholesterol to phospholipid was 0.8. Cholesteryl ester content was extremely low. ApoA-I was the major apolipoprotein, while apoE was the next most abundant protein; small quantities of apoA-II and apoCs were also present. Immunoblot analysis of the d 1.063-1.20 g/ml fraction after gradient gel electrophoresis showed that apoE was localized in the larger pore region of the gel (apparent diameter greater than 12.2 nm); the apoA-I distribution in this fraction was very broad (7.1-12.2 nm), and included a distinct band at 7.4 nm. Immunoblotting after gradient gel electrophoresis of concentrated medium revealed that a significant fraction of apoA-I in the uncentrifuged medium was in a lipid-poor or lipid-free form. This cell line may be a useful model for investigating the metabolism of newly formed HDL.     

High-carbohydrate diets affect the size and composition of plasma lipoproteins in hamsters (Mesocricetus auratus)

High-carbohydrate diets reduce plasma low-density lipoprotein (LDL)-cholesterol but also provoke the appearance of an atherogenic lipoprotein profile (ALP). Characterized by high plasma triglyceride, small dense LDL, and reduced high-density lipoprotein (HDL) cholesterol, an ALP is associated with insulin resistance. Despite extensive use of the fructose-fed hamster as a model of insulin resistance, little is known about changes that occur in the physical properties of circulating lipoproteins. Therefore, we investigated the metabolic and physical properties of lipoproteins in hamsters fed high-carbohydrate diets of varying complexity (60% carbohydrate as chow, cornstarch, or fructose) for 2 wk. Hamsters fed the high-fructose diet showed significantly increased very- low-density lipoprotein (VLDL)-triglyceride (92.3%), free cholesterol (68.6%), and phospholipid (95%), whereas apolipoprotein B levels remained unchanged. Median diameter of circulating VLDL was larger in fructose-fed hamsters (63 nm) than in cornstarch-fed hamsters. Fructose feeding induced a 42.5% increase LDL-triglyceride concurrent with a 20% reduction in LDL-cholesteryl ester. Compositional changes were associated with reduced LDL diameter. In contrast, fructose feeding caused elevations in all HDL fractions. The physical properties of apolipoprotein-B-containing lipoprotein fractions are similar between fructose-fed hamsters and humans with ALP. However, metabolism of high-density lipoprotein appears to differ in the 2 species.     

Cholesterol metabolism. Effect of 26-thiacholesterol and 26-aminocholesterol, analogues of 26-hydroxycholesterol, on cholesterol synthesis and low-density-lipoprotein-receptor binding.

1. The effects of 26-aminocholesterol and 26-thiacholesterol on cholesterol synthesis and LDL (low-density lipoprotein)-receptor activity were compared with naturally occurring 26-hydroxycholesterol utilizing both human fibroblasts and hepatoma (Hep G2) cells. 2. At equimolar concentrations (0.625 microM), down-regulation of LDL-receptor activity and cholesterol synthesis was greater with human fibroblasts than with Hep G2 cells. 3. At much higher concentrations (5-20 microM) the 26-thia analogue had little effect on either cholesterol synthesis or LDL-receptor activity.     

Dioxin alters the human low-density and very low-density lipoprotein structure with evidence for specific quenching of Trp-48 in apolipoprotein C-II

The intercellular transport of cholesterol and triglycerides via lipoproteins interacting with their receptors is a critical component in human lipid metabolism. The delivery of cholesterol to cells is accomplished primarily through low-density lipoproteins (LDLs), while the transport of fatty acids to adipose and muscle tissue is accomplished primarily through the actions of very low-density lipoproteins (VLDLs). Disruption of lipoprotein structure leading to impaired binding between these lipoproteins and their obligate receptors is a known risk factor for cardiovascular disease. Because of recent investigations linking 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in humans with coronary artery disease, investigations have been carried out by fluorescence and circular dichroism to evaluate conformational changes in LDL and VLDL structure upon binding of TCDD. These studies demonstrate that, at a molar ratio of three TCDD molecules to one lipoprotein molecule, TCDD binds and disrupts the secondary and tertiary lipoprotein structure. Circular dichroism studies show that residues within the inner core of apoC-II, which compose a four-alpha-helix bundle when this apolipoprotein is associated with VLDL, are directly affected upon binding TCDD. Fluorescence also indicates the specific interaction of Trp-48 within apoC-II upon TCDD binding. We found that the TCDD/apoC-II complex suffers a 5-fold reduction in its ability to bind lipoprotein lipase compared to untreated apoC-II. The interaction of TCDD with LDL markedly altered the secondary structure of apoB reducing its alpha-helical content. These cumulative responses in lipoprotein structure may impair the LDL and VLDL cellular uptake leading to a buildup of serum lipoproteins and fats thus hastening the development of coronary artery disease.     

Heterologous expression and purification of the soybean 7S globulin α' subunit extension region: in vitro evidence of its involvement in cell cholesterol homeostasis

In a previous paper, the biological activity of a 216-amino acid recombinant truncated form of the soybean 7S globulin α' subunit, known to control cholesterol and triglyceride homeostasis, was described. In this work, a shorter version of the polypeptide chain, spanning 142 amino acid residues from the N-terminus and thus exclusively including the so-called extension region, was cloned and overexpressed in Pichia pastoris. The yield of the recombinant polypeptide, which was termed α'E, was 8-fold greater than the previous truncated version. The α'E polypeptide was purified by simple conventional biochemical techniques to make it available for biological assays. Human hepatoma cell lines (Hep G2) were used to monitor the uptake and degradation of labeled low-density lipoproteins (LDL), according to an established procedure. The LDL uptake (+86%) and degradation (+94%) by cells tested at the highest α'E dose (2 μM) were similar to those found in cells incubated with 1 μM simvastatin, a potent inhibitor of cholesterol biosynthesis. Additionally, the cell response to α'E was found to be dose-dependent. The present findings strongly suggest that this recombinant polypeptide, or a fragment thereof, is the molecular determinant for cholesterol homeostasis and open new prospects for understanding the mechanism involved in this biological response, as a gateway to its utilization in lipid-lowering therapies.     

Acetaldehyde binding increases the catabolism of rat serum low-density lipoproteins

Acetaldehyde was found to form adducts with rat serum lipoproteins. The binding of [14C]acetaldehyde to lipoproteins was studied at low concentrations which are known to exist during ethanol oxidation. The amount of lipoprotein adducts was a linear function of acetaldehyde concentration up to 250 microM. Incubation of rat plasma low-density lipoproteins (LDL) with 200 microM acetaldehyde increased the disappearance rate of the 3H-label from the cholesterol ester moiety of LDL injected into normal rats. The data show that even low concentrations of acetaldehyde are capable of affecting LDL metabolism. These findings may provide an explanation for the low concentrations of serum LDL in alcoholics.     

Effects of compactin, mevalonate and low-density lipoprotein on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and low-density-lipoprotein-receptor activity in the human hepatoma cell line Hep G2.

Compactin, an inhibitor of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase, decreased cholesterol synthesis in intact Hep G2 cells. However, after the inhibitor was washed away, the HMG-CoA-reductase activity determined in the cell homogenate was found to be increased. Also the high-affinity association of LDL (low-density lipoprotein) to Hep G2 cells was elevated after incubation with compactin. Lipoprotein-depleted serum, present in the incubation medium, potentiated the compactin effect compared with incubation in the presence of human serum albumin. Addition of either mevalonate or LDL prevented the compactin-induced rise in activities of both HMG-CoA reductase and LDL receptor in a comparable manner. It is concluded that in this human hepatoma cell line, as in non-transformed cells, both endogenous mevalonate or mevalonate-derived products and exogenous cholesterol are able to modulate the HMG-CoA reductase activity as well as the LDL-receptor activity.     

Genetic studies of human apolipoproteins

Summary Apolipoprotein H (APO H) has recently been identified as a structural component of chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Although the precise metabolic function of APO H in lipid metabolism is not certain, it has been suggested that APO H may be involved in triglyceride (TG) metabolism. In addition to the previously described quantitative polymorphism, we have recently detected a common qualitative polymorphism at the APO H structural locus. To test the role of APO H genetic variation in determining lipoprotein and lipid levels, we have estimated the allelic effects of APO H variation on TG, VLDL, LDL, HDL, HDL3, and total cholesterol on 356 Nigerian blacks(189 males, 167 females). While no significant effect of phenotype was observed on lipoprotein levels, the effect of interaction between phenotype and gender was significant. Therefore, data on males and females were analyzed separately using analysis of variance after adjusting for age and body mass index. Logarithmic transformation of pertinent variables was done to bring the distribution of the variables closer to normality. A statistically significant effect of phenotype was observed on triglyceride levels in females only (P<0.05). Further analysis of this phenotypic effect revealed that it is due to the impact of the APO H ^* 3 allele, which raises triglycerides by 9.92 mg/dl as compared to the common allele, APO H ^* 2. These findings are in accordance with the postulated role of APO H in triglyceride metabolism. On the basis of its sex-specific effect, we propose a hypothesis that may explain the combined influence of the quantitative and qualitative polymorphisms at the APO H locus on triglyceride levels in females.     

Unique structural properties of apolipoprotein B in low-density lipoproteins produced by several human hepatoma-derived cell lines

Previous work has shown that low-density lipoproteins (LDL) secreted by hepatoma-derived cell lines have an unusual composition compared to plasma LDL; rather than cholesteryl ester, the hepatoma cell-secreted LDL have a triacylglycerol core. We have found that they also have an increased negative charge, as judged by agarose electrophoresis. Since apolipoprotein B is a glycoprotein containing carbohydrate chains terminated with negatively charged sialic acid residues, we examined whether increased glycosylation of the apolipoprotein B from three hepatoma cell lines (Hep G2, Hep 3B and Huh 7) might account for the differences in LDL charge. The weight percent carbohydrate for Hep G2, Hep 3B and Huh 7 LDL-protein (1.1 +/- 0.2; 1.7 +/- 0.8; 0.4 +/- 0.1) was found to be extremely low compared with the 2.8-9% range we found for plasma LDL-protein, while the amount of LDL-lipid associated carbohydrate from hepatoma LDL was similar to that we found in plasma LDL. Furthermore, desialation of hepatoma cell-secreted LDL with neuraminidase did not normalize the negative charge to that of neuraminidase-treated plasma LDL. Western blots of thrombin proteolytic fragments indicated that, in addition to the T1-T4 fragments seen in plasma apolipoprotein B, apolipoprotein B of hepatoma-derived LDL produced four to five new fragments (T5-T9), suggesting increased exposure of proteolytic sites. Western blotting of the new fragments with antibodies specific for known apolipoprotein B sequences suggests that many of the new cleavage sites cluster in or near the putative LDL receptor recognition site.     

Modification by acrolein, a component of tobacco smoke and age-related oxidative stress, mediates functional impairment of human apolipoprotein E

Oxidative damage to proteins such as apolipoprotein B-100 increases the atherogenicity of low-density lipoproteins (LDL). However, little is known about the potential oxidative damage to apolipoprotein E (apoE), an exchangeable antiatherogenic apolipoprotein. ApoE plays an integral role in lipoprotein metabolism by regulating the plasma cholesterol and triglyceride levels. Hepatic uptake of lipoproteins is facilitated by apoE's ability to bind with cell surface heparan sulfate proteoglycans and to lipoprotein receptors via basic residues in its 22 kDa N-terminal domain (NT). We investigated the effect of acrolein, an aldehydic product of endogenous lipid peroxidation and a tobacco smoke component, on the conformation and function of recombinant human apoE3-NT. Acrolein caused oxidative modification of apoE3-NT as detected by Western blot with acrolein-lysine-specific antibodies, and tertiary conformational alterations. Acrolein modification impairs the ability of apoE3-NT to interact with heparin and the LDL receptor. Furthermore, acrolein-modified apoE3-NT displayed a 5-fold decrease in its ability to interact with lipid surfaces. Our data indicate that acrolein disrupts the functional integrity of apoE3, which likely interferes with its role in regulating plasma cholesterol homeostasis. These observations have implications regarding the role of apoE in the pathogenesis of smoking- and oxidative stress-mediated cardiovascular and cerebrovascular diseases.     

Macrophage colony-stimulating factor rapidly enhances beta-migrating very low density lipoprotein metabolism in macrophages through activation of a Gi/o protein signaling pathway

Previous studies have examined lipoprotein metabolism by macrophages following prolonged exposure (>24 h) to macrophage colony-stimulating factor (M-CSF). Because M-CSF activates several signaling pathways that could rapidly affect lipoprotein metabolism, we examined whether acute exposure of macrophages to M-CSF alters the metabolism of either native or modified lipoproteins. Acute incubation of cultured J774 macrophages and resident mouse peritoneal macrophages with M-CSF markedly enhanced low density lipoproteins (LDL) and beta-migrating very low density lipoproteins (beta-VLDL) stimulated cholesteryl [(3)H]oleate deposition. In parallel, M-CSF treatment increased the association and degradation of (125)I-labeled LDL or beta-VLDL without altering the amount of lipoprotein bound to the cell surface. The increase in LDL and beta-VLDL metabolism did not reflect a generalized effect on lipoprotein endocytosis and metabolism because M-CSF did not alter cholesterol deposition during incubation with acetylated LDL. Moreover, M-CSF did not augment beta-VLDL cholesterol deposition in macrophages from LDL receptor (-/-) mice, indicating that the effect of M-CSF was mediated by the LDL receptor. Incubation of macrophages with pertussis toxin, a specific inhibitor of G(i/o) protein signaling, had no effect on cholesterol deposition during incubation with beta-VLDL alone, but completely blocked the augmented response promoted by M-CSF. In addition, incubation of macrophages with the direct G(i/o) protein activator, mastoparan, mimicked the effect of M-CSF by enhancing cholesterol deposition in cells incubated with beta-VLDL, but not acetylated LDL. In summary, M-CSF rapidly enhances LDL receptor-mediated metabolism of native lipoproteins by macrophages through activation of a G(i/o) protein signaling pathway. Together, these findings describe a novel pathway for regulating lipoprotein metabolism.