Regulation Of Cellular Cholesterol Metabolism Research Articles

Exercise-Induced Changes in Caveolin-1, Depletion of Mitochondrial Cholesterol, and the Inhibition of Mitochondrial Swelling in Rat Skeletal Muscle but Not in the Liver.

The reduction in cholesterol in mitochondria, observed after exercise, is related to the inhibition of mitochondrial swelling. Caveolin-1 (Cav-1) plays an essential role in the regulation of cellular cholesterol metabolism and is required by various signalling pathways. Therefore, the aim of this study was to investigate the effect of prolonged swimming on the mitochondrial Cav-1 concentration; additionally, we identified the results of these changes as they relate to the induction of changes in the mitochondrial swelling and cholesterol in rat skeletal muscle and liver. Male Wistar rats were divided into a sedentary control group and an exercise group. The exercised rats swam for 3 hours and were burdened with an additional 3% of their body weight. After the cessation of exercise, their quadriceps femoris muscles and livers were immediately removed for experimentation. The exercise protocol caused an increase in the Cav-1 concentration in crude muscle mitochondria; this was related to a reduction in the cholesterol level and an inhibition of mitochondrial swelling. There were no changes in rat livers, with the exception of increased markers of oxidative stress in mitochondria. These data indicate the possible role of Cav-1 in the adaptive change in the rat muscle mitochondria following exercise.

Abstract 524: Role of Transmembrane Protein 55B in Insulin Signaling and Hepatic Lipid Metabolism

Hepatic steatosis is the leading cause of liver disease in the United States and is associated with increased risk of cardiovascular disease. Insulin resistance appears to be a major determinant of hepatic steatosis, as improper hepatic insulin signaling stimulates lipogenesis and dysregulates lipoprotein secretion, resulting in fat accumulation in the liver. Insulin signal transduction is dependent on phosphatidylinositol-4,5-biphosphate (PIP 2 ) conversion to phosphatidylinositol-3,4,5-biphosphate (PIP 3 ), which results in activation of AKT. Transmembrane protein 55B ( TMEM55B ), a gene we recently identified as a novel regulator of cellular cholesterol metabolism, encodes a phosphatidylinositol phosphatase that converts PIP 2 to phosphatidylinositol-5-phosphate (PI5P). PI5P has also been shown to activate AKT; however, the physiological effects of this pathway are less well understood. In the first in vivo model of Tmem55b modulation, we found that hepatic Tmem55b knockdown in Western diet-fed C57BL/6 mice caused increased body weight, hepatic steatosis, hyperinsulinemia and elevated plasma cholesterol compared to controls. In addition, hepatic expression of insulin-regulated lipogenic genes, including Srebp1c and Dgat2, was increased, suggesting that Tmem55b knockdown promotes lipogenesis. In addition, two genes involved in VLDL secretion, ApoC3 and Mtp , were upregulated. Notably, we observed a significant inverse correlation between TMEM55B expression, quantified in 480 human lymphoblastoid cell lines, with plasma triglyceride, VLDL and APOC3 levels in the cell line donors. These relationships were significantly stronger in individuals with elevated plasma glucose (>110 mg/dL), suggesting that the relationship between TMEM55B and these measures may be enhanced in the insulin resistant state. Based on these findings, we hypothesize that TMEM55B modulates insulin signaling and affects hepatic lipid accumulation, potentially through stimulation of lipogenesis. With the ever-rising incidence of type 2 diabetes, elucidation of a novel pathway by which insulin resistance leads to hepatic steatosis may inform the development of therapeutics used for the prevention or treatment of this disease.

Caveolin-1 regulates the anti-atherogenic properties of macrophages

Atherosclerosis is a complex disease initiated by the vascular accumulation of lipoproteins in the sub-endothelial space, followed by the infiltration of monocytes into the arterial intima. Caveolin-1 (Cav-1) plays an essential role in the regulation of cellular cholesterol metabolism and of various signaling pathways. In order to study specifically the role of macrophage Cav-1 in atherosclerosis, we used Cav-1 (-/-) Apoe (-/-) mice and transplanted them with bone marrow (BM) cells obtained from Cav-1 (+/+) Apoe (-/-) or Cav-1 (-/-) Apoe (-/-) mice and vice versa. We found that Cav-1 (+/+) mice harboring Cav-1 (-/-) BM-derived macrophages developed significantly larger lesions than Cav-1 (+/+) mice harboring Cav-1 (+/+) BM-derived macrophages. Cav-1 (-/-) macrophages were more susceptible to apoptosis and more prone to induce inflammation. The present study provides clear evidence that the absence of Cav-1 in macrophage is pro-atherogenic, whereas its absence in endothelial cells protects against atherosclerotic lesion formation. These findings demonstrate the cell-specific role of Cav-1 during the development of this disease.

Transmembrane protein 55B is a novel regulator of cellular cholesterol metabolism.

Interindividual variation in pathways affecting cellular cholesterol metabolism can influence levels of plasma cholesterol, a well-established risk factor for cardiovascular disease. Inherent variation among immortalized lymphoblastoid cell lines from different donors can be leveraged to discover novel genes that modulate cellular cholesterol metabolism. The objective of this study was to identify novel genes that regulate cholesterol metabolism by testing for evidence of correlated gene expression with cellular levels of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) mRNA, a marker for cellular cholesterol homeostasis, in a large panel of lymphoblastoid cell lines. Expression array profiling was performed on 480 lymphoblastoid cell lines established from participants of the Cholesterol and Pharmacogenetics (CAP) statin clinical trial, and transcripts were tested for evidence of correlated expression with HMGCR as a marker of intracellular cholesterol homeostasis. Of these, transmembrane protein 55b (TMEM55B) showed the strongest correlation (r=0.29; P=4.0E-08) of all genes not previously implicated in cholesterol metabolism and was found to be sterol regulated. TMEM55B knockdown in human hepatoma cell lines promoted the decay rate of the low-density lipoprotein receptor, reduced cell surface low-density lipoprotein receptor protein, impaired low-density lipoprotein uptake, and reduced intracellular cholesterol. Here, we report identification of TMEM55B as a novel regulator of cellular cholesterol metabolism through the combination of gene expression profiling and functional studies. The findings highlight the value of an integrated genomic approach for identifying genes that influence cholesterol homeostasis.

Alternative splicing in the regulation of cholesterol homeostasis.

With the advent of whole-transcriptome sequencing, or RNA-seq, we now know that alternative splicing is a generalized phenomenon, with nearly all multiexonic genes subject to alternative splicing. In this review, we highlight recent studies examining alternative splicing as a modulator of cellular cholesterol homeostasis and as an underlying mechanism of dyslipidemia. A number of key genes involved in cholesterol metabolism are known to undergo functionally relevant alternative splicing. Recently, we have identified coordinated changes in alternative splicing in multiple genes in response to alterations in cellular sterol content. We and others have implicated several splicing factors as regulators of lipid metabolism. Furthermore, a number of cis-acting human gene variants that modulate alternative splicing have been implicated in a variety of human metabolic diseases. Alternative splicing is of importance in various types of genetically influenced dyslipidemias and in the regulation of cellular cholesterol metabolism.

RHOA Is a Modulator of the Cholesterol-Lowering Effects of Statin

Although statin drugs are generally efficacious for lowering plasma LDL-cholesterol levels, there is considerable variability in response. To identify candidate genes that may contribute to this variation, we used an unbiased genome-wide filter approach that was applied to 10,149 genes expressed in immortalized lymphoblastoid cell lines (LCLs) derived from 480 participants of the Cholesterol and Pharmacogenomics (CAP) clinical trial of simvastatin. The criteria for identification of candidates included genes whose statin-induced changes in expression were correlated with change in expression of HMGCR, a key regulator of cellular cholesterol metabolism and the target of statin inhibition. This analysis yielded 45 genes, from which RHOA was selected for follow-up because it has been found to participate in mediating the pleiotropic but not the lipid-lowering effects of statin treatment. RHOA knock-down in hepatoma cell lines reduced HMGCR, LDLR, and SREBF2 mRNA expression and increased intracellular cholesterol ester content as well as apolipoprotein B (APOB) concentrations in the conditioned media. Furthermore, inter-individual variation in statin-induced RHOA mRNA expression measured in vitro in CAP LCLs was correlated with the changes in plasma total cholesterol, LDL-cholesterol, and APOB induced by simvastatin treatment (40 mg/d for 6 wk) of the individuals from whom these cell lines were derived. Moreover, the minor allele of rs11716445, a SNP located in a novel cryptic RHOA exon, dramatically increased inclusion of the exon in RHOA transcripts during splicing and was associated with a smaller LDL-cholesterol reduction in response to statin treatment in 1,886 participants from the CAP and Pravastatin Inflamation and CRP Evaluation (PRINCE; pravastatin 40 mg/d) statin clinical trials. Thus, an unbiased filter approach based on transcriptome-wide profiling identified RHOA as a gene contributing to variation in LDL-cholesterol response to statin, illustrating the power of this approach for identifying candidate genes involved in drug response phenotypes.

Role of activated endocannabinoid system in regulation of cellular cholesterol metabolism in macrophages

Evidence from recent studies suggests that the endocannabinoid system participates in the regulation of lipid metabolism and body composition. We hypothesize that the system is activated by oxidized low-density lipoprotein (oxLDL) and regulates cellular cholesterol metabolism in macrophages. Primary peritoneal macrophages isolated from Sprague-Dawley rats and RAW264.7 mice macrophages were cultured. A liquid chromatography/mass spectrometry (LC/MS) system was used to measure the endocannabinoid anandamide (AEA), 2-arachidonoylglycerol (2-AG), and cellular cholesterol levels in macrophages. The regulatory mechanisms of cellular cholesterol metabolism were also investigated by molecular biology methods. The results showed that the endocannabinoid system in macrophages was activated by oxLDL through elevation of the AEA and 2-AG levels and the up-regulation of the cannabinoid CB1 and CB2 receptor expression. Win55,212-2, a synthetic cannabinoid, promotes cellular cholesterol accumulation in macrophages, which was associated with an increase in the expression of CD36 and a decrease in the expression of ATP-binding cassette protein A1 (ABCA1) as mediated by an up-regulated peroxisome proliferator-activated receptor gamma (PPARgamma). AM251, a selective cannabinoid CB1 receptor antagonist, impaired the abilities of Win55,212-2-treated macrophages to accumulate cholesterol by down-regulating CD36 receptor expression and up-regulating ABCA1 expression. We have demonstrated, for the first time, that the endocannabinoid system in macrophages is activated by oxLDL and that the activated endocannabinoid system promotes cellular cholesterol accumulation in macrophages. The results also indicate that selectively blocking the CB1 receptor can reduce oxLDL accumulation in macrophages, which might represent a promising therapeutic strategy for atherosclerosis.

Overexpression of A-kinase anchoring protein 12A activates sterol regulatory element binding protein-2 and enhances cholesterol efflux in hepatic cells

A-kinase anchoring protein 12 (AKAP12) is known to function as a scaffold protein and as a putative tumor suppressor. However, little is known about the biological role of AKAP12 in hepatic cells. In this study, we performed micro-array analysis to identify the downstream pathway of AKAP12A, and found that AKAP12A overexpression up-regulates the expressions of several cholesterol-associated genes including HMG-CoA reductase and LDL receptor, which have been reported to be controlled by sterol regulatory element binding protein-2 (SREBP-2). It was found that AKAP12A activates SREBP-2 in hepatic cells, as demonstrated by the presence of its cleavage product, whereas the activation of sterol regulatory element binding protein-1 was not remarkably changed. Moreover, AKAP12A-induced SREBP-2 activation was found to depend on SREBP cleavage-activating protein (SCAP), as inhibition of SCAP by RNAi or sterols blocked SREBP-2 activation in response to AKAP12A overexpression. Interestingly, the hydrophobic amine U18666A caused dramatic movement of AKAP12A from the plasma membrane to cytosol and lysosomal membranes. Moreover, cholesterol depletion from the plasma membrane (using methyl-β-cyclodextrin) caused a shift of AKAP12A from the plasma membrane to the cytoplasm. Cholesterol binding assay revealed that the N-terminal region of AKAP12A binds directly to cholesterol in vitro. Furthermore, AKAP12A overexpression enhanced [ 3H]-cholesterol efflux to extracellular acceptors, suggesting that AKAP12A may activate SREBP-2 by increasing cholesterol efflux. In conclusion, the present study suggests that AKAP12A is a novel regulator of cellular cholesterol metabolism.

The effect of sterol regulatory element-binding protein 2 polymorphism on the serum lipid in northern Chinese subjects

Sterol regulatory element-binding protein 2 (SREBP-2) is an important nuclear transcription factor in the regulation of cellular cholesterol metabolism. To determine allele frequency of the 1784G>C polymorphism at the SREBP-2 locus and investigate the relationship between this polymorphism and serum lipid levels in Chinese people, we selected 486 individuals (118 men and 368 women) from the Xicheng District of Beijing. The subjects were divided into four groups: hypercholesterolemic subjects, hypertriglyceridemic subjects, combined hyperlipidemic subjects, and normal subjects. Serum lipid profiles were measured in all subjects, and 1784G>C was analyzed using polymerase chain reaction-restriction fragment length polymorphism analysis. There was no significant difference in genotype frequencies or allele frequencies of this polymorphism between the hyperlipidemic and control groups. The serum total cholesterol (TC) and LDL cholesterol (LDL-C) levels of the individuals carrying the C allele were higher than the noncarriers in both males and females in the hypercholesterolemic group, but statistical significance was only observed in females. The results of this study indicate that the SREBP-2 polymorphism is related to elevated concentrations of serum TC and LDL-C in hypercholesterolemic subjects. Further work is necessary to confirm the role of 1784G>C in the development of hyperlipidemia.

Family of human oxysterol binding protein (OSBP) homologues: a novel member implicated in brain sterol metabolism

Oxysterol binding protein (OSBP) is a cytosolic protein that undergoes ligand-induced binding to the Golgi apparatus and has been implicated in the regulation of cellular cholesterol metabolism. In the yeast Saccharomyces cerevisiae an OSBP homologue is involved in membrane trafficking through the Golgi complex. Prompted by the multitude of OSBP-related genes in the yeast genome, we carried out a search for human expressed sequence tags (ESTs) displaying homology to the sterol-binding domain of OSBP. This revealed a minimum of six novel OSBP-related proteins, designated ORP-1 to ORP-6. ORP cDNA probes were generated by reverse transcription-PCR from human liver mRNA, and used for Northern blot analysis of human tissue transcript panels. This verified that each of them represents a different gene product and showed that they display distinct tissue-specific expression patterns. The ORP-1 and -2 mRNA expression levels were similar to or higher than that of OSBP while the ORP-3 to -6 mRNAs were detected at lower levels in specific tissues.▪ The most abundantly expressed new gene, ORP-1, was transcribed at strikingly high levels in the cortical areas of human brain and displayed sterol-regulated expression in a cultured human neuroblastoma cell line. This indicates that ORP-1 may play an important role in maintaining the sterol balance in cells of the central nervous system. Together with OSBP, the identified gene products constitute a novel human protein family that may provide a link between organellar sterol status and membrane dynamics.—Laitinen, S., V. M. Olkkonen, C. Ehnholm, and E. Ikonen. Family of human oxysterol binding protein (OSBP) homologues: a novel member implicated in brain sterol metabolism.

Effects of amlodipine on gene expression and extracellular matrix formation in human vascular smooth muscle cells and fibroblasts: implications for vascular protection

Vascular smooth muscle cells (VSMC) are involved in the pathogenesis of hypertension and coronary artery disease. Amlodipine, a calcium channel blocker of the dihydropyridine type, is widely used in the therapy of these diseases, and has been shown to reduce the progression of the underlying pathophysiological mechanisms, such as atherosclerosis and restenosis. Research on the impact of calcium channel blockers on cell behavior has revealed an antiproliferative effect on VSMC. Cell proliferation is tightly controlled by permanent interaction of cells with their surrounding microenvironment, the extracellular matrix (ECM). The ECM is subjected to a continuous turnover and implicated in (i) stabilization and compartmentalization of tissue architecture and (ii) local binding and preservation of growth factors and cytokines. These growth factors and cytokines can be released during degradation of the ECM, and can function as local inflammatory factors without de novo synthesis. In this context, we assessed the effects of amlodipine on the composition of the ECM and related factors. We investigated the effects of amlodipine on (i) the regulation of cellular cholesterol metabolism, (ii) the activation of genes encoding for inflammatory factors, (iii) gene expression and turnover of ECM compounds, and (iv) the activity of matrix-degrading enzymes. Most of these effects of calcium channel blockers require direct induction of gene expression. In this respect, we demonstrate that amlodipine increases expression of the cytokine interleukin-6 by directly activating the respective gene promoter in human VSMC.

Characterization of Chinese hamster ovary cells that are resistant to 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one inhibition of low density lipoprotein-derived cholesterol metabolism.

The pharmacological agent U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one inhibits the intracellular transport of low density lipoprotein (LDL)-derived cholesterol in Chinese hamster ovary (CHO) cells. LDL-derived cholesterol accumulates in the lysosomes of U18666A-treated cells causing delayed LDL-mediated regulation of cellular cholesterol metabolism and impaired movement of LDL-derived cholesterol to other cell membranes. As a result of impaired LDL-derived cholesterol transport, LDL-dependent growth of CHO cells is also inhibited by U18666A. By selecting for cell growth in the presence of U18666A, we have identified a CHO cell line, designated U18R, that is resistant to U18666A-inhibition of LDL-derived cholesterol trafficking. When compared to parental CHO cells, U18R cells are relatively resistant to U18666A inhibition of LDL-derived cholesterol transport as well as LDL-mediated regulation of cellular cholesterol metabolism. In cell fusion experiments, the U18666A resistance observed in U18R cells displays a dominant phenotype. Identification of the U18666A-resistant factor may provide important insights toward the understanding of intracellular LDL-derived cholesterol regulation and trafficking.

12-[(5-Iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoic acid: biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase

Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl [14C]oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent Ki of 150 microM. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid (1 mM) provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions (apparent Ki = 20 microM), and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of 125IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.

The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one

In mammalian cells, low density lipoprotein (LDL) is bound, internalized, and delivered to lysosomes where LDL-cholesteryl esters are hydrolyzed to unesterified cholesterol. The mechanisms of intracellular transport of LDL-cholesterol from lysosomes to other cellular sites and LDL-mediated regulation of cellular cholesterol metabolism are unknown. We have identified a pharmacological agent, U18666A (3-beta-[2-diethyl-amino)ethoxy]androst-5-en-17-one), which impairs the intracellular transport of LDL-derived cholesterol in cultured Chinese hamster ovary (CHO) cells. U18666A blocks the ability of LDL-derived cholesterol to stimulate cholesterol esterification, and to suppress 3-hydroxy-3-methylglutaryl-coenzyme A reductase and LDL receptor activities. However, U18666A does not impair 25-hydroxycholesterol-mediated regulation of these processes. In addition, U18666A impedes the ability of LDL-derived cholesterol to support the growth of CHO cells. However, U18666A has only moderate effects on growth supported by non-lipoprotein cholesterol. LDL binding, internalization, and lysosomal hydrolysis of LDL-cholesteryl esters are not affected by the presence of U18666A. Analysis of intracellular cholesterol transport reveals that LDL-derived cholesterol accumulates in the lysosomes of U18666A-treated CHO cells which results in impaired movement of LDL-derived cholesterol to other cell membranes.

Low density lipoprotein (LDL)-mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick type C fibroblasts.

One characteristic of type C Niemann-Pick (NPC) disease is the substantial intracellular accumulation of unesterified cholesterol. The increased cholesterol content in NPC fibroblasts which are grown in the presence of low density lipoproteins (LDL) has been postulated to be due to a deficiency in cellular cholesterol esterification. We have examined several aspects of LDL metabolism in NPC fibroblasts. We observe that LDL binding, internalization, and lysosomal hydrolysis of LDL cholesteryl esters are normal in NPC cells. As reported by Pentchev et al. (Pentchev, P. G., Comly, M. E., Kruth, H. S., Vanier, M. T., Wenger, D. A., Patel, S., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8247-8251), we find that LDL does not stimulate cholesterol esterification. However, we also show that LDL does not down-regulate cholesterol synthesis or LDL receptor activity as normal. In NPC cells, these processes are regulated normally by nonlipoprotein effectors, such as 25-hydroxycholesterol or mevalonate. Since NPC cells are not defective in lysosomal hydrolysis of LDL-derived cholesteryl esters, they must exhibit a different defect than Wolman's or cholesteryl ester storage diseases. We conclude that NPC cells are defective specifically in LDL-mediated regulation of cellular cholesterol metabolism. We suggest that the intracellular processing of LDL-derived cholesterol may be defective in NPC fibroblasts.

Receptor interactions controlling lipoprotein metabolism.

Lipoprotein receptors play a central role in lipoprotein metabolism and a major role in cholesterol homeostasis. The most completely characterized lipoprotein receptor is the LDL (low density lipoprotein) or apo-B,E(LDL) receptor. The apo-B,E(LDL) receptor is present on both hepatic and extrahepatic cells and is responsible for the metabolism of a major portion of plasma LDL. Binding and internalization of LDL particles by this receptor initiates a series of intracellular events, resulting in the regulation of cellular cholesterol metabolism. In addition to the apo-B on LDL interacting with the apo-B,E(LDL) receptor, the apo-E on apo-E-containing lipoproteins is also capable of interacting and regulating intracellular cholesterol metabolism. The liver has also been shown to contain a second distinct lipoprotein receptor that is specific for apo-E. This receptor has been demonstrated on hepatic membranes from humans, dogs, and swine and is referred to as the apo-E receptor. This receptor may be responsible for the clearance of chylomicron remnants from plasma by the liver and may participate in reverse cholesterol transport. Thus, apo-E is a major determinant in lipoprotein metabolism and cholesterol homeostasis. The receptor binding properties of apo-E are well characterized, and a series of structural variants, several with lipoprotein binding defects, have been identified. Studies of the binding activity of these receptor-defective apo-E variants have helped to define the receptor binding domain of apo-E.(ABSTRACT TRUNCATED AT 250 WORDS)

The LDL receptor and the regulation of cellular cholesterol metabolism.

The LDL receptor and the regulation of cellular cholesterol metabolism.

The origin and properties of free cholesterol potential gradients in plasma, and their relation to atherogenesis

The aqueous solubilities of most lipids in lipoproteins are so low that their transfer between lipoproteins and cell membranes depends on random (collision) or specific (receptor-mediated) physical interaction between donor and acceptor interfaces, or the activity of transfer proteins which serve to increase the solubility of lipids in the aqueous phase (1). However, the solubility of cholesterol, although small (critical micelle concentration (CMC) -3 X IO-' M) (2), is sufficient for simple Fick diffusion to account for a major part of the observed flux of cholesterol between different lipoprotein particles, or between lipoproteins and cell membranes (Table 1). A similar identity has been found for the activation energies of cholesterol transfer between cell membranes and lipoproteins, and between synthetic lipid vesicles. When bidirectional flux rates are equal (for example, between cell membranes and unfractionated native normal plasma, when plasma cholesterol content is not perturbed by esterification), there is no spontaneous net transport of cholesterol into or out of the cell (6). When 1ecithin:cholesterol acyltransferase (LCAT) in plasma is active, and by esterification reduces the effective concentration (i.e., chemical potential) of cholesterol in the plasma in the face of an unchanged efflux of cholesterol from the adjacent cell membranes (3,6), its effect overall is to catalyze a compensatory net transport of free cholesterol from the cell to the plasma medium. This is compensated in vivo by cholesterogenesis within the affected cell, and by the entry of new lipoprotein cholesterol from those cells that secrete lipoproteins into plasma, which reduces or nullifies the induced cell-toplasma chemical potential gradient of cholesterol. From the same principles, it follows that if the chemical potential of cholesterol in a plasma lipoprotein species is higher than that in the other lipoproteins or in the cell membranes, cholesterol will flow down its gradient to such cells or lipoproteins until their free cholesterol content reaches its new steady state. If the acceptor lipoproteins in plasma (by the activity of LCAT) or cells (by the action of acyl CoA:cholesterol acyltransferase, ACAT) remove the cholesterol transferred from the equilibrium by the synthesis of esterified cholesterol, the flow of cholesterol will continue indefinitely, as long as the supply of cholesterol-rich donor lipoproteins continues. This process will also be governed by the thermodynamic parameters shown in Table 1. Two arguments in particular can be advanced against a significant role for thermodynamic (i.e., non-receptor) processes in cholesterol transport between plasma lipoproteins and cells, and the regulation of cellular cholesterol metabolism, particularly in relation to the cholesterol accumulation that occurs in atherosclerosis. i) In the plasma of many of those at risk for atherosclerosis, e.g., hyperbetalipoproteinemics (7), the composition, and, in particular, the cholesterol content, of the lipoproteins is normal. ii) Only that lipoprotein cholesterol interiorized by receptor mechanisms is effective in mediating changes in cellular cholesterol metabolism (8). Neither argument, however, is likely to be valid.

Interaction of lipoproteins with macrophages.

The suggestion that the cholesterol accumulation in cardiovascular lesions is caused by an interaction of serum lipoproteins with arterial cells has been supported by numerous investigations. The lipid-loaden foam cells which accumulate cholesterol in atheroma are thought to arise from either smooth muscle cells [1–4] or monocytes-macrophages [1,2,5,6]. At the present time, however, it has not been possible to demonstrate that smooth muscle cells can be overloaded with cholesterol by any of the naturally occurring or cholesterol-induced lipoproteins. These cells, like fibroblasts, lymphocytes and endothelial cells, can prevent excessive uptake and accumulation of lipoprotein-cholesterol due to the presence of specific cell surface receptors for plasma lipoproteins [7]. The binding of the lipoproteins to these receptors initiates a number of intracellular events, including internalization and degradation of the lipoproteins and the subsequent regulation of cellular cholesterol metabolism. The receptors interact with both low density lipoproteins (LDL), which contain the B-apoprotein, and certain high density lipoproteins (HDL), which contain the E-apoprotein, and are, therefore, designated as apo B, E receptors [8, 9].

Desialized Low‐Density Lipoprotein Regulates Cholesterol Metabolism in Receptor‐Deficient Fibroblasts

Binding of homologous low density lipoproteins (LDL) to specific cell‐membrane receptors initiates regulation of cholesterol metabolism in normal human fibroblasts, but minor regulatory effects are observed in receptor‐deficient cells. After enzymic removal of sialic acid residues, however, the desialized LDL aquires the ability to regulate cholesterol biosynthesis and cholesterol esterification in receptor‐deficient cells as effective as native LDL in normal cells. This is indicated by a suppression of hydroxymethylglutaryl‐CoA reductase activity, by a reduced incorporation of [14C]acetate into cholesterol and by a stimulation of the [3H]oleate incorporation into cholesterol esters in both normal and receptor deficient cells. Competitive binding and internalisation experiments revealed that the action of desialized LDL in normal and receptor‐deficient cells is receptor‐specific.The results suggest that desialisation of LDL facilitates its binding to cell membrane receptors, its internalisation and subsequent regulation of cellular cholesterol metabolism, this effect becoming significant in fibroblasts with reduced number of LDL receptors.