Increased LDL Receptor Activity Research Articles

Leu22_Leu23 Duplication at the Signal Peptide of PCSK9 Promotes Intracellular Degradation of LDLr and Autosomal Dominant Hypercholesterolemia.

PCSK9 (Proprotein convertase subtilisin/kexin type 9) regulates LDL-C (low-density lipoprotein cholesterol) metabolism by targeting LDLr (LDL receptor) for lysosomal degradation. PCSK9 gain-of-function variants cause autosomal dominant hypercholesterolemia by reducing LDLr levels, the D374Y variant being the most severe, while loss-of-function variants are associated with low LDL-C levels. Gain-of-function and loss-of-function activities have also been attributed to variants occurring in the PCSK9 signal peptide. Among them, L11 is a very rare PCSK9 variant that seems to increase LDL-C values in a moderate way causing mild hypercholesterolemia. Using molecular biology and biophysics methodologies, activities of L8 and L11 variants, both located in the leucine repetition stretch of the signal peptide, have been extensively characterized in vitro. L8 variant is not associated with increased LDLr activity, whereas L11 activity is increased by ≈20% compared with wt PCSK9. The results suggest that the L11 variant reduces LDLr levels intracellularly by a process resulting from impaired cleavage of the signal peptide. This would lead to less efficient LDLr transport to the cell membrane and promote LDLr intracellular degradation. Deletion of a leucine in the signal peptide in L8 variant does not affect PCSK9 activity, whereas the leucine duplication in the L11 variant enhances LDLr intracellular degradation. These findings highlight the importance of deep in vitro characterization of PCSK9 genetic variants to determine pathogenicity and improve clinical diagnosis and therapy of inherited familial hypercholesterolemia disease.

Abstract 630: Pcsk9 Deficiency Reduces Atherosclerosis, Apolipoprotein B Secretion, Lipogenesis and Endothelial Dysfunction

Objective: Proprotein convertase subtilisin kexin type 9 (PCSK9) interacts directly with cytoplasmic apolipoprotein B (apoB) and prevents its degradation via the autophagosome/lysosome pathway. This process affects VLDL and LDL production and influences atherogenesis. Here, we investigated the molecular machinery by which PCSK9 modulates autophagy and affects atherogenesis. Approach and Results: We backcrossed Pcsk9-/- mice with atherosclerosis prone LDb ( Ldlr-/-Apobec1-/-) mice to generate LTp ( Ldlr-/-Apobec1-/-Pcsk9-/- ). Deletion of PCSK9 resulted in decreased hepatic apoB secretion, increased autophagic flux and autophagy signaling, and decreased plasma levels of IDL and LDL particles. The LDLs from LTp mice (LTp-LDL) were less atherogenic and contained less cholesteryl ester and phospholipids than LDb-LDL. Deficiency of PCSK9 affected SREBP-1c lipogenesis pathway. Moreover LTp-LDL induced lower endothelial expression of the genes encoding TLR2, Lox-1, ICAM-1, CCL2, CCL7, IL-6, IL-1β, Beclin-1, p62 , and TRAF6 . Collectively, these effects were associated with substantially less atherosclerosis development (> 4-fold) in LTp mice. Conclusions: The absence of PCSK9 in Ldlr-/-Apobec1-/- mice results in decreased lipid and apoB levels, less atherogenic LDLs, and marked reduction of atherosclerosis. The effect on atherogenesis may be mediated in part by the effects of modified LDLs on endothelial cell receptors, and proinflammatory and autophagy molecules. These findings suggest that there may be clinical benefits of PCSK9 inhibition due to mechanisms unrelated to increased LDL receptor activity.

PCSK9 deficiency reduces atherosclerosis, apolipoprotein B secretion, and endothelial dysfunction

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interacts directly with cytoplasmic apoB and prevents its degradation via the autophagosome/lysosome pathway. This process affects VLDL and LDL production and influences atherogenesis. Here, we investigated the molecular machinery by which PCSK9 modulates autophagy and affects atherogenesis. We backcrossed Pcsk9−/− mice with atherosclerosis-prone Ldlr−/−Apobec1−/− (LDb) mice to generate Ldlr−/−Apobec1−/−Pcsk9−/− (LTp) mice. Deletion of PCSK9 resulted in decreased hepatic apoB secretion, increased autophagic flux, and decreased plasma levels of IDL and LDL particles. The LDLs from LTp mice (LTp-LDLs) were less atherogenic and contained less cholesteryl ester and phospholipids than LDb-LDLs. Moreover LTp-LDLs induced lower endothelial expression of the genes encoding TLR2, Lox-1, ICAM-1, CCL2, CCL7, IL-6, IL-1β, Beclin-1, p62, and TRAF6. Collectively, these effects were associated with substantially less atherosclerosis development (>4-fold) in LTp mice. The absence of PCSK9 in LDb mice results in decreased lipid and apoB levels, fewer atherogenic LDLs, and marked reduction of atherosclerosis. The effect on atherogenesis may be mediated in part by the effects of modified LDLs on endothelial cell receptors and proinflammatory and autophagy molecules. These findings suggest that there may be clinical benefits of PCSK9 inhibition due to mechanisms unrelated to increased LDL receptor activity.

Abstract 12151: PCSK9 Mediates Atherogenesis

Background: Proprotein convertase subtilisin kexin type 9 (PCSK9) negatively regulates the low-density lipoprotein (LDL) receptor (LDLR), resulting in increased LDL cholesterol levels. We have suggested that PCSK9 regulates apolipoprotein B (apoB) metabolism via an autophagic process that is independent of the LDLR. Furthermore, whether PCSK9 plays a causal role in atherogenesis independent of LDLR has been controversial. Here, we investigated the molecular mechanisms by which PCSK9 modulates autophagy and the LDLR-independent effects of PCSK9 on atherogenesis. Methods and Results: Deletion of the PCSK9 gene from atherosclerosis prone LDb ( Ldlr-/-Apobec1-/-) mice resulted in substantially less atherosclerosis (> 4-fold), as well as a substantial reduction of plasma apoB, lipids, and triglyceride-rich lipoproteins, but increased HDL particles. The triple knockout mice had a decreased rate of apoB secretion resulting from increased autophagic flux. The deletion of PCSK9 also had profound influences on aortic endothelial cell response to exposure to LDL. LDL associated with PCSK9 increased the expression of TLR2, Lox-1, ICAM-1, CCL2, CCL7, IL-6, IL-1β, Beclin-1, p62, and TRAF6, compared to LDL without PCSK9. Conclusion: Deletion of PCSK9 markedly decreases apoB production by increasing autophagic flux. The absence of PCSK9 results in decreased lipid levels and marked reduction of atherosclerosis, independent of LDLR. The influence on atherogenesis is in part due to the direct effects of PCSK9 on endothelial cell receptors and on proinflammatory and autophagy molecules. These findings suggest that there may be clinical benefits of PCSK9 inhibition due to mechanisms unrelated to increased LDLR activity.

Deferoxamine stimulates LDLR expression and LDL uptake in HepG2 cells.

Iron overload contributes to the pathogenesis of atherosclerosis and iron chelators are beneficial through their antioxidant properties. Hepatic iron loading increases cholesterol synthesis. Whether iron depletion could affect hepatic cholesterol metabolism is unknown. We examined the effect of the iron chelator deferoxamine (DFO) on mRNA expression of genes involved in cholesterol metabolism and/or cholesterol uptake. Our results revealed that DFO increases LDL receptor (LDLR) mRNA levels in human hepatocyte-derived cell lines HepG2 and Huh7 cells, and in K562 cells. In HepG2 cells, we observed that DFO increases (i) LDLR-mRNA levels in a time- and dose-dependent manner, (ii) LDLR-protein levels; (iii) cell surface LDLR; and (iv) LDL uptake. In contrast, the mRNA levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol regulatory element-binding proteins, and the mRNA/protein levels of proprotein convertase subtilisin-kexin 9 were not modulated by DFO, suggesting that the LDLR regulation by DFO is not at the transcriptional or posttranslational levels. Since LDLR-mRNA was stabilized by DFO, a posttranscriptional mechanism is suggested for the DFO-mediated upregulation of LDLR. DFO induced an increase in LDLR expression by a posttranscriptional mechanism resulting in an enhancement of LDL uptake in HepG2 cells, suggesting increased LDLR activity as one of the underlying causes of the hypocholesterolemic effect of iron reduction.

Role of Nutraceuticals in Hypolipidemic Therapy.

Nutraceuticals are food components or active ingredients present in foods and used in therapy. This article analyzes the characteristics of the molecules with a lipid-lowering effect. The different nutraceuticals may have different mechanisms of action: inhibition of cholesterol synthesis primarily through action on the enzyme HMG-CoA reductase (policosanol, polyphenols, garlic and, above all, red yeast rice), increase in LDL receptor activity (berberine), reduction of intestinal cholesterol absorption (garlic, plant sterols, probiotics), and also the ability to interfere with bile metabolism (probiotics, guggul). Based on the different mechanisms of action, some nutraceuticals are then able to enhance the action of statins. Nutraceuticals are often used without relevant evidence: mechanisms of action are not clearly confirmed; most of clinical data are derived from small, uncontrolled studies, and finally, except for fermented red rice, there are no clinical trials which may document the relationship between these interventions and the reduction of clinical events. Therefore, among all nutraceuticals, it is necessary to extrapolate those having a really documentable efficacy. However, these kinds of treatments are usually well-tolerated by patients. Overall, subjects with a middle or low cardiovascular risk are the best indication of nutraceuticals, but they may also be useful for patients experiencing side effects during classical therapies. Finally, in consideration of the additive effect of some nutraceuticals, a combination therapy with classical drugs may improve the achievement of clinical targets. Thus, nutraceuticals may be a helpful alternative in hypolipidemic treatment and, if properly used, might represent a valid strategy of cardiovascular prevention.

Clinically used selective oestrogen receptor modulators increase LDL receptor activity in primary human lymphocytes.

Treatment with selective oestrogen receptor modulators (SERMs) reduces low-density lipoprotein (LDL) cholesterol levels. We assessed the effect of tamoxifen, raloxifene and toremifene and their combinations with lovastatin on LDL receptor activity in lymphocytes from normolipidaemic and familial hypercholesterolaemic (FH) subjects, and human HepG2 hepatocytes and MOLT-4 lymphoblasts. Lymphocytes were isolated from peripheral blood, treated with different compounds, and 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (DiI)-labelled LDL uptake was analysed by flow cytometry. Tamoxifen, toremifene and raloxifene, in this order, stimulated DiI-LDL uptake by lymphocytes by inhibiting LDL-derived cholesterol trafficking and subsequent down-regulation of LDL receptor expression. Differently to what occurred in HepG2 and MOLT-4 cells, only tamoxifen consistently displayed a potentiating effect with lovastatin in primary lymphocytes. The SERM-mediated increase in LDL receptor activity was not altered by the anti-oestrogen ICI 182,780 nor was it reproduced by 17β-oestradiol. However, the tamoxifen-active metabolite endoxifen was equally effective as tamoxifen. The SERMs produced similar effects on LDL receptor activity in heterozygous FH lymphocytes as in normal lymphocytes, although none of them had a potentiating effect with lovastatin in heterozygous FH lymphocytes. The SERMs had no effect in homozygous FH lymphocytes. Clinically used SERMs up-regulate LDL receptors in primary human lymphocytes. There is a mild enhancement between SERMs and lovastatin of lymphocyte LDLR activity, the potentiation being greater in HepG2 and MOLT-4 cells. The effect of SERMs is independent of oestrogen receptors but is preserved in the tamoxifen-active metabolite endoxifen. This mechanism may contribute to the cholesterol-lowering action of SERMs.

Insights Into PCSK9, Low-Density Lipoprotein Receptor, and Low-Density Lipoprotein Cholesterol Metabolism

The identification of PCSK9 in 20031 has resulted in substantial revision to previous knowledge regarding cholesterol homeostasis, provided new insights into low-density lipoprotein (LDL) metabolism and LDL receptor (LDLR) function, and presented a new compelling therapeutic target to reduce LDL cholesterol (LDL-C). PCSK9 is a serine protease expressed predominantly in the liver, intestine, and kidney and regulates plasma LDL-C levels by binding to the epidermal growth factor A of the LDLR, targeting it for degradation rather than for recycling to the cell surface.2 Article see p 2403 Plasma levels of LDL-C and PCSK9 are related because overexpression or gain-of-function mutations of PCSK9 promote degradation of mainly hepatic LDLR, whereas loss-of-function mutations promote increased LDLR activity through enhanced recycling. The relationships between PCSK9, the LDLR, and plasma LDL-C have been the subject of intense study over the last decade, helped by transgenic mice models that can be made to express human PCSK9 and then studied in mice with and without LDLR defects. Although these studies have provided significant insights, the data have at times been inconsistent and confusing, particularly in 3 main areas that have potential clinical relevance: the role of PCSK9 in adrenal function, whether PCSK9 has an independent role in lipid metabolism other than via the LDLR, and the role of PCSK9 during statin therapy. In a series of elegant studies reported in this issue of Circulation , Tavori et al3 use these mouse models, supplemented with additional in vitro and in vivo experiments, to further elucidate the relationship between these parameters and provide some potential applications and extrapolations to human which may answer these questions and prove useful for current therapeutics in clinical development. The LDLR−/− mouse model provides a unique system in which to study the metabolism of both LDL and PCSK9 in …

More on Cholesterol Trafficking in the Body!

More on Cholesterol Trafficking in the Body!

Regulation of macrophage apoE secretion and sterol efflux by the LDL receptor

Factors that regulate apolipoprotein E (apoE) secretion by macrophages will have important effects on vessel wall lipid flux and atherosclerosis. Macrophages express the LDL receptor, which binds apoE with high affinity and could thereby affect the net secretion of apoE from macrophages. In these studies, we demonstrate that treatment of J774 macrophages transfected to constitutively express a human apoE3 cDNA with simvastatin, to increase LDL receptor activity, reduces the secretion of apoE. To further examine the relationship between LDL receptor expression and apoE secretion from macrophages, mouse peritoneal macrophages (MPMs) were isolated from mice with constitutively high expression of human LDL receptor to increase overall LDL receptor expression by 2- to 3-fold. Cells with increased LDL receptor expression also showed reduced apoE secretion compared with MPMs with basal LDL receptor expression. The effect of changes in LDL receptor expression on apoE secretion was isoform-specific, with greater reduction of apoE4 compared with apoE3 secretion and no reduction of apoE2 secretion, paralleling the known affinity of each isoform for LDL receptor binding. The effect of the LDL receptor on apoE secretion for each isoform was further reflected in LDL receptor-dependent changes in apoE-mediated cholesterol efflux. These results establish a regulatory interaction between two branches of macrophage sterol homeostatic pathways that could facilitate a rapid response to changes in macrophage sterol content relative to need.

Impact of Dietary Patterns and Interventions on Cardiovascular Health

Dietary recommendations are a key element in the management of cardiovascular disease. Evidence is mounting that certain dietary patterns can influence cardiovascular health by modifying risk factors such as obesity, dyslipidemia, and hypertension, as well as factors involved in systemic inflammation, insulin sensitivity, oxidative stress, endothelial function, thrombosis, and cardiac rhythm.1,2 In recent years, numerous dietary fads have emerged, in part as a response to the rising prevalence of obesity in the United States.3 In the present study, we review the various dietary portfolios that have emerged in the literature and the major studies that investigated their effectiveness in modifying cardiovascular risk. Currently, the typical American diet is estimated to derive 49% of its calories from carbohydrates, 34% from fat, and 12% to 16% from protein.4 Proposals to alter the proportions and/or types of macronutrients in this diet have been made for weight loss and cardiovascular health (Table 1).5–12 For weight management, for example, the strategy recommended by most medical groups entails the intake of a low-calorie, low-fat diet. The concept of fat restriction for weight management stems from traditional calorimetric measurements, which assign greater energy values to fat (&9 kcal/g) and less to carbohydrate and protein (&4 kcal/g). The low-calorie concept, on the other hand, is an intuitive technique to induce negative energy balance and has been adopted by some commercialized weight loss programs such as Weight Watchers International. View this table: TABLE 1. Various Dietary Patterns, Including Those Popularized Commercially and Those Investigated by Observational Studies and Clinical Trials One alternative proposed for weight loss is the low-carbohydrate diet. This was first described by William Banting13 in the 1860s and recently has received much attention in the form of the Atkins’, Stillman, Protein Power Lifeplan, and Zone diets. The Atkins’ diet begins with a weight-loss …

Red Grape Juice Polyphenols Alter Cholesterol Homeostasis and Increase LDL-Receptor Activity in Human Cells In Vitro

Red grape juice (RGJ) polyphenols have been shown to reduce circulating levels of LDL cholesterol and to increase LDL receptor activity. To explore the effect of RGJ-derived polyphenols on intracellular cholesterol homeostasis, human hepatocarcinoma HepG2 and promyelocytic HL-60 cell lines were incubated in serum-free medium, with or without LDL, in the presence or absence of RGJ. In the presence of LDL, RGJ increased both the activity and cell surface expression of the LDL receptor, and increased the cell total cholesterol content. In cells exposed to LDL, RGJ also increased levels of the active form of sterol regulatory element-binding protein-1 and mRNA expression of the LDL receptor and hydroxymethylglutaryl-CoA reductase. In contrast, RGJ caused a marked reduction in the expression of CYP7A1, apolipoprotein B, ABCA1, and ABCG5. Experiments using the acyl-CoA cholesterol acyltransferase inhibitor S-58035 indicated that no measurable free cholesterol from endocytosed LDL reaches the endoplasmic reticulum in cells treated with RGJ. Finally, fluorescence microscopy revealed that in RGJ-treated cells, DiI-labeled LDL did not colocalize with CD63, a protein localized at steady state in the internal vesicles of late endosomes. These results indicate that RGJ polyphenols disrupt or delay LDL trafficking through the endocytic pathway, thus preventing LDL cholesterol from exerting regulatory effects on intracellular lipid homeostasis.

Effects of atorvastatin versus fenofibrate on apoB-100 and apoA-I kinetics in mixed hyperlipidemia

Kinetics of apo B and apo AI were assessed in 8 patients with mixed hyperlipidemia at baseline and after 8 weeks of atorvastatin 80 mg q.d. and micronised fenofibrate 200 mg q.d. in a cross-over study. Both increased hepatic production and decreased catabolism of VLDL accounted for elevated cholesterol and triglyceride concentrations at baseline. Atorvastatin significantly decreased triglyceride, total, VLDL and LDL cholesterol and apo B concentrations (-65%, -36%, -57%, -40% and -33%, respectively, P<0.05). Kinetic analysis revealed that atorvastatin stimulated the catabolism of apo B containing lipoproteins, enhanced the delipidation of VLDL1 and decreased VLDL1 production. Fenofibrate lowered triglycerides and VLDL cholesterol (-57% and -64%, respectively, P<0.05) due to enhanced delipidation of VLDL1 and VLDL2 and increased VLDL1 catabolism. Changes of HDL particle composition accounted for the increase of HDL cholesterol during atorvastatin and fenofibrate (18% and 23%, P<0.01). Only fenofibrate increased apo AI concentrations through enhanced apo AI synthesis (45%, P<0.05). We conclude that atorvastatin exerts additional beneficial effects on the metabolism of apo B containing lipoproteins unrelated to an increase in LDL receptor activity. Fenofibrate but not atorvastatin increases apo AI production and plasma turnover.

Proteins of White Lupin Seed, a Naturally Isoflavone-Poor Legume, Reduce Cholesterolemia in Rats and Increase LDL Receptor Activity in HepG2 Cells

White lupin (Lupinus albus, L.), a widely cultivated crop that has been consumed for many years in Western Europe, may provide a useful alternative for individuals wishing to substitute animal with plant proteins for cardiovascular disease prevention. Lupin seeds have a very low content of isoflavones, and lupin protein isolates are essentially isoflavone free. In rats fed a casein-based cholesterol + cholic acid diet, a relatively low daily intake (50 mg/d by gavage for 2 wk) of total lupin protein extract reduced plasma total and VLDL + LDL cholesterol concentrations by 21 and 30%, respectively (both P<0.001). In an attempt to elucidate the lipid-lowering mechanism, LDL receptor activity was evaluated in a human hepatoma cell line (HepG2). In this model, the lupin total protein extract was essentially inactive, whereas one purified minor protein component, conglutin gamma, had a remarkable upregulatory effect, with maximal increases of 53 and 21% (both P<0.05) for LDL uptake and degradation, respectively. This initial study indicates that lupin, although isoflavone free, has hypocholesterolemic activity similar to that of other leguminous proteins in an established animal model. Further, the cholesterol reduction appears to be associated with stimulation of LDL receptors by a well-defined protein component of the lupin seeds as demonstrated by in vitro studies.

Effect of atorvastatin on apolipoprotein B 48 metabolism and low-density lipoprotein receptor activity in normolipidemic patients with coronary artery disease

We aimed to examine postprandial dyslipidemia in normolipidemic patients with coronary artery disease (CAD) and the effects of treatment with an hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor (atorvastatin). Subjects with angiographicaly established CAD were randomized to treatment for 12 weeks with 80 mg/d atorvastatin or placebo and the effects on markers of postprandial lipoproteins and low-density lipoprotein (LDL)-receptor binding determined. LDL-receptor binding was determined in mononuclear cells, as a surrogate for hepatic activity. Fasting levels of cholesterol ( P < .001), LDL-cholesterol ( P < .001), apolipoprotein (apo)B 48 ( P = .019), remnant-like particle-cholesterol (RLP-C) ( P = .032), and total postprandial apoB 48 area under the curve (AUC) ( P = .013) significantly decreased with atorvastatin compared with placebo. Atorvastatin also significantly increased LDL-receptor binding activity ( P < .001), and this was correlated with changes in fasting apoB 48 ( r = .80, P = .01). We report that aberrations in chylomicron metabolism in normolipidemic CAD subjects are correctable with atorvastatin by a mechanism involving increased LDL-receptor activity. This effect may, in part, explain the cardiovascular benefit of statins used in clinical trials of CAD patients with normal lipid levels.

Red Wine Polyphenolics Increase LDL Receptor Expression and Activity and Suppress the Secretion of ApoB100 from Human HepG2 Cells

Epidemiologic studies suggest that the consumption of red wine may lower the risk of cardiovascular disease. The cardioprotective effect of red wine has been attributed to the polyphenols present in red wine, particularly resveratrol (a stilbene, with estrogen-like activity), and the flavonoids, catechin, epicatechin, quercetin and phenolic acids such as gallic acid. At present, very little is known about the mechanisms by which red wine phenolic compounds benefit the cardiovascular system. Therefore, the aim of this study was to elucidate whether red wine polyphenolics reduce lipoprotein production and clearance by the liver. Cultured HepG2 cells were incubated in the presence of dealcoholized red wine, alcohol-containing red wine and atorvastatin for 24 h. The apolipoprotien B100 (apoB100) protein (marker of hepatic lipoproteins) was quantified on Western blots with an anti-apoB100 antibody and the enhanced chemiluminescence detection system. Apolipoprotein B100 levels in the cells and that secreted into the media were significantly reduced by 50% in liver cells incubated with alcohol-stripped red wine compared with control cells. This effect of dealcoholized red wine on apoB100 production in HepG2 cells was similar to the effect of atorvastatin. Apo B100 production was significantly attenuated by 30% in cells incubated with alcoholized red wine, suggesting that the alcohol was masking the effect of red wine polyphenolics. Apo B100 production was significantly attenuated by 45% with the polyphenolic compounds resveratrol and quercertin. In addition, dealcoholized and alcoholized red wine and atorvastatin significantly increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA and LDL receptor binding activity relative to controls. Dealcoholized red wine also increased LDL receptor gene expression. Collectively, this study suggests that red wine polyphenolics regulate major pathways involved in lipoprotein metabolism.

Effect of estrogen on scavenger receptor BI expression in the rat.

High-dose 17alpha-ethinyl estradiol treatment is associated with increased adrenal and decreased hepatic levels of scavenger receptor class B type 1 (SR-BI) in rats. In this paper we explored the mechanisms responsible for the differential regulation of SR-BI by estrogen in these two tIssues. Previously it was shown that estrogen-treated rats are profoundly hypolipidemic due to increased hepatic low density lipoprotein receptor (LDLR) activity, and that this effect is not maintained with hypophysectomy. To determine if the reduction in hepatic SR-BI was a direct or indirect effect of estrogen, we treated hypophysectomized rats with high-dose estrogen; the levels of SR-BI expression did not change in the livers or adrenals of these animals. To determine if the absence of response to estrogen in the adrenals of hypophysectomized animals was due to the absence of adrenocorticotropic hormone (ACTH), we examined the effect of estrogen treatment on SR-BI expression in animals treated with dexamethasone, which inhibits endogenous ACTH production. The administration of dexamethasone completely inhibited the increase in SR-BI expression in the adrenals of estrogen-treated rats. From these studies we conclude that estrogen does not have a direct effect on SR-BI expression in either the liver or the adrenals. In the liver, the decrease in SR-BI is dependent on the estrogen-induced increase in LDLR activity, and in the adrenal glands, ACTH is required for the estrogen-associated increase in expression of SR-BI.

Troglitazone upregulates LDL receptor activity in HepG2 cells.

The aim of this in vitro study was to investigate the effect of troglitazone, a new oral antidiabetic agent, on LDL catabolism. HepG2 cells, which are cells from a well-differentiated cell line of hepatoma cells, were cultured and used to study LDL catabolism. Different concentrations of troglitazone, all within the therapeutic range for humans, were incubated in culture medium with 125I-labeled LDL to measure cell-associated and degraded 125I-LDL. Troglitazone increased cell-associated and degraded 125I-LDL by approximately 30%. We also investigated if this effect occurred through a LDL receptor-mediated pathway or a non-LDL receptor pathway. By using dextran sulfate, a substance known to release bound LDL from its receptor, we found that troglitazone upregulated LDL receptor activity by approximately 35%. In addition, we found that troglitazone increased the expression of the LDL receptor mRNA. The effect of troglitazone was comparable with that of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, fluvastatin, with troglitazone having an upregulatory effect similar to that of fluvastatin. Insulin within human physiological concentrations also increased LDL receptor activity. We found that troglitazone and insulin had an additive effect on LDL catabolism. Also, the effect of troglitazone on LDL catabolism was studied in the presence of cyclosporine, an immunosuppressant drug that reduces LDL catabolism mainly by decreasing LDL receptor activity. The results showed that troglitazone can compensate for the reduced LDL receptor activity induced by cyclosporine, but that cyclosporine had a residual effect on the action of troglitazone. Thus troglitazone enhanced LDL binding, cell association, and degradation by increasing LDL receptor mRNA expression, with a subsequent increase in LDL receptor activity.

Saturated fatty acids and LDL receptor modulation in humans and monkeys

It has been known for 40 years that dietary saturated fat (SAT FAT) increases plasma cholesterol, including LDL-C and HDL-C. In humans, where LDL-C is typically > 90 mg/dl this SAT FAT effect largely reflects changes in LDL-C pool size. The original human studies suggested that LDL-C expansion during SAT FAT consumption reflected reduced LDL clearance (LDL receptor activity) in hyperlipemics and increased LDL production rates in normolipemics (LDL-C < 100 mg/dl). This dual explanation is supported by data from several animal models where specific saturated fatty acids (SFAs) have been the focus. However, the situation is complicated by the fact that polyunsaturated fatty acids (PUFAs) oppose SFAs, i.e. PUFAs decrease LDL-C and increase LDL receptor (LDL r) activity, so the effect of SAT FAT intake may represent the combined influence of increased SFAs and decreased PUFAs. In fact, careful scrutiny of primate data suggests a negligible effect of saturated fat on LDL clearance (and receptor activity) in the absence of dietary cholesterol when PUFA intake is adequate (5–10%en) and the lipoprotein profile is relatively normal (LDL-C < 90 mg/dl), i.e. normolipemic situations at the time of dietary intervention. In such cases increases in LDL-C due to SFAs (particularly 12:0+14:0) appear to reflect LDL overproduction associated with a shift in cholesterol from tissues to the plasma cholesteryl ester (CE) pool (both LDL-C and HDL-C) without altering whole-body cholesterol balance. The reason for this shift, which is accompanied by an increase in the plasma oleic/linoleic CE ratio, is unknown but may reflect a decreased rate of CE hydrolysis by the liver. When individuals or animals are rendered hyperlipemic by other factors (e.g. chronic caloric and dietary cholesterol excesses in humans or by cholesterol feeding in animals) specific SFAs (particularly 16:0) can contribute to decreased LDL r activity initiated by a primary factor, such as dietary cholesterol. However, LDL r down-regulation by dietary cholesterol greatly exceeds any contribution from SFAs.

Increased Uptake of LDL by Oxidized Macrophages is the Result of an Initial Enhanced LDL Receptor Activity and of a Further Progressive Oxidation of LDL

Iron ions were recently shown to induce cellular lipid peroxidation in macrophages, and these oxidized cells can convert native low-density lipoprotein (LDL) to oxidized LDL (Ox-LDL). The present study demonstrates that deoxycholic acid (DCA) and angiotensin II (ANG-II) can also induce oxidative modification of macrophages via metal ions independent mechanisms. Furthermore, incubation of LDL (200 μg of protein/ml) for 24 h at 37°C with DCA, ANG-II, as well as FeSO 4-induced oxidized macrophages, resulted in oxidative modification of the lipoprotein as evidenced by increased TBARS formation in LDL (by 50, 105, and 258%, respectively), decreased TNBS reactivity (by 45, 56, and 42%, respectively), and increased cellular uptake (by 60, 166, and 230%, respectively). A positive correlation ( n = .88) was found between the extent of the cellular lipid peroxidation and the increment in the cellular uptake of the LDL. The oxidative modification of LDL by oxidized macrophages was found to be a progressive process. Incubation of LDL with oxidized macrophages for increasing periods of time up to 24 h resulted in progressive increment in: (1) the electrophoretic mobility of the LDL; (2) the TBARS formation in LDL; (3) the cellular uptake of LDL by the oxidized macrophages via the Ox-LDL receptor. Upon fractionation on a heparin-sepharose column of LDL that was incubated for different periods of time with oxidized macrophages, a gradual increment in the unbound LDL fraction was obtained, up to 72% after 24 h of incubation. During the first hour of LDL incubation with the oxidized macrophages a twofold increase in the cellular uptake of LDL by these cells was detected, although no significant oxidation of the lipoprotein occurred during this short time period. This effect could be attributed to an increased number of LDL receptors on the cell surface of the oxidized macrophages. In conclusion, increased uptake of LDL by oxidized macrophages results from two routes: (1) enhanced uptake via the LDL receptor due to increased LDL receptor activity; (2) lipoprotein uptake via the Ox-LDL receptors due to cellular modification of LDL. Both of these processes lead to macrophage cholesterol accumulation and foam cell formation, and thus contribute to accelerated atherosclerosis under oxidative stress. © 1997 Elsevier Science Inc.