Down-regulation Of Low Density Lipoprotein Receptors Research Articles

Activation of lipophagy ameliorates cadmium-induced neural tube defects via reducing low density lipoprotein cholesterol levels in mouse placentas

Neural tube defects (NTDs) represent a prevalent and severe category of congenital anomalies in humans. Cadmium (Cd) is an environmental teratogen known to cause fetal NTDs. However, its underlying mechanisms remain elusive. This study aims to investigate the therapeutic potential of lipophagy in the treatment of NTDs, providing valuable insights for future strategies targeting lipophagy activation as a means to mitigate NTDs.We successfully modeled NTDs by Cd exposure during pregnancy. RNA sequencing was employed to investigate the transcriptomic alterations and functional enrichment of differentially expressed genes in NTD placental tissues. Subsequently, pharmacological/genetic (Atg5-/- placentas) experiments confirmed that inducing placental lipophagy can alleviate Cd induced-NTDs. We found that Cd exposure caused NTDs. Further analyzed transcriptomic data from the placentas with NTDs which revealed significant downregulation of low-density lipoprotein receptor associated protein 1(Lrp1) gene expression responsible for positive regulation of low-density lipoprotein cholesterol (LDL-C) transport. Correspondingly, there was an increase in maternal serum/placenta/amniotic fluid LDL-C content. Subsequently, we have discovered that Cd exposure activated placental lipophagy. Pharmacological/genetic (Atg5-/- placentas) experiments confirmed that inducing placental lipophagy can alleviate Cd induced-NTDs. Furthermore, our findings demonstrate that activation of placental lipophagy effectively counteracts the Cd-induced elevation in LDL-C levels. Lipophagy serves to mitigate Cd-induced NTDs by reducing LDL-C levels within mouse placentas.

Overexpression of low-density lipoprotein receptor prevents neurotoxic polarization of astrocytes via inhibiting NLRP3 inflammasome activation in experimental ischemic stroke.

JOURNAL/nrgr/04.03/01300535-202502000-00027/figure1/v/2024-05-28T214302Z/r/image-tiff Neurotoxic astrocytes are a promising therapeutic target for the attenuation of cerebral ischemia/reperfusion injury. Low-density lipoprotein receptor, a classic cholesterol regulatory receptor, has been found to inhibit NLR family pyrin domain containing protein 3 (NLRP3) inflammasome activation in neurons following ischemic stroke and to suppress the activation of microglia and astrocytes in individuals with Alzheimer's disease. However, little is known about the effects of low-density lipoprotein receptor on astrocytic activation in ischemic stroke. To address this issue in the present study, we examined the mechanisms by which low-density lipoprotein receptor regulates astrocytic polarization in ischemic stroke models. First, we examined low-density lipoprotein receptor expression in astrocytes via immunofluorescence staining and western blotting analysis. We observed significant downregulation of low-density lipoprotein receptor following middle cerebral artery occlusion reperfusion and oxygen-glucose deprivation/reoxygenation. Second, we induced the astrocyte-specific overexpression of low-density lipoprotein receptor using astrocyte-specific adeno-associated virus. Low-density lipoprotein receptor overexpression in astrocytes improved neurological outcomes in middle cerebral artery occlusion mice and reversed neurotoxic astrocytes to create a neuroprotective phenotype. Finally, we found that the overexpression of low-density lipoprotein receptor inhibited NLRP3 inflammasome activation in oxygen-glucose deprivation/reoxygenation injured astrocytes and that the addition of nigericin, an NLRP3 agonist, restored the neurotoxic astrocyte phenotype. These findings suggest that low-density lipoprotein receptor could inhibit the NLRP3-meidiated neurotoxic polarization of astrocytes and that increasing low-density lipoprotein receptor in astrocytes might represent a novel strategy for treating cerebral ischemic stroke.

LDLR promotes autophagy-mediated cisplatin resistance in ovarian cancer associated with the PI3K/AKT/mTOR signaling pathway.

Autophagy is one of the underlying causes of resistance to many antitumor drugs, including cisplatin (DDP). The low-density lipoprotein receptor (LDLR) is a regulator of ovarian cancer (OC) progression. However, whether LDLR regulates DDP resistance in OC via autophagy-related pathways remains unclear. LDLR expression was measured by quantitative real-time PCR, western blot (WB) and IHC staining. A Cell Counting Kit 8 assay was employed to evaluate DDP resistance and cell viability, and flow cytometry was used to assess apoptosis. WB analysis was employed to evaluate the expression of autophagy-related proteins and PI3K/AKT/mTOR signaling pathway proteins. The autophagolysosomes and the fluorescence intensity of LC3 were observed by transmission electron microscopy and immunofluorescence staining, respectively. A xenograft tumor model was established to explore the role of LDLR in vivo. LDLR was highly expressed in OC cells, which was correlated with disease progression. In DDP-resistant OC cells, high LDLR expression was related to DDP resistance and autophagy. Downregulation of LDLR repressed autophagy and growth in DDP-resistant OC cell lines by activating the PI3K/AKT/mTOR pathway, and these effects were eliminated by an mTOR inhibitor. In addition, LDLR knockdown also reduced OC tumor growth by suppressing autophagy associated with the PI3K/AKT/mTOR pathway. LDLR promoted autophagy-mediated DDP resistance in OC associated with the PI3K/AKT/mTOR pathway, indicating that LDLR might be a new target to prevent DDP resistance in OC patients.

Identification of the Transcription Factor ATF3 as a Direct and Indirect Regulator of the LDLR.

Coronary artery disease (CAD) is a complex, multifactorial disease caused, in particular, by inflammation and cholesterol metabolism. At the molecular level, the role of tissue-specific signaling pathways leading to CAD is still largely unexplored. This study relied on two main resources: (1) genes with impact on atherosclerosis/CAD, and (2) liver-specific transcriptome analyses from human and mouse studies. The transcription factor activating transcription factor 3 (ATF3) was identified as a key regulator of a liver network relevant to atherosclerosis and linked to inflammation and cholesterol metabolism. ATF3 was predicted to be a direct and indirect (via MAF BZIP Transcription Factor F (MAFF)) regulator of low-density lipoprotein receptor (LDLR). Chromatin immunoprecipitation DNA sequencing (ChIP-seq) data from human liver cells revealed an ATF3 binding motif in the promoter regions of MAFF and LDLR. siRNA knockdown of ATF3 in human Hep3B liver cells significantly upregulated LDLR expression (p < 0.01). Inflammation induced by lipopolysaccharide (LPS) stimulation resulted in significant upregulation of ATF3 (p < 0.01) and subsequent downregulation of LDLR (p < 0.001). Liver-specific expression data from human CAD patients undergoing coronary artery bypass grafting (CABG) surgery (STARNET) and mouse models (HMDP) confirmed the regulatory role of ATF3 in the homeostasis of cholesterol metabolism. This study suggests that ATF3 might be a promising treatment candidate for lowering LDL cholesterol and reducing cardiovascular risk.

TRIB1 regulates LDL metabolism through CEBPα-mediated effects on the LDL receptor in hepatocytes.

Genetic variants near the TRIB1 gene are highly significantly associated with plasma lipid traits and coronary artery disease. While TRIB1 is likely causal of these associations, the molecular mechanisms are not well understood. Here we sought to investigate how TRIB1 influences low density lipoprotein cholesterol (LDL-C) levels in mice. Hepatocyte-specific deletion of Trib1 (Trib1Δhep) in mice increased plasma cholesterol and apoB and slowed the catabolism of LDL-apoB due to decreased levels of LDL receptor (LDLR) mRNA and protein. Simultaneous deletion of the transcription factor CCAAT/enhancer-binding protein alpha (CEBPα) with TRIB1 eliminated the effects of TRIB1 on hepatic LDLR regulation and LDL catabolism. Using RNA-seq, we found that activating transcription factor 3 (Atf3) was highly upregulated in the livers of Trib1Δhep but not Trib1Δhep CebpaΔhep mice. ATF3 has been shown to directly bind to the CEBPα protein, and to repress the expression of LDLR by binding its promoter. Blunting the increase of ATF3 in Trib1Δhep mice reduced the levels of plasma cholesterol and partially attenuated the effects on LDLR. Based on these data, we conclude that deletion of Trib1 leads to a posttranslational increase in CEBPα, which increases ATF3 levels, thereby contributing to the downregulation of LDLR and increased plasma LDL-C.

A Mechanism-Based Pharmacokinetic/Pharmacodynamic Model for Bococizumab, a Humanized Monoclonal Antibody Against Proprotein Convertase Subtilisin/Kexin Type 9, and Its Application in Early Clinical Development.

Bococizumab (RN316/PF-04950615), a humanized monoclonal antibody, binds to secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) and prevents its downregulation of low-density lipoprotein receptor, leading to improved clearance and reduction of low-density lipoprotein cholesterol (LDL-C) in plasma. A mechanism-based drug-target binding model was developed, accounting for bococizumab, PCSK9, and LDL-C concentrations and the effects of concomitant administration of statins. This model was utilized to better understand the pharmacokinetic/pharmacodynamic (PK/PD) data obtained from 3 phase 1 and 2 phase 2a clinical studies. First, simulations performed with this model demonstrated that the conventional method of the area-under-the-curve ratio for bioavailability determination underestimated the subcutaneous bioavailability of bococizumab due to its target-mediated disposition. Second, a covariate model component for statin effects on bococizumab PK/PD was characterized, including a description of the decreased baseline LDL-C, increased baseline PCSK9, and increased LDL-C lowering with concomitant use of statins. Last, the impact of the dosing regimens with and without a dose holiday on bococizumab's LDL-C-lowering effectiveness was shown to be predictable due to the well-characterized PK-PD relationship.

Characterization of a quantitative method to measure free proprotein convertase subtilisin/kexin type 9 in human serum

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that plays an important role in the regulation of serum low-density lipoprotein (LDL) cholesterol by downregulation of LDL receptor, and as such is considered a novel target in cholesterol lowering therapy. In support of the drug development program for Evolocumab, a fully human IgG2 antibody that targets PCSK9, a quantitative ELISA to measure free PCSK9 in human serum was developed. PCSK9 serves as a biomarker of pharmacological response during treatment, and measuring levels of the free ligand post-dosing was of interest as an aid to establishing the pharmacokinetic and pharmacodynamic properties of the therapeutic. Given the complexities associated with the measurement of free ligand in the presence of high concentrations of circulating drug, it was important to challenge the method with experiments designed to assess ex vivo conditions that have the potential to affect the binding equilibrium of drug and ligand within test samples during routine sampling handling and assay conditions. Herein, we report results of experiments that were conducted to characterize the assay in alignment with regulatory guidance and industry standards, and to establish evidence that the method is measuring the free ligand in circulation at the time serum was collected. A robust supporting data package was generated that demonstrates the method specifically and reproducibly measures the free ligand, and is suitable for its intended use.

Cholesterol-induced mammary tumorigenesis is enhanced by adiponectin deficiency: role of LDL receptor upregulation

Adiponectin is an adipokine that can suppress the proliferation of various human carcinoma cells. Although its anti-tumor activities have been suggested by many clinical investigations and animal studies, the underlying mechanisms are not fully characterized. In MMTV-polyomavirus middle T antigen (MMTV-PyVT) transgenic mice models, reduced- or complete loss-of-adiponectin expression promotes mammary tumor development. The present study demonstrated that while tumor development in control MMTV-PyVT mice is associated with a progressively decreased circulating cholesterol concentration, adiponectin deficient MMTV-PyVT mice showed significantly elevated total- and low density lipoprotein (LDL)-cholesterol levels. Cholesterol contents in tumors derived from adiponectin deficient mice were dramatically augmented. High fat high cholesterol diet further accelerated the tumor development in adiponectin deficient PyVT mice. The protein levels of LDL receptor (LDLR) were found to be upregulated in adiponectin-deficient tumor cells. In human breast carcinoma cells, treatment with LDL-cholesterol or overexpressing LDLR elevates nuclear beta-catenin activity and facilitates tumor cell proliferation. On the other hand, adiponectin decreased LDLR protein expression in breast cancer cells and inhibited LDL-cholesterol-induced tumor cell proliferation. Both in vivo and in vitro evidence demonstrated a stimulatory effect of adiponectin on autophagy process, which mediated the down-regulation of LDLR. Adiponectin-induced reduction of LDLR was blocked by treatment with a specific inhibitor of autophagy, 3-methyladenine. In conclusion, the study demonstrates that adiponectin elicits tumor suppressive effects by modulating cholesterol homeostasis and LDLR expression in breast cancer cells, which is at least in part attributed to its role in promoting autophagic flux.

Proprotein Convertase Substilisin/Kexin Type 9 Antagonism Reduces Low-Density Lipoprotein Cholesterol in Statin-Treated Hypercholesterolemic Nonhuman Primates

Proprotein convertase substilisin/kexin type 9 (PCSK9) promotes the degradation of low-density lipoprotein (LDL) receptor (LDLR) and thereby increases serum LDL-cholesterol (LDL-C). We have developed a humanized monoclonal antibody that recognizes the LDLR binding domain of PCSK9. This antibody, J16, and its precursor mouse antibody, J10, potently inhibit PCSK9 binding to the LDLR extracellular domain and PCSK9-mediated down-regulation of LDLR in vitro. In vivo, J10 effectively reduces serum cholesterol in C57BL/6 mice fed normal chow. J16 reduces LDL-C in healthy and diet-induced hypercholesterolemic cynomologous monkeys, but does not significantly affect high-density lipoprotein-cholesterol. Furthermore, J16 greatly lowered LDL-C in hypercholesterolemic monkeys treated with the HMG-CoA reductase inhibitor simvastatin. Our data demonstrate that anti-PCSK9 antibody is a promising LDL-C-lowering agent that is both efficacious and potentially additive to current therapies.

The unique role of proprotein convertase subtilisin/kexin 9 in cholesterol homeostasis

The LDL receptor (LDLR) plays an essential role in the regulation of plasma (LDL) cholesterol concentrations by virtue of its ability to clear plasma LDL. Down-regulation of the LDLR by proprotein convertase subtilisin/kexin 9 (PCSK9) has recently emerged as a regulatory mechanism that controls plasma LDL cholesterol concentrations. Studies in which PCSK9 is over-expressed in mice, have demonstrated that PCSK9, by enhancing hepatic LDLR degradation, decreases the availability of the LDLR for LDL uptake, resulting in increased plasma LDL cholesterol levels. However, PCSK9 has also recently been shown to mediate down-regulation of surface receptors other than the LDLR, suggesting that it may have much broader roles than initially thought.

Chondroitin sulfate-modified LDL induces increased cholesteryl ester synthesis and down-regulation of LDL receptors in smooth muscle cells and macrophages

AbstractHypercholesterolemia induces increased transcytosis and accumulation of plasma lipoproteins in the arterial intima, where they interact with matrix proteins and become modified and reassembled lipoproteins. Chondroitin 6-sulfate-modified LDL (CS-mLDL) induces migration, proliferation, and lipid accumulation in human aortic smooth muscle cells (SMCs). To search for the mechanism(s) responsible for lipid accumulation, cultured SMC and macrophages were exposed to CS-mLDL, minimally modified LDL (mmLDL), and native LDL (as a control). Then the cellular uptake, degradation and expression of the LDL receptor (LDL-R) was determined using radioiodinated ligands, ACAT activity assay, fluorescence microscopy and RT-PCR. The uptake of CS-mLDL was 2-fold higher in SMC and 3-to 4-fold higher in macrophages as compared to LDL and mmLDL; the lysosomal degradation of CS-mLDL was slower in SMCs and considerably diminished in macrophages. Compared with LDL, CS-mLDL induced increased synthesis and accumulation of esterified cholesterol in SMCs (∼2-fold) and macrophages (∼10-fold) within an expanded acidic compartment. CS-mLDL and mmLDL down-regulate the gene expression of the LDL-R in the both cell types. Mechanisms of CS-mLDL-induced lipid accumulation in SMC and macrophages involve increased cellular uptake, and diminished cellular degradation that stimulates cholesterol ester synthesis and accumulation in cytoplasmic inclusions and in the lysosomal compartment in an undegraded form; modified lipoproteins induce down-regulation of LDL-R.

Regulation of lipoprotein trafficking in the kidney: role of inflammatory mediators and transcription factors.

Inflammation and dyslipidaemia both play important roles in the development of glomerular atherosclerosis in renal diseases. We have demonstrated that inflammatory mediators induced Scr (scavenger receptor) expression and the formation of foam cells, and that AP-1 (activator protein 1)/ets were necessary transcriptional factors for Scr induction in HMCs (human kidney mesangial cells). Most cells are protected from excessive native LDL (low-density lipoprotein) accumulation by tight feedback regulation of the LDLr (LDL receptor). However, we observed that HMCs formed foam cells via the LDLr pathway when incubated with IL-1beta (interleukin-1beta; 5 ng/ml) and unmodified LDL (200 microg/ml), suggesting that inflammatory mediators may disrupt the cholesterol-mediated feedback regulation. This feedback involves cholesterol-mediated down-regulation of LDLr controlled by SCAP [SREBP (sterol responsive element-binding protein) cleavage-activating protein]. We have also demonstrated that both tumour necrosis factor alpha and IL-1beta increased nuclear SREBP-1 levels by increasing SCAP mRNA expression, even in the presence of a high concentration of LDL. Since intracellular lipid content is governed by both influx and efflux mechanisms, we set out to examine the impact of inflammatory cytokines on cholesterol efflux, a process mediated by the protein ABCA1 (ATP binding cassette A1). IL-1beta inhibited [(3)H]cholesterol efflux from HMCs by inhibition of the peroxisome-proliferator-activated receptor/LXR (liver X receptor)/ABCA1 pathway. Taken together, our results suggest that inflammatory mediators increase lipid accumulation in HMCs not only by promoting increased lipoprotein uptake by Scr and LDLr, but also by inhibiting ABCA1-mediated cholesterol efflux to high-density lipoprotein.

Implication of cholesterol in cyclosporine pharmacodynamics in minimal change nephrotic syndrome.

High blood cholesterol concentrations in patients with minimal change nephrotic syndrome may affect cyclosporine (INN, ciclosporin) pharmacodynamics and its clinical efficacy, but few attempts have been carried out to disclose this problem. We evaluated the cellular pharmacodynamics of cyclosporine in 24 patients with minimal change nephrotic syndrome. In vitro cyclosporine concentrations yielding 50% inhibition (IC(50)) of blastogenesis of peripheral blood mononuclear cells (PBMCs) stimulated with concanavalin A were calculated. The relationships between the IC(50) values and laboratory data including serum total cholesterol, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol concentrations were examined. Clinical cyclosporine efficacy was assessed by a decreasing rate (percentage) of urinary protein 1 week after cyclosporine therapy. Percentages of LDL receptor-positive or CD3-positive PBMCs were evaluated with flow cytometry in 7 patients and 15 healthy subjects. The cyclosporine IC(50) values negatively correlated with the clinical cyclosporine efficacy assessed by a decreasing rate of urinary protein (r = -0.708, P =.0006). Cyclosporine IC(50) values significantly correlated with either serum total cholesterol (r = 0.681, P =.0003) or LDL cholesterol (r = 0.751, P =.0034) concentrations. Furthermore, serum total or LDL cholesterol levels significantly correlated negatively with clinical cyclosporine efficacy (r = -0.613, P =.0057, and r = -0.773, P =.0399, respectively). In 7 patients, serum total and LDL cholesterol concentrations were significantly higher than those of 15 healthy subjects (P <.005), whereas the percentages of LDL receptor-expressing cells in CD3-enriched PBMCs were not significantly different between these patients and healthy subjects. In addition, the cyclosporine IC(50) values and the percentages of LDL receptor-expressing PBMCs did not negatively correlate in either the patients or the healthy subjects. The data raised the possibility that hypercholesterolemia in patients with minimal change nephrotic syndrome attenuates cellular and clinical cyclosporine pharmacodynamics. Down-regulation of LDL receptor in T cells was not observed in these patients, and individual deviation of PBMC response to cyclosporine does not appear to be related to the difference of LDL receptor-positive cell numbers.

Oxidized LDL through LOX-1 modulates LDL-receptor expression in human coronary artery endothelial cells

Experimental studies have shown that oxidized low-density lipoprotein (ox-LDL) up-regulates its receptor LOX-1. Both ox-LDL and LOX-1 are expressed in atherosclerotic plaques. Native LDL concentrations are elevated in atherosclerosis, suggesting a reduction in LDL-receptors. We hypothesized that ox-LDL via LOX-1 could influence the expression of LDL-receptors. This study was designed to examine the interaction between ox-LDL, LOX-1, and LDL-receptors in human coronary artery endothelial cells (HCAECs). HCAECs were incubated with ox-LDL (10–80 μg/ml) for 3–24 h. Ox-LDL decreased the expression of LDL-receptor in a concentration- and time-dependent fashion. The effects of ox-LDL were mediated by its endothelial receptor LOX-1, since pretreatment of HCAECs with a blocking antibody to LOX-1 (JTX92, 10 μg/ml) prevented the effect of ox-LDL on LDL-receptor expression. The role of LOX-1 was further confirmed by the use of an antisense to LOX-1 mRNA, which also blocked the effect of ox-LDL in LDL-receptor expression. In other experiments, ox-LDL as expected induced superoxide anion generation; and pretreatment of HCAECs with the anti-oxidants trolox and α-tocopherol (each 10 μM) inhibited the formation of superoxide anions as well as the down-regulation of LDL-receptor in response to ox-LDL. These studies provide the first evidence that ox-LDL via LOX-1 modulates LDL-receptor expression in HCAECs. The generation of free radicals elicited by ox-LDL may be a key step in this process.

Dietary Fatty Acids and the Regulation of Plasma Low Density Lipoprotein Cholesterol Concentrations ,

Epidemiologic studies over the past 25 years have shown that the level of dietary fat intake is positively correlated with the average serum cholesterol value and mortality from coronary heart disease (CHD). A number of different investigators demonstrated that in addition to total fat, the fatty acid composition of diets influenced serum total cholesterol (TC) in humans. In general, saturated fatty acids were found to elevate the serum cholesterol concentration, and unsaturated fatty acids were found to decrease this value. The lipoprotein fraction most affected was the level of cholesterol carried in low density lipoprotein (LDL-C). It has now been demonstrated that the steady-state level of LDL-C is predominantly dictated by metabolic events in the liver. As the amount of dietary cholesterol entering the body is increased, there is expansion of the sterol pool in the liver cell and down regulation of LDL receptors (LDLR) that are primarily responsible for clearing LDL-C from the blood stream. When dietary cholesterol intake is kept constant, however, long-chain saturated fatty acids further suppress hepatic LDLR activity, whereas several unsaturated fatty acids have the opposite effect. These regulatory events depend upon the availability of the various fatty acids to shift intracellular cholesterol between a regulatory and storage pool of cholesterol, and this effect is mediated by the enzyme acyl-CoA:cholesterol acyltransferase (ACAT). J. Nutr. 128: 444S-448S, 1998.

Regulation of low-density lipoprotein receptors and assessment of their functional role in Burkitt's lymphoma cells

The status of low-density lipoprotein receptors (LDLR) in Daudi Burkitt's lymphoma (BL) cells was examined using a flow cytometric assay employing the fluorescent ligand DiI-LDL, and a radioligand-binding assay using [ 125I]LDL. The binding is concentration-and time-dependent; and is specific, as judged by its competitive displacement in the presence of unlabeled LDL, and inhibition by heparin, EGTA, and 4°C incubation. The regulation of the receptor and its functional role were then explored. Our results suggest the following: (a) In sharp contrast to normal peripheral blood lymphocytes, the LDLR levels in BL cells are basally elevated when cultured in fetal bovine serum (FBS) medium. (b) In accord with normal peripheral blood lymphocytes, incubation in lipoprotein-deficient serum (LPDS) medium further up-regulates the level of the receptor in BL cells, and co-incubation with LDL or 25-hydroxycholesterol down-regulates the receptor level. The magnitude of the up-regulation is significantly smaller than in normal peripheral blood lymphocytes. (c) Northern blots using a plasmid-DNA probe for LDLR mRNA point to a similar pattern for message regulation as is observed in direct binding studies. (d) Although the LDLR level is constitutively high in BL cells, availability of LDL, unlike transferrin, is not a growth requirement since incubation of cells in LPDS medium does not prevent proliferation of these cells. (e) In contrast to anti-transferrin receptor antibody which results in apoptosis upon binding, anti-LDLR antibody does not inhibit growth or induce apoptosis. Our results suggest LDLR is expressed at a significantly higher level in BL cells than in normal peripheral blood lymphocytes. Although up-regulation and down-regulation of LDLR are observed, this applies only to a small population of LDLR. The bulk receptor population is significantly resistant to down-regulation. Furthermore, notable differences in the functional role of the LDLR are found relative to the transferrin receptor which is also up-regulated in BL cells.

Soy consumption and cholesterol reduction: review of animal and human studies.

Animal proteins such as casein are more hypercholesterolemic than soy protein or other plant proteins when fed to rabbits in low-fat, cholesterol-free, semipurified diets. A casein-amino acid mixture produces a hypercholesterolemia similar to that of casein. This appears to be mainly due to lysine and methionine, although other essential amino acids probably contribute to the effect. Arginine appeared to counteract the hypercholesterolemic effects of other essential amino acids. Soy protein gave a lower level of serum cholesterol in rabbits than did a soy protein-amino acid mixture, suggesting the presence of factors in soy protein that counteract the effects of hypercholesterolemic amino acids. Soy protein is also less hypercholesterolemic than casein in other animal species, particularly when the diet contains cholesterol, and substitution of soy protein for animal protein in the diet reduces the concentration of serum cholesterol in humans. This effect is somewhat variable but is generally greater in hypercholesterolemic than in normocholesterolemic subjects. The differing effects of dietary proteins on serum cholesterol concentrations in humans and in rabbits are primarily due to changes in LDL cholesterol, and the hypercholesterolemia produced by dietary casein is associated with down-regulation of hepatic LDL receptors.

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

The regulation of low-density-lipoprotein(LDL)-receptor activity by 4 beta-phorbol 12-myristate 13-acetate (PMA) and LDL was investigated in the human hepatoma cell line Hep G2. Treatment of Hep G2 cells for 22h with PMA results in an 18.6-fold increase in the amount of LDL-binding sites on the cell surface. The rate of turnover of LDL receptors was not significantly altered upon PMA treatment. Treatment of cultured rat parenchymal cells and human parenchymal cells with PMA did not lead to increased binding of LDL to these cells, suggesting that protein-kinase-C-mediated regulation of LDL-receptor activity is specific for Hep G2 cells and that in this aspect of regulation of LDL receptors, Hep G2 cells do not reflect human hepatocytes. The down-regulation of LDL receptors by a 22-h prior incubation with LDL in PMA-treated Hep G2 cells, in which LDL receptors are upregulated, is more effective than in non-treated cells. Prior incubation of control Hep G2 cells with an excess of LDL caused a partial down-regulation to 33% of the initial level of receptor binding. In PMA-treated Hep G2 cells an excess of non-labeled LDL, led to a down-regulation to 13% of the PMA-induced level. Prior incubation of Hep G2 cells with LDL in the presence of PMA led to a 2.3-fold increase of intracellular cholesteryl esters and a 9.1-fold increase in acyl-CoA:cholesterol-acyltransferase (ACAT) activity. In control cells, LDL prior incubation led to a 1.6-fold increase in intracellular cholesteryl esters and a 1.8-fold increase of ACAT activity. It is concluded that in Hep G2 cells LDL itself can be an effective suppressor of the expression of LDL receptors, provided that the initial amount of receptor allows an adequate intracellular delivery of cholesterol to its sterol-regulatory site.

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.

Decreased receptor-mediated LDL catabolism in casein-fed rabbits precedes the increase in plasma cholesterol levels

The effect of dietary protein on, in vivo, receptor-dependent and receptor-independent low density lipoprotein (LDL) catabolism was assessed in rabbits fed semipurified cholesterol-free diets containing casein or soy protein for 300 days. Plasma cholesterol (7.37 ± 0.59 versus 1.84 ± 0.28 mmol/L; P < 0.001), LDL cholesterol (2.66 ± 0.41 versus 0.67 ± 0.10 mmol/L; P < 0.001) and LDL apo B concentrations (1.39 ± 0.40 versus 0.54 ± 0.12 g/ L plasma; P < 0.001) were significantly higher in rabbits fed casein. Kinetic parameters calculated following the simultaneous injection of radiolabeled native and methylated LDL showed that in casein-fed rabbits, total LDL apo B fractional catabolic rate (FCR) was significantly lower (0.64 ± 0.11 versus 1.42 ± 0.20 pools/d; P < 0.02), and this was due exclusively to a four-fold reduction in receptor-dependent LDL apo B FCR (0.21 ± 0.07 versus 0.86 ± 0.10 pools/d; P < 0.002), since receptor-independent LDL apo B FCR was similar between groups (0.44 ± 0.05 versus 0.56 ± 0.12 pools/d). In groups of rabbits transferred from a soy protein diet to a casein diet, total LDL apo B FCR was decreased significantly in rabbits fed casein diet, 5 days after being switched from the soy protein diet (1.03 ± 0.08 versus 1.83 ± 0.13 pools/d; P < 0.05). This decrease in total LDL apo B FCR was due to decreases in receptor-mediated catabolism (0.56 ± 0.06 versus 1.03 ± 0.14 pools/d; P < 0.001) and receptor-independent catabolism (0.47 ± 0.09 versus 0.80 ± 0.10 pools/d; P < 0.05). However, the plasma cholesterol in these casein-fed rabbits was not significantly different from that observed in soy protein-fed rabbits (2.33 ± 0.52 versus 2.02 ± 0.39 mmol/L). Compared to soy protein-fed rabbits, the plasma cholesterol was significantly higher when the casein had been fed for 10 to 25 days, and in these rabbits total LDL apo B FCR was also significantly reduced, due exclusively to decreased receptor-mediated catabolism. There was a significant inverse correlation in all rabbits between plasma cholesterol and both total LDL apo B FCR ( R = 0.66; P < 0.001) and receptor-dependent LDL apo B FCR ( R = 0.72; P < 0.001). These studies show that down-regulation of LDL receptors in vivo, precedes the subsequent elevation in plasma cholesterol levels induced by a casein diet.