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Introduction: This study aimed to evaluate the possible role of urolithin A (UA) and urolithin B (UB) on the mRNA expression levels of LDL receptor (LDLR) and PSCK9 genes, and also of the uptake of LDL particles in HepG2 cells. Material and methods: The potential role of UA and UB on the induction of LDL uptake and the expression of its regulatory genes was explored using HepG2 cells and curcumin (20 µM), berberine (50 µM), UA (80 µM), and UB (80 µM) as the treatments in the experimental tests. Results: The LDL uptake and cell-surface LDLR were higher in cells treated with UA in comparison with cells treated with UB, and even in relation to the cells treated with curcumin and berberine as positive controls. In addition, cells treated with UB showed approximately 2 times greater LDLR expression levels compared with curcumin (FC = 2.144, p = 0.013) and berberine (FC = 2.761, p = 0.006). However, UA treatment resulted in significantly lower expression levels of LDLR compared with curcumin (FC = 0.274, p < 0.001) and berberine (FC = 0.352, p = 0.009). UB demonstrated approximately 8 times higher LDLR expression levels when compared with UA (FC = 7.835, p = 0.001). Compared with UB, as well as curcumin and berberine as positive controls, UA was more efficient in reducing PCSK9 expression levels. Although UB did not show any significant differences compared with curcumin and berberine, it showed higher levels of PCSK9 expression when compared with the UA group (FC = 3.694, p < 0.001). Conclusions: The present results suggest that UA was more effective than UB in promoting LDL uptake as well as cell surface LDLR in HepG2 cells. This effect seems to be mostly mediated through the suppression of PCSK9 expression but not the induction of LDLR expression. [ABSTRACT FROM AUTHOR]

Introduction This study aimed to evaluate the possible role of urolithin A (UA) and urolithin B (UB) on the mRNA expression levels of LDL receptor (LDLR) and PSCK9 genes, and also of the uptake of LDL particles in HepG2 cells. Material and methods The potential role of UA and UB on the induction of LDL uptake and the expression of its regulatory genes was explored using HepG2 cells and curcumin (20 µM), berberine (50 µM), UA (80 µM), and UB (80 µM) as the treatments in the experimental tests. Results The LDL uptake and cell-surface LDLR were higher in cells treated with UA in comparison with cells treated with UB, and even in relation to the cells treated with curcumin and berberine as positive controls. In addition, cells treated with UB showed approximately 2 times greater LDLR expression levels compared with curcumin (FC = 2.144, p = 0.013) and berberine (FC = 2.761, p = 0.006). However, UA treatment resulted in significantly lower expression levels of LDLR compared with curcumin (FC = 0.274, p < 0.001) and berberine (FC = 0.352, p = 0.009). UB demonstrated approximately 8 times higher LDLR expression levels when compared with UA (FC = 7.835, p = 0.001). Compared with UB, as well as curcumin and berberine as positive controls, UA was more efficient in reducing PCSK9 expression levels. Although UB did not show any significant differences compared with curcumin and berberine, it showed higher levels of PCSK9 expression when compared with the UA group (FC = 3.694, p < 0.001). Conclusions The present results suggest that UA was more effective than UB in promoting LDL uptake as well as cell surface LDLR in HepG2 cells. This effect seems to be mostly mediated through the suppression of PCSK9 expression but not the induction of LDLR expression.

Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused mainly by substitutions in the low-density lipoprotein receptor (LDLR) gene, leading to an increased risk of premature cardiovascular diseases. Tremendous advances in sequencing techniques have resulted in the discovery of more than 3000 variants of the LDLR gene, but not all of them are clinically relevant. Therefore, functional studies of selected variants are needed for their proper classification. Here, a single-cell, kinetic, fluorescent LDL uptake assay was applied for the functional analysis of LDLR variants in a model of an LDLR-deficient human cell line. An LDLR-defective HEK293T cell line was established via a CRISPR/Cas9-mediated luciferase–puromycin knock-in. The expressing vector with the LDLR gene under the control of the regulated promoter and with a reporter gene has been designed to overproduce LDLR variants in the host cell. Moreover, an LDLR promoter–luciferase knock-in reporter system has been created in the human cell line to study transcriptional regulation of the LDLR gene, which can serve as a simple tool for screening and testing new HMG CoA reductase-inhibiting drugs for atherosclerosis therapy. The data presented here demonstrate that the obtained LDLR-deficient human cell line HEK293T-ldlrG1 and the dedicated pTetRedLDLRwt expression vector are valuable tools for studying LDL internalization and functional analysis of LDLR and its genetic variants. Using appropriate equipment, LDL uptake to a single cell can be measured in real time. Moreover, the luciferase gene knock-in downstream of the LDLR promotor allows the study of promoter regulation in response to diverse conditions or drugs. An analysis of four known LDLR variants previously classified as pathogenic and benign was performed to validate the LDLR-expressing system described herein with the dedicated LDLR-deficient human cell line, HEK293T-ldlrG1. [ABSTRACT FROM AUTHOR]

Pyranoanthocyanins have been reported to possess better chemical stability and bioactivities than monomeric anthocyanins in some aspects. The hypocholesterolemic activity of pyranoanthocyanins is unclear. In view of this, this study was conducted to compare the cholesterol-lowering activities of Vitisin A with the anthocyanin counterpart Cyanidin-3-O-glucoside(C3G) in HepG2 cells and to investigate the interaction of Vitisin A with the expression of genes and proteins associated with cholesterol metabolism. HepG2 cells were incubated with 40 μM cholesterol and 4 μM 25-hydroxycholeterol with various concentrations of Vitisin A or C3G for 24 h. It was found that Vitisin A decreased the cholesterol levels at the concentrations of 100 μM and 200 μM with a dose–response relationship, while C3G exhibited no significant effect on cellular cholesterol. Furthermore, Vitisin A could down-regulate 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) to inhibit cholesterol biosynthesis through a sterol regulatory element-binding protein 2 (SREBP2)-dependent mechanism, and up-regulate low-density lipoprotein receptor (LDLR) and blunt the secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein to promote intracellular LDL uptake without LDLR degradation. In conclusion, Vitisin A demonstrated hypocholesterolemic activity, by inhibiting cholesterol biosynthesis and enhancing LDL uptake in HepG2 cells. [ABSTRACT FROM AUTHOR]

Introduction: Endothelial cells (ECs) form the inner lining of all blood vessels of the body play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in vitro investigations of ECs' physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal-derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications. Method: Human induced pluripotent stem cells (hiPSCs) are cultured under serum-free conditions and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 h. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt monohydrate (8Bro) and melatonin (Mel) for 48 h. Result: This combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two-dimensional (2D) monolayer into three-dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von Willebrand factor and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4). Conclusion: The differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at high efficiency, hence providing a scalable source of hiPSC-ECs. [ABSTRACT FROM AUTHOR]

Autosomal Dominant Hypercholesterolemia (ADH) is a genetic disorder caused by pathogenic variants in LDLR, APOB, PCSK9 and APOE genes. We sought to identify new candidate genes responsible for the ADH phenotype in patients without pathogenic variants in the known ADH-causing genes by focusing on a French family with affected and non-affected members who presented a high ADH polygenic risk score (wPRS). Linkage analysis, whole exome and whole genome sequencing resulted in the identification of variants p.(Pro398Ala) in CYP7A1, p.(Val1382Phe) in LRP6 and p.(Ser202His) in LDLRAP1. A total of 6 other variants were identified in 6 of 160 unrelated ADH probands: p.(Ala13Val) and p.(Aps347Asn) in CYP7A1; p.(Tyr972Cys), p.(Thr1479Ile) and p.(Ser1612Phe) in LRP6; and p.(Ser202LeufsTer19) in LDLRAP1. All six probands presented a moderate wPRS. Serum analyses of carriers of the p.(Pro398Ala) variant in CYP7A1 showed no differences in the synthesis of bile acids compared to the serums of non-carriers. Functional studies of the four LRP6 mutants in HEK293T cells resulted in contradictory results excluding a major effect of each variant alone. Within the family, none of the heterozygous for only the LDLRAP1 p.(Ser202His) variant presented ADH. Altogether, each variant individually does not result in elevated LDL-C; however, the oligogenic combination of two or three variants reveals the ADH phenotype. [ABSTRACT FROM AUTHOR]

Autosomal recessive hypercholesterolemia (ARH) is a rare monogenic disorder caused by pathogenic variants in the low-density lipoprotein receptor (LDLR) adaptor protein 1 (LDLRAP1) gene, encoding for the LDLRAP1 protein, which impairs internalization of hepatic LDLR. There are variable responses of ARH patients to treatment and the pathophysiological mechanism(s) for this variability remains unclear. This is in part caused by absence of reliable cellular models to evaluate the effect of LDLRAP1 mutations on the LDLRAP1 protein function and its role in LDLR internalization. Here, we aimed to validate patient-specific induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) as an appropriate tool to model ARH disease. Fibroblasts from an ARH patient carrying the recently reported nonsense mutation, c.649G>T, were reprogrammed into hiPSCs using Sendai viral vectors. In addition, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to create an LDLRAP1 gene (also known as ARH) knockout in two different human iPSC lines. ARH patient-derived iPSCs, ARH-knockout iPSC lines, and control iPSCs were efficiently differentiated into HLCs. Western blot analysis demonstrated the absence of LDLRAP1 in HLCs derived from patient and knockout iPSCs, and this was associated with a decreased low-density lipoprotein cholesterol (LDL-C) uptake in ARH-mutant/knockout HLCs compared to control HLCs. In conclusion, we determined that the recently described c.649G>T point mutation in LDLRAP1 induces absence of the LDLRAP1 protein, similar to what is seen following LDLRAP1 knockout. This causes a decreased, although not fully absent, LDL-uptake in ARH-mutant/knockout HLCs. As knockout of LDLRAP1 or presence of the c.649G>T point mutation results in absence of LDLRAP1 protein, residual LDL uptake might be regulated by LDLRAP1-independent internalization mechanisms. Patient-specific iPSC-derived HLCs can therefore be a powerful tool to further decipher LDLRAP1 mutations and function of the protein. [ABSTRACT FROM AUTHOR]

Autosomal Dominant Hypercholesterolemia (ADH) is a genetic disorder caused by pathogenic variants in LDLR, APOB, PCSK9 and APOE genes. We sought to identify new candidate genes responsible for the ADH phenotype in patients without pathogenic variants in the known ADH-causing genes by focusing on a French family with affected and non-affected members who presented a high ADH polygenic risk score (wPRS). Linkage analysis, whole exome and whole genome sequencing resulted in the identification of variants p.(Pro398Ala) in CYP7A1, p.(Val1382Phe) in LRP6 and p.(Ser202His) in LDLRAP1. A total of 6 other variants were identified in 6 of 160 unrelated ADH probands: p.(Ala13Val) and p.(Aps347Asn) in CYP7A1; p.(Tyr972Cys), p.(Thr1479Ile) and p.(Ser1612Phe) in LRP6; and p.(Ser202LeufsTer19) in LDLRAP1. All six probands presented a moderate wPRS. Serum analyses of carriers of the p.(Pro398Ala) variant in CYP7A1 showed no differences in the synthesis of bile acids compared to the serums of non-carriers. Functional studies of the four LRP6 mutants in HEK293T cells resulted in contradictory results excluding a major effect of each variant alone. Within the family, none of the heterozygous for only the LDLRAP1 p.(Ser202His) variant presented ADH. Altogether, each variant individually does not result in elevated LDL-C; however, the oligogenic combination of two or three variants reveals the ADH phenotype.

Although the hypocholesterolaemic activity of soy foods has been well known for decades, its molecular mechanism is still poorly understood. The aim of this study was a detailed molecular characterization of the activity of IAVPGEVA, IAVPTGVA, and LPYP, three peptides from soy glycinin hydrolysis, which in the past had been shown to be competitive inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoAR). HepG2 cells were treated with these peptides and the samples were analysed with a combination of molecular techniques. The experiments demonstrated that IAVPGEVA, IAVPTGVA, and LPYP are able to interfere with the catalytic activity of HMGCoAR and to modulate the cholesterol metabolism, through the activation of the LDLR-SREBP2 pathway, increasing the ability of HepG2 cells to uptake the LDL. This also involves the activation of AMPK and ERK 1/2. For the first time, this study provides a characterization of the molecular mechanism through which peptides derived from soy glycinin exert their cholesterol-lowering activity.

High lipid levels are associated with the increase tendency of atherosclerosis formation. In the pathogenesis of atherosclerosis, increase in low density lipoprotein cholesterol (LDL-c) concentration has been identified as the main culprit in many cardiovascular disease (CVD) incidents. Both quercetin and kaempferol are flavonoids that most abundantly found in fruits and vegetables. Several studies have dictated that both compounds exhibit CVD protective effects through the regulation of lipid levels. In the present study, the hypolipidemic potential of quercetin and kaempferol through LDL-c uptake were tested on HepG2 cells. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in order to study the cytotoxicity effect quercetin and kaemperol on cell proliferation. The present study demonstrated that quercetin and kaempferol at low concentration of 15 µM, possess the highest hypolipidemic effects via LDL-c uptake in HepG2 cells (p<0.05). Interestingly, quercetin and kaempferol combination at 1:1 ratio possesses the best effect on LDL-c uptake in HepG2 cells as compared to other ratios. It is suggested that there is a possibility of synergistic effects of quercetin and kaempferol that enhance the LDL uptake more effectively than its single compounds alone. The decrease in cell viability was higher in mixture combinations of quercetin and kaemperol (1:1, 2:1 and 1:2) than to individually treated quercetin and kaempferol (1:0 and 0:1). Further studies should be conducted on primary human liver cells on the LDL uptake and cell viability to further justify the significance of both quercetin and kaempferol as lipid lowering agents as normal LDL-c uptake occurs in healthy cells, rather than a tumour cells like HepG2. [ABSTRACT FROM AUTHOR]

This review integrates historical biochemical and modern genetic findings that underpin our understanding of the low-density lipoprotein (LDL) dyslipidemias that bear on human disease. These range from life-threatening conditions of infancy through severe coronary heart disease of young adulthood, to indolent disorders of middle- and old-age. We particularly focus on the biological aspects of those gene mutations and variants that impact on sterol absorption and hepatobiliary excretion via specific membrane transporter systems (NPC1L1, ABCG5/8); the incorporation of dietary sterols (MTP) and of de novo synthesized lipids (HMGCR, TRIB1) into apoB-containing lipoproteins (APOB) and their release into the circulation (ANGPTL3, SARA2, SORT1); and receptor-mediated uptake of LDL and of intestinal and hepatic-derived lipoprotein remnants (LDLR, APOB, APOE, LDLRAP1, PCSK9, IDOL). The insights gained from integrating the wealth of genetic data with biological processes have important implications for the classification of clinical and presymptomatic diagnoses of traditional LDL dyslipidemias, sitosterolemia, and newly emerging phenotypes, as well as their management through both nutritional and pharmaceutical means.

This research aimed to investigate the effect of silver carp hydrolysates (SCHs) on hypercholesterolemia and enterohepatic cholesterol metabolism. Results showed that in vitro gastrointestinal digestion products of Alcalase-SCH (GID-Alcalase) exhibited the highest inhibitory activity of cholesterol absorption mainly through downregulating the expression of essential genes related to cholesterol transport in a Caco-2 monolayer. After being absorbed by the Caco-2 monolayer, GID-Alcalase increased the low-density lipoprotein (LDL) uptake in HepG2 cells by enhancing the protein level of the LDL receptor (LDLR). The in vivo experiment showed that long-term intervention of Alcalase-SCH ameliorated hypercholesterolemia in ApoE -/- mice fed with a Western diet (WD). After transepithelial transport, four novel peptides (TKY, LIL, FPK, and IAIM) were identified, and these peptides possessed dual hypocholesterolemic functions including inhibition of cholesterol absorption and promotion of peripheric LDL uptake. Our results indicated for the first time the potential of SCHs as functional food ingredients for the management of hypercholesterolemia.

Although the hypocholesterolaemic activity of soy foods has been well known for decades, its molecular mechanism is still poorly understood. The aim of this study was a detailed molecular characterization of the activity of IAVPGEVA, IAVPTGVA, and LPYP, three peptides from soy glycinin hydrolysis, which in the past had been shown to be competitive inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoAR). HepG2 cells were treated with these peptides and the samples were analysed with a combination of molecular techniques. The experiments demonstrated that IAVPGEVA, IAVPTGVA, and LPYP are able to interfere with the catalytic activity of HMGCoAR and to modulate the cholesterol metabolism, through the activation of the LDLR-SREBP2 pathway, increasing the ability of HepG2 cells to uptake the LDL. This also involves the activation of AMPK and ERK 1/2. For the first time, this study provides a characterization of the molecular mechanism through which peptides derived from soy glycinin exert their cholesterol-lowering activity. [ABSTRACT FROM AUTHOR]

Background Resistance of cancer cells towards chemotherapeutics represents a major cause of therapy failure. The objective of our study was to evaluate cellular defense strategies in response to the novel vacuolar H+-ATPase inhibitor, archazolid B.Experimental approach: The effects of archazolid B on T24 bladder carcinoma cells were investigated by combining "omics" technologies (transcriptomics (mRNA and miRNA) and proteomics). Free cholesterol distribution was determined by filipin staining using flow cytometry and fluorescence microscopy. Flow cytometry was performed for LDLR surface expression studies. Uptake of LDL cholesterol was visualized by confocal microscopy. SREBP activation was determined performing Western Blotting. The efficiency of archazolid B/fluvastatin combination was tested by cytotoxicity assays. Results Archazolid B led to accumulation of free cholesterol within intracellular compartments and drastic disturbances in cholesterol homeostasis resulting in activation of SREBP-2 (sterol regulatory element-binding protein 2) and up-regulation of target genes including HMGCR (HMG-CoA reductase), the key enzyme of cholesterol biosynthesis. LDLR surface expression was reduced and LDL uptake was completely inhibited after 24h, indicating newly synthesized cholesterol to be the main source of cholesterol in archazolid B treated cells. By combining archazolid B with the HMGCR inhibitor fluvastatin, cholesterol was reduced and cell viability decreased by about 20% compared to archazolid B treatment alone. Conclusions Our study revealed cholesterol biosynthesis as an important resistance mechanism in T24 cells after archazolid B treatment. The combination of archazolid B with statins may be an attractive strategy to potentiate archazolid B induced cell killing by affecting cholesterol biosynthesis. [ABSTRACT FROM AUTHOR]

Amyloid precursor protein (APP) ubiquitously expressed. Studies in neuronal cells have implicated APP or its fragments as negative regulators of cholesterol metabolism. In the current study, APP acted, via its o~-cleavage, as a positive regulator of sterol regulatory element-binding protein-2 (SREBP2) signaling in human astrocytic cells (U251MG), hepatic cells (HepG2), and primary fibroblasts, leading to an approximate 30% increase in SRE-dependent gene expression and, consequently, enhanced cholesterol biosynthesis and LDL receptor levels. This effect was mediated via the secretory ectodomain APPsα. The β-cleaved ectodomain, in turn, repressed SRE-dependent gene expression by up to ~30%. This resulted in decreased cholesterol synthesis and LDL receptor content, establishing a physiological feedback loop in cholesterol-loaded cells, where APP undergoes preferential β-cleavage. Patients with familial Alzheimer's disease had decreased circulating lathosterol, reflecting hepatic cholesterol synthesis, and their fibroblasts had reduced LDL receptor content, which was alleviated decreasing β-cleavage. These results show that APP regulates cholesterol metabolism in cells relevant for whole-body cholesterol balance and reveal that APP α- and β-cleavages produce opposing paracrine regulators of SREBP2 signaling. [ABSTRACT FROM AUTHOR]

Abstract: Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL [1] Abbreviations: CHO, cholesterol; CHOE, cholesteryl ester; DRM, lipid microdomains or detergent-resistant membrane; LDL, low density lipoprotein; 125I-LDL, LDL labeled with 125I; 3H-CHO-LDL, LDL with 3H-Cholesterol incorporated; TR-LDL, LDL with Texas-Red-Phosphatidylethanolamine associated. FITC-LDL, LDL with fluorescein 5’-isothyocianate associated; MBCD, methyl-β-cyclodextrin. 1 particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-β-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Elevated levels of low-density-lipoprotein cholesterol (LDL-C) in the plasma are a well-established risk factor for the development of coronary heart disease. Plasma LDL-C levels are in part determined by the rate at which LDL particles are removed from the bloodstream by hepatic uptake. The uptake of LDL by mammalian liver cells occurs mainly via receptor-mediated endocytosis, a process which entails the binding of these particles to specific receptors in specialised areas of the cell surface, the subsequent internalization of the receptor–lipoprotein complex, and ultimately the degradation and release of the ingested lipoproteins’ constituent parts. We formulate a mathematical model to study the binding and internalization (endocytosis) of LDL and VLDL particles by hepatocytes in culture. The system of ordinary differential equations, which includes a cholesterol-dependent pit production term representing feedback regulation of surface receptors in response to intracellular cholesterol levels, is analysed using numerical simulations and steady-state analysis. Our numerical results show good agreement with in vitro experimental data describing LDL uptake by cultured hepatocytes following delivery of a single bolus of lipoprotein. Our model is adapted in order to reflect the in vivo situation, in which lipoproteins are continuously delivered to the hepatocyte. In this case, our model suggests that the competition between the LDL and VLDL particles for binding to the pits on the cell surface affects the intracellular cholesterol concentration. In particular, we predict that when there is continuous delivery of low levels of lipoproteins to the cell surface, more VLDL than LDL occupies the pit, since VLDL are better competitors for receptor binding. VLDL have a cholesterol content comparable to LDL particles; however, due to the larger size of VLDL, one pit-bound VLDL particle blocks binding of several LDLs, and there is a resultant drop in the intracellular cholesterol level. When there is continuous delivery of lipoprotein at high levels to the hepatocytes, VLDL particles still out-compete LDL particles for receptor binding, and consequently more VLDL than LDL particles occupy the pit. Although the maximum intracellular cholesterol level is similar for high and low levels of lipoprotein delivery, the maximum is reached more rapidly when the lipoprotein delivery rates are high. The implications of these results for the design of in vitro experiments is discussed. [ABSTRACT FROM AUTHOR]

Individuals with elevated levels of plasma low density lipoprotein (LDL) cholesterol (LDL-C) are considered to be at risk of developing coronary heart disease. LDL particles are removed from the blood by a process known as receptor-mediated endocytosis, which occurs mainly in the liver. A series of classical experiments delineated the major steps in the endocytotic process; apolipoprotein B-100 present on LDL particles binds to a specific receptor (LDL receptor, LDL-R) in specialized areas of the cell surface called clathrin-coated pits. The pit comprising the LDL-LDL-R complex is internalized forming a cytoplasmic endosome. Fusion of the endosome with a lysosome leads to degradation of the LDL into its constituent parts (that is, cholesterol, fatty acids, and amino acids), which are released for reuse by the cell, or are excreted. In this paper, we formulate a mathematical model of LDL endocytosis, consisting of a system of ordinary differential equations. We validate our model against existing in vitro experimental data, and we use it to explore differences in system behavior when a single bolus of extracellular LDL is supplied to cells, compared to when a continuous supply of LDL particles is available. Whereas the former situation is common to in vitro experimental systems, the latter better reflects the in vivo situation. We use asymptotic analysis and numerical simulations to study the longtime behavior of model solutions. The implications of model-derived insights for experimental design are discussed. [ABSTRACT FROM AUTHOR]

Previous studies have shown that people infected with schistosomiasis have lower levels of serum cholesterol than uninfected controls. To better understand the impact of this parasitic infection on serum cholesterol levels and on atherosclerotic lesion development induced by hypercholesterolemia, apolipoprotein E (ApoE)-deficient mice were chronically exposed to the eggs of Schistosoma mansoni over a period of 16 weeks. Total serum cholesterol and low-density lipoprotein (LDL) were reduced in egg-exposed ApoE-deficient mice fed a diet high in cholesterol compared to unexposed controls. However, exposure to eggs had no effect on atherosclerotic lesion size or progression in ApoE-deficient mice. Macrophages isolated from egg-exposed mice had an enhanced ability to take up LDL but not acetylated LDL (acLDL). This study suggests that schistosome eggs alone may alter serum lipid profiles through enhancing LDL uptake by macrophages, but these changes do not ultimately affect atherosclerotic lesion development. [ABSTRACT FROM AUTHOR]

Explores the role of Chlamydia pneumoniae infection in macrophage expression of lipoprotein lipase gene. Influence of Chlamydia pneumoniae on the upregulation of lipoprotein lipase messenger RNA gene expression; Impact of calcium influx signals on lipoprotein lipase gene expression; Assessment of lipoprotein lipase activity.

In the present study, the binding, internalization and degradation of low-density lipoprotein (LDL) was investigated in Hep-G2 cells treated with 18:0, 18:1, 18:2 and 18:3. In non-treated control cells, the surface binding (heparin-releasable) of 125I-LDL progressed in a saturable manner reaching equilibrium within 2 h, amounting 24.0 ± 1.1, 29.5 ± 1.3 and 31.4 ± 2.8 (ng/mg cell protein) at 1, 2 and 4 h, respectively. The cells rapidly internalized 125I-LDL reaching a plateau at 2 h (72.4 ± 6.3/1 h, 96.7 ± 4.3/2 h and 100.8 ± 4.6 ng/mg protein/4 h, respectively). The degradation of internalized LDL progressed slowly during the first hour of incubation reflecting the time required to an uptake and delivery of LDL to the cellular lysosomes. The levels of degraded LDL discharged into the medium then increased rapidly in a linear manner after the initial lag period, amounting 16.8 ± 1.2, 51.8 ± 7.0 and 118.2 ± 5.7 ng/mg protein at 1, 2 and 4 h, respectively. The treatment of cells with of 1.0 mM of fatty acids for 4 h resulted in a significant increase in the surface binding of 125I-LDL compared to the control (34.9 ± 3.0), but it was significantly lower in cells exposed to 18:0 (48.2 ± 2.0) than to 18:1 (56.8 ± 5.1), 18:2 (56.0 ± 3.5) and 18:3 (57.8 ± 6.0 ng/mg protein/4 h) (P < 0.05). The levels of degraded LDL in cells remained nearly the same regardless of fatty acid treatments, but degraded LDL levels in the medium were much higher in cells exposed to 18:1 (167.6 ± 10.1), 18:2 (159.8 ± 7.7) and 18:3 (165.1 ± 14.7) than to 18:0 (142.1 ± 8.4) and the control (121.2 ± 3.4 ng/mg protein/4 h) (P < 0.05). The present finding that 18:1 is equally effective in enhancing the receptor-mediated LDL uptake and its degradation as those of 18:2 and 18:3 suggests that the major action of 18:1 in lowering LDL-cholesterol levels also involves an increased clearance of LDL via hepatic LDL-receptors. [Copyright &y& Elsevier]

Systematic insight into cellular dysfunction can improve understanding of disease etiology, risk assessment, and patient stratification. We present a multiparametric high-content imaging platform enabling quantification of low-density lipoprotein (LDL) uptake and lipid storage in cytoplasmic droplets of primary leukocyte subpopulations. We validate this platform with samples from 65 individuals with variable blood LDL-cholesterol (LDL-c) levels, including familial hypercholesterolemia (FH) and non-FH subjects. We integrate lipid storage data into another readout parameter, lipid mobilization, measuring the efficiency with which cells deplete lipid reservoirs. Lipid mobilization correlates positively with LDL uptake and negatively with hypercholesterolemia and age, improving differentiation of individuals with normal and elevated LDL-c. Moreover, combination of cell-based readouts with a polygenic risk score for LDL-c explains hypercholesterolemia better than the genetic risk score alone. This platform provides functional insights into cellular lipid trafficking and has broad possible applications in dissecting the cellular basis of metabolic disorders., Competing Interests: A patent application covering the use of the here-suggested patient stratification methods has been filed (application: FI 20206284), in which University of Helsinki is the applicant and E.I. and S.G.P. are the inventors., (© 2022 The Authors.)

A previous study of low density lipoprotein metabolism by cultured cells focused on the metabolism of normal lipoproteins in vitro by fibroblasts isolated from diabetic patients. No abnormalities were found. We have followed the opposite approach. Using normal human fibroblasts as test cells we compared the metabolism in vitro of low density lipoproteins isolated from diabetic patients before and after metabolic control. We found a significant decrease (p<0.02) in internalization and degradation of low density lipoproteins isolated from diabetic patients before metabolic control when compared with those isolated from normal control subjects or from the same patients after metabolic control. The observed changes were mainly apparent in intracellular degradation. To evaluate whether the observed differences in low density lipoprotein behaviour were correlated with lipid or apolipoprotein composition, we measured cholesterol, triglyceride, apolipoprotein B and total protein levels in the low density lipoproteins tested. A significant decrease (p<0.05) of the triglyceride/protein ratio was found in post-control low density lipoproteins suggesting that a high triglyceride content may interfere with low density lipoprotein metabolism. The present study represents the first observation that metabolic control in diabetes mellitus can alter low density lipoprotein-cell interaction and suggests a possible mechanism for the enhanced incidence of atherosclerosis in diabetic patients. [ABSTRACT FROM AUTHOR]

The aim of the study was to investigate the effect of zinc depletion on the susceptibility of Wistar rat low-density lipoproteins (LDL) to peroxidation and their uptake by macrophages, before and after in vitro oxidation. The rats were fed for 7 wk a Zn-adequate diet (100 ppm) ad libitum (AL), a Zn-deficient diet (0.2 ppm) ad libitum (ZD), or a Zn-adequate diet according to the pair-feeding method (PF). Zinc status was determined and, for each group, blood was pooled, and LDL were isolated and labeled withIodine. An aliquot of each LDL sample was oxidized using Fe 10 μ M/ascorbate 250 μ M. Oxidized and nonoxidized (native) LDL were incubated with P 388 D1 macrophages, and their rates of uptake and degradation by macrophages were measured. Before oxidation, LDL uptake and degradation were not modified by the diet, suggesting that Zn deficiency did not modify rat LDL in vivo. After oxidation, both LDL uptake and degradation were significantly enhanced in the three groups. Nevertheless, we did not observe a significant effect of Zn deficiency. This observation suggests that, in our experimental conditions, Zn deficiency did not modify LDL catabolism. [ABSTRACT FROM AUTHOR]

Amyloid precursor protein (APP) is ubiquitously expressed. Studies in neuronal cells have implicated APP or its fragments as negative regulators of cholesterol metabolism. In the current study, APP acted, via its α-cleavage, as a positive regulator of sterol regulatory element-binding protein-2 (SREBP2) signaling in human astrocytic cells (U251MG), hepatic cells (HepG2), and primary fibroblasts, leading to an approximate 30% increase in SRE-dependent gene expression and, consequently, enhanced cholesterol biosynthesis and LDL receptor levels. This effect was mediated via the secretory ectodomain APPsα. The β-cleaved ectodomain, in turn, repressed SRE-dependent gene expression by up to ∼ 30%. This resulted in decreased cholesterol synthesis and LDL receptor content, establishing a physiological feedback loop in cholesterol-loaded cells, where APP undergoes preferential β-cleavage. Patients with familial Alzheimer's disease had decreased circulating lathosterol, reflecting hepatic cholesterol synthesis, and their fibroblasts had reduced LDL receptor content, which was alleviated by decreasing β-cleavage. These results show that APP regulates cholesterol metabolism in cells relevant for whole-body cholesterol balance and reveal that APP α- and β-cleavages produce opposing paracrine regulators of SREBP2 signaling.