LDL Receptor mRNA Levels Research Articles

Epigallocatechin gallate induces an up-regulation of LDLR accompanied by a reduction of idol in Hepg2 cells

AbstractEpigallocatechin gallate (EGCG), the primary catechin in green tea, has improved cholesterol metabolism. However, the molecular mechanisms of EGCG underlying these functions are not fully understood. In this study, we aimed to investigate the molecular mechanisms underlying EGCG’s effect on low-density lipoprotein (LDL) in HepG2 cells. Real-time PCR and Western blot analysis were used to determine the mRNA and protein levels in the human hepatoma cell line (HepG2). LDL uptake assay was used to quantify the low-density lipoprotein receptor (LDLR) function. EGCG induced significantly up-regulated LDLR protein and mRNA levels in HepG2 cells (P < 0.05). Both at the transcriptional level and at the protein level, EGCG can significantly (P < 0.05) down-regulate the elevated expression levels of liver X receptor α (LXRα) and inducible degrader of the LDLR (Idol) due to 25-OHC. Fluorescence results showed that EGCG induction could also significantly increase LDL uptake (P < 0.05). EGCG regulates LDL uptake through the LXRα-LDLR pathway, and EGCG can effectively improve the abnormal expression of protein and mRNA induced by 25-OHC. Graphical abstract

Flavonoids regulate LDLR through different mechanisms tied to their specific structures

Flavonoids, polyphenolic compounds found in plant-based diets, are associated with reduced risk of cardiovascular disease and longevity. These components are reported to reduce plasma levels of low-density lipoprotein (LDL) through an upregulation of the LDL receptor (LDLR), but the mechanism is still largely unknown. In this study, we have systematically screened the effect of 12 flavonoids from six different flavonoid subclasses on the effect on LDLR. This paper provides an in-depth analysis on how these flavonoids affect LDLR regulation and functionality. We found that most but not all of the tested flavonoids increased LDLR mRNA levels. Surprisingly, this increase was attributed to different regulatory mechanisms, such as enhanced LDLR promoter activity, LDLR mRNA stabilization, or LDLR protein stabilization, of which specific effectual parts of the flavonoid molecular structure could be assigned. These types of comparative analysis of various flavonoids enhance clarity and deepen the understanding of how the different structures of flavonoids affect LDLR regulation. Our data offer useful insights that may guide future research in developing therapeutic approaches for cardiovascular health.

Hypercholesterolemia induced by spontaneous oligogenic mutations in rhesus macaques (Macaca mulatta).

A rhesus macaque with the fourth highest plasma cholesterol (CH) levels of 501 breeding macaques was identified 22 years ago. Seven offspring with gene mutations causing hypercholesterolemia were obtained. Activity of low-density lipoprotein receptor (LDLR), plasma CH levels and mRNA expression levels of LDLR were measured after administration of 0.1% (0.27 mg/kcal) or 0.3% CH. Activity of p. (Cys82Tyr) of LDLR was 71% and 42% in the heterozygotes and a homozygote, respectively. The mRNA expression level of LDLR in the p. (Val241Ile) of membrane-bound transcription factor protease, site 2 (MBTPS2, S2P protein) was 0.83 times lower than normal levels. LDLR mRNA levels were increased for up to 4 weeks by administration of 0.3% CH before suddenly decreasing to 80% of the baseline levels after 6 weeks. Oligogenic mutations of p. (Cys82Tyr) in LDLR and p. (Val241Ile) in MBTPS2 (S2P) caused hypercholesterolemia exceeding cardiovascular risk levels under a 0.1% CH diet.

Turnover Rates of the Low-Density Lipoprotein Receptor and PCSK9: Added Dimension to the Cholesterol Homeostasis Model.

We measured the turnover rates of the LDLR (low-density lipoprotein receptor) and PCSK9 (proprotein convertase subtilisin/kexin type 9) in mice by metabolic labeling with heavy water and mass spectrometry. Approach and Results: In liver of mice fed high-cholesterol diets, LDLR mRNA levels and synthesis rates were markedly lower with complete suppression of cholesterol synthesis and higher cholesterol content, consistent with the Brown-Goldstein model of tissue cholesterol homeostasis. We observed markedly lower PCSK9 mRNA levels and synthesis rates in liver and lower concentrations and synthesis rates in plasma. Hepatic LDLR half-life (t½) was prolonged, consistent with an effect of reduced PCSK9, and resulted in no reduction in hepatic LDLR content despite reduced mRNA levels and LDLR synthesis rates. These changes in PCSK9 synthesis complement and expand the well-established model of tissue cholesterol homeostasis in mouse liver, in that reduced synthesis and levels of PCSK9 counterbalance lower LDLR synthesis by promoting less LDLR catabolism, thereby maintaining uptake of LDL cholesterol into liver despite high intracellular cholesterol concentrations. Lower hepatic synthesis and secretion of PCSK9, an SREBP2 (sterol response element binding protein) target gene, results in longer hepatic LDLR t½ in response to cholesterol feeding in mice in the face of high intracellular cholesterol content. PCSK9 modulation opposes the canonical lowering of LDLR mRNA and synthesis by cholesterol surplus and preserves LDLR levels. The physiological and therapeutic implications of these opposing control mechanisms over liver LDLR are of interest and may reflect subservience of hepatic cholesterol homeostasis to whole body cholesterol needs.

Cigarette smoke extract stimulates PCSK9 production in HepG2 cells via ROS/NF‑κB signaling.

Cigarette smoke (CS) exposure is a risk factor for dyslipidemia and atherosclerosis. Reduced expression of low-density lipoprotein receptor (LDLR) in hepatocytes may be one of the underlying mechanisms for these disorders. The aim of the present study was to investigate the molecular mechanism underlying the regulatory effect of CS extract (CSE) on proprotein convertase subtilisin/kexin type 9 (PCSK9) and low LDLR expression in HepG2 cells. PCSK9 and LDLR mRNA and protein expression levels in HepG2 cells were evaluated after CSE treatment via reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. In addition, total intracellular reactive oxygen species (ROS) production was determined via 2,7-dichlorofluorescein diacetate fluorescence. CSE significantly increased PCSK9 expression and inhibited LDLR expression in a time- and concentration-dependent manner. Furthermore, CSE significantly induced ROS production and nuclear factor κB (NF-κB) activation. However, pretreatment with a ROS scavenger or an NF-κB inhibitor significantly attenuated the CSE-induced changes in PCSK9 and LDLR expression. In addition, pretreatment with melatonin markedly reduced ROS production, NF-κB activation and PCSK9 expression, and increased LDLR expression in the CSE-treated cells. These data suggest that melatonin inhibits CSE-regulated PCSK9 and LDLR production in HepG2 cells via ROS/NF-κB signaling.

Beneficial effects of primidone in Niemann-Pick disease type C (NPC)-model cells and mice: Reduction of unesterified cholesterol levels in cells and extension of lifespan in mice

Niemann-Pick disease type C (NPC) is caused by a loss of function of either NPC1 or NPC2 protein, resulting in the accumulation of unesterified, free-cholesterol (free-C) in cells/tissues and thus leading to cell/tissue damage. In the brain of patients/animals with NPC, as a consequence of the accumulation of free-C in late endosomes/lysosomes (LE/LY) in cells, multiple lipids including complex sphingolipids are accumulated, and almost all patients/animals ultimately develop progressive/fatal neurodegeneration. Several reagents that are considered to act in the brain show beneficial effects on NPC-model animals. In the present study, we investigated the effects of antiepileptic drugs, such as primidone and valproic acid, on the accumulation of free-C in NPC1-null CHO cells and NPC1* fibroblasts, human fibroblasts established from a patient with NPC1 mutation. Like valproic acid, treatment with primidone reduced free-C levels in LE/LY in NPC1-null/mutant cells. Down-regulation of cholesterol ester levels in NPC1-null cells and up-regulation of HMG-CoA reductase and low-density lipoprotein receptor mRNA levels in NPC1* cells were partially recovered by primidone treatment. Thus, primidone was suggested to enhance free-C trafficking from LE/LY to endoplasmic reticulum in NPC1-null/mutant cells. In NPC1-null mice, oral application of primidone (100 mg/kg/day) extended lifespan by approximately 5 days, although the first days showing ataxia, a typical symptom of neuromotor dysfunction, were not affected. Our findings suggest the potential of primidone for the treatment of NPC.

Impaired cholesterol metabolism in the mouse model of cystic fibrosis. A preliminary study.

This study aims to investigate cholesterol metabolism in a mouse model with cystic fibrosis (CF) by the comparison of affected homozygous versus wild type (WT) mice. In particular, we evaluated the effects of a diet enriched with cholesterol in both mice groups in comparison with the normal diet. To this purpose, beyond serum and liver cholesterol, we analyzed serum phytosterols as indirect markers of intestinal absorption of cholesterol, liver lathosterol as indirect marker of de novo cholesterol synthesis, liver cholestanol (a catabolite of bile salts synthesis) and the liver mRNA levels of LDL receptor (LDLR), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR), acyl CoA:cholesterol acyl transferase 2 (ACAT2), cytochrome P450 7A1 (CYP7A1) and tumor necrosis factor alpha (TNFα). CF mice showed lower intestinal absorption and higher liver synthesis of cholesterol than WT mice. In WT mice, the cholesterol supplementation inhibits the synthesis of liver cholesterol and enhances its catabolism, while in CF mice we did not observe a reduction of LDLR and HMG-CoAR expression (probably due to an altered feed-back), causing an increase of intracellular cholesterol. In addition, we observed a further increase (5-fold) in TNFα mRNA levels. This preliminary study suggests that in CF mice there is a vicious circle in which the altered synthesis/secretion of bile salts may reduce the digestion/absorption of cholesterol. As a result, the liver increases the biosynthesis of cholesterol that accumulates in the cells, triggering inflammation and further compromising the metabolism of bile salts.

Aqueous extracts of lingonberry and blackberry leaves identified by high-content screening beneficially act on cholesterol metabolism.

Decreasing circulating low-density lipoprotein (LDL) cholesterol levels leads to decreased risk of cardiovascular diseases. Natural compounds are capable of lowering LDL-cholesterol even on top of lifestyle modification or medication. To identify novel plant-derived compounds to lower plasma LDL cholesterol levels, we performed high-content screening based on the transcriptional activation of the promoter of the LDL receptor (LDLR). The identified hits were thoroughly validated in human hepatic cell lines in terms of increasing LDLR mRNA and protein levels, lowering cellular cholesterol levels and increasing cellular LDL uptake. By means of this incremental validation process in vitro, aqueous extracts prepared from leaves of lingonberries (Vaccinium vitis-idaea) as well as blackberries (Rubus fruticosus) were found to have effects comparable to lovastatin, a prototypic cholesterol-lowering drug. When applied in vivo in mice, both extracts induced subtle increases in hepatic LDLR expression. In addition, a significant increase in high-density lipoprotein (HDL) cholesterol was observed. Taken together, aqueous extracts from lingonberry or blackberry leaves were identified and characterized as strong candidates to provide cardiovascular protection.

Hydrogen sulfide accumulates LDL receptor precursor via downregulating PCSK9 in HepG2 cells

Endogenous hydrogen sulfide (H2S) affects cholesterol homeostasis and liver X receptor α (LXRα) expression. However, whether low-density lipoprotein (LDL) receptor (LDLR), a key player in cholesterol homeostasis, is regulated by exogenous H2S through LXRα signaling has not been determined. We investigated the effects of sodium hydrosulfide (NaHS, H2S donor) on LDLR expression in the presence or absence of LXR agonists, T0901317 or GW3965 in HepG2 cells. We found that H2S strongly accumulated LDLR precursor in the presence of T0901317. Hence, LDLR transcription and the genes involved in LDLR precursor maturation and degradation were studied. T0901317 increased the LDLR mRNA level, whereas H2S did not affect LDLR transcription. H2S had no significant effect on the expression of LXRα and inducible degrader of LDLR (IDOL). H2S and T0901317 altered mRNA levels of several enzymes for N- and O-glycosylation and endoplasmic reticulum (ER) chaperones assisting LDLR maturation, but did not affect their protein levels. H2S decreased proprotein convertase subtilisin/kexin type 9 (PCSK9) protein levels and its mRNA level elevated by T0901317. T0901317 with PCSK9 siRNA also accumulated LDLR precursor as did T0901317 with H2S. High glucose increased PCSK9 protein levels and attenuated LDLR precursor accumulation induced by T0901317 with H2S. Taken together, H2S accumulates LDLR precursor by downregulating PCSK9 expression but not through the LXRα-IDOL pathway, LDLR transcriptional activation, or dysfunction of glycosylation enzymes and ER chaperones. These results also indicate that PCSK9 plays an important role in LDLR maturation in addition to its well-known effect on the degradation of LDLR mature form.

Differential Effects of TGF-β2 on the Low-Density Lipoprotein Receptor Expression in Three Types of Human Subconjunctival Fibroblasts

ABSTRACT Purpose: To investigate whether TGF-β2 had a different effect on the expression levels of low-density lipoprotein receptor (LDLr) in the subconjunctival fibroblasts from glaucoma patients who underwent a reoperation (RGSFs) compared with those from glaucoma patients who underwent first filtering surgery (GSFs) and control patients with cataracts (HSFs). Methods: Human subconjunctival fibroblasts were obtained from the three groups of patients. Different concentrations of TGF-β2 were added to the fibroblasts for 1, 3, and 5 days. The proliferation of the fibroblasts was determined by CCK-8 assays. Real-time PCR and western blotting were performed to analyze the mRNA and protein levels of LDLr. The uptake of DiI-labeled LDL was determined by confocal microscopy. Results: The results revealed that under TGF-β2 exposure, fibroblast proliferation was positively correlated with LDLr expression (all p < .001). The LDLr mRNA and protein levels were affected by TGF-β2 in a concentration-dependent and time-dependent manner in the RGSFs, GSFs and HSFs. The maximal expression of LDLr after TGF-β2 stimulation was consistent with the peak uptake of DiI-LDL, which was obviously highest in the RGSFs, followed by the GSFs, and then the HSFs (all p < .05). All 3 groups took up DiI-LDL in a similar time-dependent manner, with maximal uptake at 6 h following DiI-LDL incubation (all p < .05). In addition, there were significant differences in the LDLr protein levels in the subconjunctival tissues isolated from the glaucoma patients during reoperation, the glaucoma patients during first filtering surgery and the control patients at day 3 (p < .05). The highest protein expression of LDLr was observed in the RG group. Conclusion: These data suggested that the RGSFs had the highest LDLr expression and the highest peak uptake of LDL among three groups. The LDLr-drug-LDL delivery system could potentially be used for targeted delivery of antimetabolite agents in anti-scarring therapy for glaucoma patients after filtering surgery.

Hyperlipidemia: Insights into Mechanisms Involved in Modulation of Drug Pharmacokinetics and Response

Hyperlipidemia is associated with disturbances in plasma lipoproteins. These changes were shown to modify the pharmacokinetics of some lipophilic lipoprotein-bound drugs with some of these changes being clinically significant. In this manuscript potential mechanisms that might be involved in hyperlipidemia-induced changes in drug pharmacokinetics such as modulation of p-glycoproteins and liver metabolising enzymes activities and/or expression were investigated. P-glycoprotein gene expression (MDR1A/B) and activity was assessed in porcine (LLC-PK1) and murine (NRK-52E) proximal tubular renal epithelial cells that were maintained in normolipidemic- or hyperlipidemic-serum containing media and was compared to expression levels in serum-deprived cells. Gene expression of low density lipoprotein receptor (LDLr) and MDR1A/B along with cytochrome P450 CYP3A2 and 2C11 were also investigated in hepatocytes from normolipidemic and hyperlipidemic rats using qRT-PCR. Hyperlipidemic serum did not affect MDR1A/B gene expression in LLC-PK1 and NRK-52E cells. However, it significantly reduced p-glycoprotein activity in rifampin-treated and non-treated LLC-PK1 cells. Interestingly, p-glycoprein gene expression was induced in LLC-PK1 and NRK-52E cell lines upon serum starvation. Moreover, hepatocytes from hyperlipidemic rat livers had reduced LDLr, MDR1A/B, and CYP2C11 mRNA levels compared to normolipidemic rats. It is concluded that hyperlipidemia-induced changes in drug pharmacokinetics might be attributed to modulation of drug efflux/uptake, metabolism and/or excretion. Further, p-glycoprotein gene expression is potentially inducible by serum starvation in renal proximal tubular cells, which might be a novel detoxification mechanism by which these cells adapt to stressful conditions.

Inhibitors of AKT kinase increase LDL receptor mRNA expression by two different mechanisms.

Protein kinase B (AKT) is a serine/threonine kinase that functions as an important downstream effector of phosphoinositide 3-kinase. We have recently shown that MK-2206 and triciribine, two highly selective AKT inhibitors increase the level of low density lipoprotein receptor (LDLR) mRNA which leads to increased amount of cell-surface LDLRs. However, whereas MK-2206 induces transcription of the LDLR gene, triciribine stabilizes LDLR mRNA, raising the possibility that the two inhibitors may actually affect other kinases than AKT. In this study, we aimed to ascertain the role of AKT in regulation of LDLR mRNA expression by examining the effect of five additional AKT inhibitors on LDLR mRNA levels. Here we show that in cultured HepG2 cells, AKT inhibitors ARQ-092, AKT inhibitor VIII, perifosine, AT7867 and CCT128930 increase LDLR mRNA levels by inducing the activity of LDLR promoter. CCT128930 also increased the stability of LDLR mRNA. To study the role of AKT isoforms on LDLR mRNA levels, we examined the effect of siRNA-mediated knockdown of AKT1 or AKT2 on LDLR promoter activity and LDLR mRNA stability. Whereas knockdown of either AKT1 or AKT2 led to upregulation of LDLR promoter activity, only knockdown of AKT2 had a stabilizing effect on LDLR mRNA. Taken together, these results provide strong evidence for involvement of AKT in regulation of LDLR mRNA expression, and point towards the AKT isoform specificity for upregulation of LDLR mRNA expression.

Relationship between porcine miR-20a and its putative target low-density lipoprotein receptor based on dual luciferase reporter gene assays.

ObjectiveMutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a.MethodsBioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3′-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3′-UTR and pGL3 Control LDLR mutant-3′-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3′-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR.ResultsGel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3′-UTR-driven (p<0.01), but not mutant LDLR-3′-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = −0.656, p<0.05).ConclusionLDLR is a potential target of miR-20a, which might directly bind the LDLR 3′-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

Chitosan oligosaccharides enhance lipid droplets via down-regulation of PCSK9 gene expression in HepG2 cells

Chitosan oligosaccharides (COS), linear polymers of N-acetyl-D-glucosamine and deacetylated glucosamine, exhibit diverse pharmacological effects such as antimicrobial, antitumor, antioxidant and anti-inflammatory activities. Here, we explored their hypocholesterolemic effects in vivo and the molecular mechanisms of COS in hepatic cells. Our in vivo study of dyslipidemic ApoE-/- male mice showed that COS treatment of 500 mg kg−1 d−1 for 4 weeks clearly reduced the lipid deposits in the aorta and significantly decreased the hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) protein levels versus HFD groups (p < 0.05). To elucidate the mechanisms behind these effects, the HepG2 cell line was treated with COS. We found that COS (200 μg/ml) increased the amount of cell-surface low-density lipoprotein receptor (LDLR) and enhanced the lipid droplets in HepG2 cells (p < 0.05). The mRNA levels of LDLR and HMG-CoA protein levels were not altered, and the mRNA levels of PCSK9 were down-regulated by COS treatment for 24 h. We also observed that the expression levels of SREBP-2 (125 kD) and HNF-1α were increased in total cell lysates, but nuclear SREBP-2 (nSREBP-2, 68 kD, the active subunit of SREBP-2) levels were decreased and FOXO3a levels increased in nuclear lysates after COS treatment for 24 h. We demonstrated that one of the reasons for regulation of lipid transfer with COS is that FOXO3a levels are up-regulated by COS, leading to a reduction in the PCSK9 promoter binding capacity of HNF-1α and thus suppressing PCSK9 gene expression, up-regulating LDLR levels, and enhancing the lipid droplets in HepG2 cells. In addition, decreased expression of the PCSK9 gene was also contributed to by down-regulation of SREBP-2 by COS. We further confirmed the effect of suppression of PCSK9 expression by COS by utilizing RNA interference to silence HNF-1α and SREBP-2. Finally, to the best of our knowledge, we are the first to demonstrate that PCSK9 expression and LDLR activity are synergistically changed by a combination of HNF-1α and SREBP-2 after COS treatment. Our findings indicate that COS may regulate PCSK9 to modulate hepatic LDLR abundance and activity.

Non-Native Conformational Isomers of the Catalytic Domain of PCSK9 Induce an Immune Response, Reduce Lipids and Increase LDL Receptor Levels.

PCSK9 (Proprotein convertase subtilisin/kexin type 9) increases plasma cholesterol levels by promoting LDL receptor degradation. Current antibody inhibitors block the interaction between PCSK9 and LDL receptors, significantly decrease plasma cholesterol levels, and provide beneficial clinical outcomes. To reduce the action of PCSK9 in plasma, a novel strategy that will produce a panel of non-native, conformationally-altered isomers of PCSK9 (X-PCSK9) to develop active immunotherapy targeting of native PCSK9 and inhibiting/blocking the interaction of PCSK9 with LDL receptor, thus decreasing plasma cholesterol levels is proposed. The authors used the scrambled disulfide bond technique to generate conformationally-altered isomers of the catalytic domain of mouse PCSK9. The focus was on the immune response of four X-isomers and their effects on plasma cholesterol and triglyceride levels in both C57BL/6J and Apoe−/− mice. The authors showed that the four immunogens produced significant immunogenicity against native PCSK9 to day 120 after immunization of C57BL/6J and Apoe−/− mice. This resulted in significantly decreased plasma cholesterol levels in C57BL/6J mice, and to a lesser degree in Apoe−/− mice. The X-PCSK9-B1 treated mice had increased LDL receptor mRNA and protein levels at day 120 after treatment. Thus, this study provides a new, potentially promising approach that uses long-term immunotherapy for a treatment of hypercholesterolemia.

Xanthohumol Suppresses Mylip/Idol Gene Expression and Modulates LDLR Abundance and Activity in HepG2 Cells.

Xanthohumol, a prenylated flavonoid found in hops (Humulus lupulus L.), exhibits multiple biological activities such as antiatherosclerosis and hypolipidemic activities. In this study, we aim to investigate the hypocholesterolemic effects and molecular mechanisms of xanthohumol in hepatic cells. We found that xanthohumol (10 and 20 μM) increased the amount of cell-surface low-density lipoprotein receptor (LDLR) from 100.0 ± 2.1% to 115.0 ± 1.3% and 135.2 ± 2.7%, and enhanced the LDL uptake activity from 100.0 ± 0.9% to 139.1 ± 13.2% in HepG2 cells (p < 0.01). The mRNA levels of LDLR, HMGCR, and PCSK9 were not altered. Xanthohumol (20 μM) reduced the expression of inducible degrader of the LDL receptor (Mylip/Idol) mRNA and protein by approximately 45% (p < 0.01), which was reported to be associated with increases of LDLR level. We demonstrated that xanthohumol suppressed hepatic Mylip/Idol expression via counteracting liver X receptor (LXR) activation. The molecular docking results predicted that xanthohumol has a high binding affinity to interact with the LXRα ligand-binding domain, which may result in attenuation of LXRα-induced Mylip/Idol expression. Finally, we demonstrated that the Mylip/Idol expression and LDLR activity were synergistically changed by a combination of xanthohumol and simvastatin treatment. Our findings indicated that xanthohumol may regulate the LXR-Mylip/Idol axis to modulate hepatic LDLR abundance and activity.

Overexpression of low-density lipoprotein receptors stimulated by vascular endothelial growth factor in fibroblasts from pterygium

The activation of subconjunctival fibroblasts is believed to be responsible for the pathogenesis of pterygium. Vascular endothelial growth factor (VEGF) appears to be the most potent stimulator of formation and progression of pterygium. Pterygium excision is a common procedure, although the recurrence rates remain high. Various postoperative adjuvant therapies are now attempted to lower the recurrence rate, with severe side effects. To offer a greater therapeutic effect and lower side effects, it’s necessary to discover a constant nanoparticle drug delivery targeting to subconjunctival fibroblasts in pterygium (PSFs). This study was designed to investigate the expression of low-density lipoprotein receptor (LDLr) stimulated by VEGF in PSFs. We found that after exposure to VEGF, mRNA and protein levels of LDLr were both increased significantly in PSFs, assessed using relative quantitative real-time RT-PCR and Western blot. Moreover, it’s demonstrated that the expression of LDLr were positively correlated with the cells proliferation. Uptake of DiI–LDL via live PSFs was increased with time, estimated by confocal microscopy. The protein expression of LDLr in pterygium subconjunctival tissues was significantly higher than in normal subconjunctival tissues. These results suggest that LDLr in the activated PSFs may become a novel target receptor for controlled drug delivery to lower postsurgical recurrence rate.

Apelin attenuates hypoxia induced pulmonary hypertension of mice through regulation of lipid metabolism

To observe the role of apelin in the prevention of pulmonary hypertension induced by hypoxia in mice. Adult male apoE gene knockout (apoE-KO) mice were exposed to isobaric hypoxic chamber (9%~11% O2, regular chow feed, 23 h/d)for 3 weeks to establish hypoxia-induced pulmonary hypertension. Thirty apoE-KO mice were randomly divided into normoxia group, hypoxia group and hypoxic with apelin (10 nmol/(kg·d), ip) group. The concentrations of high density lipoprotein (HDL), low density lipoprotein (LDL)and total cholesterol in plasma were detected by Elisa method. The mRNA levels of ATP-binding cassette transporter A1(ABCA1), low density lipoprotein receptor (LDLR), scavenger receptor class B1 (SR-B1), and HMG-CoA reductase (HMGCR)in liver were measured by real-time PCR. The protein level of peroxisome proliferators-activated receptor gamma (PPARγ) in lung was measured by Western blot. ①The right ventricular systolic pressure (RVSP) and the weight ratio of right ventricle (RV) to left ventricle plus septum (LV+S) of hypoxia group were significantly higher than those of normoxia group by 87% and 85% (P<0.05), respectively. RVSP and RV/(LV+S) of apelin group were significantly lower than those of hypoxia group by 39% and 33%(P<0.05), respectively. ②The plasma concentration of HDL and HDL/LDL of apelin group were significantly higher than those of hypoxia group by 21% and 20%(P<0.05), respectively. ③The mRNA levels of LDLR, SR-B1 and ABCA1 in liver of apelin group were significantly up-regulated than those of hypoxia group by 241%, 112% and 69% (P<0.05), respectively, while the mRNA level of HMGCR was down-regulated by 45% (P<0.05). ④The protein level of PPARγ in lung of apelin group was significantly up-regulated than that of hypoxia group by 47% (P<0.05). Apelin attenuates hypoxia-induced pulmonary hypertension of mice through regulation of lipid metabolism.

Enhanced pro-protein convertase subtilisin/kexin type 9 expression by C-reactive protein through p38MAPK-HNF1α pathway in HepG2 cells.

Plasma C‐reactive protein (CRP) concentration is associated positively with cardiovascular risk, including dyslipidemia. We suggested a regulating role of CRP on pro‐protein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of low‐density lipoprotein (LDL) metabolism, and demonstrated the PCSK9 as a pathway linking CRP and LDL regulation. Firstly, experiments were carried out in the presence of human CRP on the protein and mRNA expression of PCSK9 and LDL receptor (LDLR) in human hepatoma cell line HepG2 cells. Treatment with CRP (10 μg/ml) enhanced significantly the mRNA and protein expression of PCSK9 and suppressed the expression of LDLR. Of note, a late return of LDLR mRNA levels occurred at 12 hrs, while the LDLR protein continued to decrease at 24 hrs, suggesting that the late decrease in LDLR protein levels was unlikely to be accounted for the decrease in LDL mRNA. Secondly, the role of PCSK9 in CRP‐induced LDLR decrease and the underlying pathways were investigated. As a result, the inhibition of PCSK9 expression by small interfering RNA (siRNA) returned partly the level of LDLR protein and LDL uptake during CRP treatment; CRP‐induced PCSK9 increase was inhibited by the p38MAPK inhibitor, SB203580, resulting in a significant rescue of LDLR protein expression and LDL uptake; the pathway was involved in hepatocyte nuclear factor 1α (HNF1α) but not sterol responsive element‐binding proteins (SREBPs) preceded by the phosphorylation of p38MAPK. These findings indicated that CRP increased PCSK9 expression by activating p38MAPK‐HNF1α pathway, with a certain downstream impairment in LDL metabolism in HepG2 cells.

Role of hesperetin in LDL-receptor expression in hepatoma HepG2 cells.

BackgroundHigh plasma concentration of low-density lipoprotein cholesterol (LDL-c) plays a significant role in the incidence of atherosclerosis and coronary heart diseases. The aim of this study was to investigate the mechanism by which the citrus flavonoid, hesperetin, regulates the LDL receptor (LDLr) gene in the human liver using the human hepatoma cell line, HepG2.MethodsLuciferase reporter gene assays were performed (in the absence of lipoprotein) to measure the activity of the LDLr promoter and the promoters of the sterol regulatory element binding protein (SREBP) transcription factors that control the LDLr promoter.ResultsOnly SREBP-1 promoter activity was significantly increased 4 h after exposure to 200 μM hesperetin. However, after 24 h incubation with 200 μM hesperetin, the activities of all the promoter-constructs, SREBP-1a, -1c, -2 and LDLr, were significantly increased. The effects of 200 μM hesperetin on elevating LDLr mRNA levels were possibly due to regulation of LDLr gene transcription by SREBP-la and SREBP-2.ConclusionsWe conclude that 200 μM hesperetin was likely to have stimulated LDLr gene expression in human hepatoma HepG2 cells via increased phosphorylation of PI3K andERK1/2, which increased SREBP-1a and SREBP-2 mRNA levels and enhanced the maturation of the encoded proteins. This may lead to lower plasma LDL cholesterol; therefore, diets supplemented with hesperidin might provide cardio-protective effects and reduce mortality and morbidity from coronary heart diseases.