Liver Low-density Lipoprotein Research Articles

Quantitative Proteomic Analysis of PCSK9 Gain of Function in Human Hepatic HuH7 Cells

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis, mediating degradation of the liver low-density lipoprotein receptor (LDLR). In fact, gain- and loss-of-function PCSK9 variations in human populations associate with hyper- or hypo- cholesterolemia, respectively. Exactly how PCSK9 promotes degradation of the LDLR, the identity of the other biomolecules involved in this process, and the global effect of PCSK9 on other proteins has not been thoroughly studied. Here we employ stable isotope labeling with amino acids in cell culture (SILAC) to present the first quantitative, subcellular proteomic study of proteins affected by the stable overexpression of a gain-of-function PCSK9 membrane-bound chimera (PCSK9-V5-ACE2) in comparison to control, empty vector transfections in a human hepatocyte (HuH7) cell line. The expression level of 327 of 5790 peptides was modified by PCSK9-V5-ACE2 overexpression. Immunoblotting was carried out for the control transferrin receptor, shown to be unaffected in cells overexpressing PCSK9-V5-ACE2, thus validating our SILAC results. We also used immunoblotting to confirm the novel SILAC results of up- and down-regulation of several proteins in cells overexpressing PCSK9-V5-ACE2. Moreover, we documented the novel down-regulation of the EH domain binding protein-1 (EHBP1) in a transgenic PCSK9 mouse model and its up-regulation in a PCSK9 knockout mouse model.

Importance of Proprotein Convertase Subtilisin/Kexin Type 9 in the Hormonal and Dietary Regulation of Rat Liver Low-Density Lipoprotein Receptors

Hormonal or dietary challenge can stimulate hepatic low-density lipoprotein receptor (LDLR) expression through posttranscriptional mechanisms. We here tested whether such observations may be due to regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9). Treatment with glucagon resulted in a 2-fold increase in hepatic LDLR protein expression, whereas its mRNA levels were reduced; this occurred simultaneously with a 70% reduction in PCSK9 expression. Insulin treatment resulted in responses opposite to those seen by treatment with glucagon. Furthermore, high-dose ethinylestradiol treatment reduced PCSK9 expression by half. Finally, feeding of rats with dietary cholesterol reduced PCSK9 expression, resulting in an increased number of hepatic LDLRs despite a reduction of LDLR mRNA levels. Regulation of PCSK9 occurred in part through sterol regulatory element binding protein-2, but changes in this cholesterol-controlled transcription factor could not explain all hormonal effects seen. We conclude that the hormonal and dietary regulation of hepatic LDLRs also involves posttranscriptional regulation by PCSK9. The identification of PCSK9 regulation by these various treatments is important in understanding of the physiological function of this protein and points to new targets for therapeutic treatments to increase hepatic LDLR numbers.

The Proprotein Convertase PCSK9 Induces the Degradation of Low Density Lipoprotein Receptor (LDLR) and Its Closest Family Members VLDLR and ApoER2

The proprotein convertase PCSK9 gene is the third locus implicated in familial hypercholesterolemia, emphasizing its role in cardiovascular diseases. Loss of function mutations and gene disruption of PCSK9 resulted in a higher clearance of plasma low density lipoprotein cholesterol, likely due to a reduced degradation of the liver low density lipoprotein receptor (LDLR). In this study, we show that two of the closest family members to LDLR are also PCSK9 targets. These include the very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. Our results show that wild type PCSK9 and more so its natural gain of function mutant D374Y can efficiently degrade the LDLR, VLDLR, and ApoER2 either following cellular co-expression or re-internalization of secreted human PCSK9. Such PCSK9-induced degradation does not require its catalytic activity. Membrane-bound PCSK9 chimeras enhanced the intracellular targeting of PCSK9 to late endosomes/lysosomes and resulted in a much more efficient degradation of the three receptors. We also demonstrate that the activity of PCSK9 and its binding affinity on VLDLR and ApoER2 does not depend on the presence of LDLR. Finally, in situ hybridization show close localization of PCSK9 mRNA expression to that of VLDLR in mouse postnatal day 1 cerebellum. Thus, this study demonstrates a more general effect of PCSK9 on the degradation of the LDLR family that emphasizes its major role in cholesterol and lipid homeostasis as well as brain development.

Inhibitors of Acyl-Coenzyme A: Cholesterol Acyltransferase

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesterol esters from cholesterol and fatty acyl-coenzyme A. Animal experiments showed that ACAT inhibitors reduce plasma cholesterol levels by suppressing absorption of dietary cholesterol and by suppressing the assembly and secretion of apolipoprotein B-containing lipoproteins such as very low density lipoprotein in liver and chylomicron in intestine. Moreover, ACAT inhibitors were shown to prevent formation of macrophage-derived foam cells in the arterial walls. However, a recent double-blind, placebo-controlled, randomized trial of a potent ACAT inhibitor, avasimibe, failed to show significant beneficial effects on coronary atherosclerosis assessed by intravascular ultrasound. For clinical application of ACAT inhibitors, development of more potent compounds and improvements of the methods to evaluate their clinical efficacy are strongly needed.

Ubiquitin-proteasome-dependent degradation of apolipoprotein B100 in vitro

Apolipoprotein B100 (apoB) is a large secretory protein that forms very low density lipoprotein in liver. An in vitro degradation assay was developed using rabbit reticulocyte (RR) lysate in order to investigate the mechanism of intracellular degradation of newly synthesized apoB by the ubiquitin-proteasome pathway. [ 3H]apoB, isolated from [ 3H]leucine pulsed/chased Hep G2 cells, was degraded 51% when incubated for 2 h at 37°C in an assay mixture that included RR lysate (source of the ubiquitin conjugation system and proteasome) and an exogenous ATP regenerating system. ApoB degradation was ATP-dependent and degradation fragments were not observed suggesting that the very large apoB molecule was extensively degraded. ApoB degradation was decreased to 50% when potent proteasome inhibitors, clasto-lactacystin β-lactone (10 μM) or MG-132 (50 μM), were added to the reaction mixture, but was not affected by the cysteine protease inhibitor, E-64, or the serine protease inhibitor, phenylmethylsulfonyl fluoride. ApoB degradation was inhibited by the mutant ubiquitin protein K48R and by ubiquitin aldehyde, an inhibitor of ubiquitin-protein isopeptidases. During incubation ubiquitination of apoB increased even as apoB was being degraded. These results suggest that in vitro degradation of apoB, a large secretory protein that is normally found in the endoplasmic reticulum (ER) lumen or associated with the ER membrane, was proteasome-dependent and involved both ubiquitination and deubiquitination steps.

Soybean Protein Products as Regulators of Liver Low-Density Lipoprotein Receptors. I. Identification of Active β-Conglycinin Subunits

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionNEXTSoybean Protein Products as Regulators of Liver Low-Density Lipoprotein Receptors. I. Identification of Active β-Conglycinin SubunitsMaria Rosa Lovati, Cristina Manzoni, Elisabetta Gianazza, and Cesare R. SirtoriCite this: J. Agric. Food Chem. 1998, 46, 10, 4464Publication Date (Web):September 24, 1998Publication History Published online24 September 1998Published inissue 1 October 1998https://doi.org/10.1021/jf9809277Copyright © 1998 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views162Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (14 KB) Get e-AlertsSUBJECTS:Anatomy,Food,Peptides and proteins,Plant derived food,Receptors Get e-Alerts

Soybean Protein Products as Regulators of Liver Low-Density Lipoprotein Receptors. I. Identification of Active β-Conglycinin Subunits

The ability of the different soy globulins to upregulate low-density lipoprotein (LDL) uptake and metabolism in human hepatoma cells (Hep G2) was investigated, in an attempt to identify peptide components responsible for the upregulation of the LDL receptor. In parallel, the metabolism of soy globulins, added to the culture medium, was investigated by two-dimensional electrophoresis. After addition of soy globulins, there were no marked changes in the cell protein pattern, as evaluated by general protein staining. By immunodetection, intact 7S components were observed both free in the culture medium and bound to plasma membranes. Inside cells, α + α‘ subunits in their native forms were not detectable, whereas most of the β chain was found unchanged. Largely unmodified soybean proteins were detected in a lysate of 11S-treated cells. Incubation of Hep G2 cell with purified α + α‘ from 7S sharply increased uptake and degradation of 125I-LDL added to the culture medium, whereas the β chains were ineffective; ...

Soybean Protein Products as Regulators of Liver Low-Density Lipoprotein Receptors. II. α−α‘ Rich Commercial Soy Concentrate and α‘ Deficient Mutant Differently Affect Low-Density Lipoprotein Receptor Activation

Activation of low-density lipoprotein (LDL) receptors has been decribed in a human hepatoma cell line (Hep G2) exposed to the 7S globulin from soy (Lovati et al. J. Agric. Food Chem. 1998, 46, 2474−2480). This in vitro model supports a direct activity of soy protein component(s) in lowering LDL cholesterolemia. To ascertain whether α or α‘ subunits from 7S globulin, or both, are responsible for the biochemical effect, a mutant soy cultivar, devoid of the α‘ subunit (Keburi), was evaluated in the same model. A commercial isoflavone-poor, heat-hydrolyzed soy preparation found effective in human hypercholesterolemia (Croksoy) was also tested. While Croksoy proved to have a similar activity as the whole 7S globulin, the α‘-free Keburi variant showed no effect on LDL receptor activation. These findings support the reliability of the human liver cell model in predicting a human hypocholesterolemic activity and suggest that specific peptide(s) may be responsible. Furthermore, they indicate that some soy cultivar...

Independent responsiveness of frog liver low-density lipoprotein receptor and HMGCoA reductase to estrogen treatment.

Cholesterol metabolism in the female frog exhibits circannual modifications which parallel plasma estrogen fluctuations. Estrogens enhance production and lipidation of the yolk precursor vitellogenin by inducing the transcription of its gene and by stimulating the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. The time dependence of the effects that these hormones have on HMGCoA reductase and the low-density lipoprotein (LDL) receptor in the liver of Rana esculenta complex has been investigated. Following estrogen treatment, the levels of LDL receptor mRNA and protein gradually increased, with a maximum concentration observed at 3 days. The HMGCoA reductase protein level increased progressively, while the mRNA level was not significantly modified. Thus the LDL receptor and HMGCoA reductase in frog behave independently after estrogen stimulation, as already reported to occur in the rat. This suggests a uncoordinated regulation that might be even partially related with the changes in cellular cholesterol content.

Tri-iodothyronine prevents the amiodarone-induced decrease in the expression of the liver low-density lipoprotein receptor gene.

Treatment with amiodarone, a potent antiarrhythmic drug, is associated with a dose-dependent increase in plasma cholesterol resulting from a decreased number of liver low-density lipoprotein (LDL) receptors. Similar changes occur in hypothyroidism, and it has been suggested that amiodarone acts via induction of a local 'hypothyroid-like' state in extrathyroidal tissues. The present study was designed to evaluate whether exogenous tri-iodothyronine (T3) could prevent the effects of amiodarone on LDL cholesterol. Rats were treated for 14 days with water, amiodarone 10 mg/100 g body weight (BW), or amiodarone and 2.5, 5 or 10 micrograms T3/100 g BW respectively. Relative to controls, amiodarone increased plasma LDL cholesterol by 31% and decreased liver LDL receptor mRNA by 56% and protein by 45%; liver T3 content was reduced by 21%. Addition of T3 to the treatment with amiodarone dose-dependently reversed all these changes, with a return to control values of plasma cholesterol and the number of liver LDL receptors, although LDL receptor mRNA remained slightly lower. Treatment of rats for 14 days with T3 alone (5 micrograms/100 g BW) decreased plasma LDL cholesterol by 19% and increased liver LDL receptor mRNA by 41%. In conclusion, T3 prevents the amiodarone-induced changes in plasma LDL cholesterol and liver LDL receptor gene expression. These findings suggest that the inhibitory effect of amiodarone on LDL receptor gene expression is mediated by T3-dependent pathways.

Cholesterol and fatty acid metabolism in piglets fed sow milk or infant formula with or without addition of cholesterol

Several studies have reported that plasma cholesterol and phospholipid (PL) levels of arachidonic acid (20:4n−6) are lower and PL levels of linoleic acid (18:2n−6) are higher in infants fed formula than in infants fed human milk. Plasma cholesterol level and possibly the dietary intake of cholesterol could be related to plasma PL n−6 fatty acid metabolism because plasma PL 18:2n−6 is used for esterification of plasma free cholesterol. Whether the low cholesterol content of infant formula as compared with human milk is related to the difference in plasma n−6 fatty acid levels between infants fed human milk and infants fed formula is not known. This study determined the effect of feeding formula with 0.05 mmol cholesterol/L, formula with 1.09 mmol cholesterol/L, or sow milk with 0.34 mmol cholesterol/L on plasma, liver, and bile lipid fatty acid levels and liver low-density lipoprotein (LDL) receptor mass in piglets. Liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity and plasma lathosterol were assayed as indices of liver and body cholesterol synthesis, respectively. Formula with or without cholesterol added, or sow milk, was fed from birth to 18 days of age. Providing cholesterol in the formula did not correct the significantly lower plasma cholesterol or plasma and liver PL 20:4n−6 levels associated with formula feeding. The liver total cholesterol and cholesteryl esters (CE), biliary bile acid, and PL concentrations were significantly higher and the liver HMG CoA reductase activity and plasma lathosterol:cholesterol ratio were significantly lower in piglets fed the formula with cholesterol than in piglets fed the formula without cholesterol added. No evidence of fewer hepatic LDL receptors was found in piglets fed sow milk compared with piglets fed formula. The results show marked differences in hepatic and bile n−6 fatty acid concentrations between artificially and naturally fed piglets that were not explained by the difference in dietary cholesterol intake alone. The significance of the changes in bile lipid composition due to formula feeding should be studied further.

Fibrates influence the expression of genes involved in lipoprotein metabolism in a tissue-selective manner in the rat.

The influence of different fibrates on apolipoprotein metabolism was investigated. Administration of fenofibrate provoked a dose-dependent decrease in plasma cholesterol concentration that was already evident after 1 day. Intestinal apolipoprotein (apo) A-I and apo A-IV mRNA levels remained fairly constant. In contrast, liver apo A-I, apo A-II, and apo A-IV mRNA levels decreased in a dose-dependent fashion, which was associated with a lower transcription rate of the apo A-I but not the apo A-II gene. The decline in hepatic apo A-I, apo A-II, and apo A-IV mRNA had already started after 1 day and was associated with a drop in plasma apo A-I and apo A-IV concentrations. Plasma apo E had already decreased after 1 day of fenofibrate, whereas apo B initially remained constant and increased only after 14 days of fenofibrate at the highest dose. Hepatic and intestinal apo B mRNA contents and liver, heart, kidney, and testis apo E mRNA contents were only marginally affected after treatment with fenofibrate. Liver low density lipoprotein receptor mRNA levels rose slightly after a 3-day administration of the highest dose of fenofibrate. Both clofibrate and gemfibrozil had effects comparable to those of fenofibrate on liver and intestinal apolipoprotein mRNA levels except for liver apo A-II mRNA, which decreased only marginally. Compared with fenofibrate, clofibrate caused similar changes in plasma cholesterol, apo A-I, apo A-IV, and apo E concentrations, whereas gemfibrozil increased plasma cholesterol and apo E without changing apo A-I and apo A-IV concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased removal of beta-very low density lipoproteins after ethinyl estradiol is associated with increased mRNA levels for hepatic lipase, lipoprotein lipase, and the low density lipoprotein receptor in Watanabe heritable hyperlipidemic rabbits.

The mechanism by which ethinyl estradiol (EE) decreases the concentration of lipids in the d less than 1.019 g/ml fraction (beta-very low density lipoprotein [beta-VLDL]) of homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits was studied. Treatment with EE increased the activity of hepatic lipase (HL) twofold to threefold in postheparin plasma and in liver biopsies. Postheparin plasma and adipose tissue lipoprotein lipase (LPL) activities were also increased twofold to fourfold after EE. The effects of EE on HL and LPL activities were associated with a threefold to sixfold elevation in liver HL mRNA and a fourfold elevation in adipose tissue LPL mRNA steady-state levels, pointing to an effect of EE on HL and LPL gene transcription. EE also increased liver low density lipoprotein (LDL) receptor mRNA levels threefold to fivefold. These results suggest a concerted action of LPL, HL, and the LDL receptor in the removal of beta-VLDL in homozygous WHHL rabbits with a defective LDL receptor. In addition, the content of apolipoprotein E in the d less than 1.019 g/ml fraction changed toward normal after EE. Because the remaining particles contained apolipoprotein B-100 almost exclusively, it is likely that apolipoprotein E-containing beta-VLDLs are preferentially removed. This may be the result of the increased activity of LPL and HL influencing the conformation of apolipoprotein E on the beta-VLDL particle in such a way that it is directly removed from the circulation, possibly by the induced LDL receptor.

The relationship between hepatic low-density lipoprotein receptor activity and serum cholesterol level in the human fetus

We found that the binding of 125I-low-density lipoprotein to fetal liver low-density lipoprotein receptor rose progressively with the increase in fetal age. During this period, total cholesterol and low-density lipoprotein-cholesterol levels in fetal serum declined significantly. The correlation coefficients between fetal age and concentration of serum total cholesterol and low-density lipoprotein cholesterol were -0.80 (p less than 0.001) and -0.77 (p less than 0.001), respectively. A significant inverse correlation also existed between the liver low-density lipoprotein receptor activity and the serum total cholesterol (r = -0.96, p less than 0.001) and low-density lipoprotein cholesterol (r = -0.95, p less than 0.001) but not high-density lipoprotein cholesterol. It is suggested that the low-density lipoprotein receptors in human fetal liver may play a key role in the regulation of the serum cholesterol levels during gestation.

The relationship between hepatic low-density lipoprotein receptor activity and serum cholesterol level in the human fetus

We found that the binding of 125I-low-density lipoprotein to fetal liver low-density lipoprotein receptor rose progressively with the increase in fetal age. During this period, total cholesterol and low-density lipoprotein-cholesterol levels in fetal serum declined significantly. The correlation coefficients between fetal age and concentration of serum total cholesterol and low-density lipoprotein cholesterol were −0.80 (p < 0.001) and −0.77 (p < 0.001), respectively. A significant inverse correlation also existed between the liver low-density lipoprotein receptor activity and the serum total cholesterol (r = −0.96, p < 0.001) and low-density lipoprotein cholesterol (r = −0.95, p < 0.001) but not high-density lipoprotein cholesterol. It is suggested that the low-density lipoprotein receptors in human fetal liver may play a key role in the regulation of the serum cholesterol levels during gestation. (HEPATOLOGY 1991;13:852–857.)

Effect of plasmapheresis on the liver uptake of ApoB-lipoproteins labeled with technetium-99m.

To study liver low density lipoprotein (LDL)-receptor activity before and after plasmapheresis, [99mTc] very low density lipoprotein (VLDL) was used. Autologous VLDL was labeled, sterilized by filtration, and administered intravenously to patients under a gamma camera. The uptake of lipoproteins in the liver was measured by scintiscanning. Liver activity curves were generated for each patient. The liver activity in patients with the heterozygous form of familial hypercholesterolemia (FH) and in patients with symptomatic atherosclerosis (SA) without hereditary deficit of LDL receptors was reduced as compared to healthy people. Plasmapheresis enhanced the liver uptake of the 99mTc-labeled lipoproteins in atherosclerotic patients. Thus, labeled metabolites could presumably be of use in assessing the effect of plasmapheresis on liver function.

The distribution of elements in the tissues of Watanabe Heritable Hyperlipidemic rabbits

A deficiency or an excess of some elements in the diet is reported to modify the concentration of cholesterol in plasma, and, conversely, a reduction of cholesterol in the diet decreases zinc in plasma. We have studied the distribution of elements Na, K, Ca, Mg, Fe, Cu, Zn, S, P, and Mn in the tissues, plasma, heart, aorta, lung, liver, spleen, kidney, thymus, and brain of New Zealand White rabbits (NZW) and of Watanabe Heritable Hyperlipidemic rabbits (WHHL). The WHHL rabbits had a massive hypercholesterolemia (7.45 +/- 1.2 g/L) induced by a lack of liver low density lipoprotein receptors. The concentrations of elements in the tissues of the control NZW rabbits were very similar to those found in the normal rat. In WHHL, compared to NZW, besides the very important increase of total phosphorus in plasma explained by the augmentation of phospholipids, there was an increase of plasma copper (+44%) and zinc (+36%). The other noticeable changes were an increase of iron in heart (+19%), sulfur, and zinc in liver (+15% and +18%). The other changes observed in WHHL rabbits were, besides the increase of ceruloplasmin, the increase of vit E (+468%) and MDA (+62%). In conclusion, despite a massive increase of lipids in plasma, there was no major disturbance of element distribution in WHHL rabbits.