Low Density Lipoprotein Receptor Binding Activity Research Articles

A Green Tea Catechin Extract Upregulates the Hepatic Low‐Density Lipoprotein Receptor in Rats

Green tea extracts have hypocholesterolaemic properties in epidemiological and animal intervention studies. Upregulation of the low-density lipoprotein (LDL) receptor may be one mechanism to explain this as it is the main way cholesterol is removed from the circulation. This study aimed to determine if a green tea extract could upregulate the hepatic LDL receptor in vivo in the rat. A green tea extract (GTE) enriched in its anti-oxidant constituents, the catechins, was fed to rats (n = 6) at concentrations of either 0, 0.5, 1.0 or 2.0% (w/w) mixed in with their normal chow along with 0.25% (w/w) cholesterol for 12 days. Administration of the GTE had no effect on plasma total or LDL cholesterol concentrations but high-density lipoprotein significantly increased (41%; p < 0.05). Interestingly, there was a significant increase in LDL receptor binding activity (2.7-fold) and LDL receptor protein (3.4-fold) in the 2% (w/w) treatment group compared to controls. There were also significant reductions in liver total and unesterified cholesterol (40%). Administration of the GTE significantly reduced cholesterol absorption (24%) but did not affect cholesterol synthesis. These results show that, despite no effect on plasma cholesterol, the GTE upregulated the LDL receptor in vivo. This appears to be via a reduction in liver cholesterol concentration and suggests that the green tea extract was able to increase the efflux of cholesterol from liver cells.

Alpha-tocopherol modulates the low density lipoprotein receptor of human HepG2 cells.

The aim of this study was to determine the effects of vitamin E (α-tocopherol) on the low density lipoprotein (LDL) receptor, a cell surface protein which plays an important role in controlling blood cholesterol. Human HepG2 hepatoma cells were incubated for 24 hours with increasing amounts of α, δ, or γ-tocopherol. The LDL receptor binding activity, protein and mRNA, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA, cell cholesterol and cell lathosterol were measured. The effect of α-tocopherol was biphasic. Up to a concentration of 50 μM, α-tocopherol progressively increased LDL receptor binding activity, protein and mRNA to maximum levels 2, 4 and 6-fold higher than control, respectively. The HMG-CoA reductase mRNA and the cell lathosterol concentration, indices of cholesterol synthesis, were also increased by 40% over control by treatment with 50 μM α-tocopherol. The cell cholesterol concentration was decreased by 20% compared to control at 50 μM α-tocopherol. However, at α-tocopherol concentrations higher than 50 μM, the LDL receptor binding activity, protein and mRNA, the HMG-CoA reductase mRNA and the cell lathosterol and cholesterol concentrations all returned to control levels. The biphasic effect on the LDL receptor was specific for α-tocopherol in that δ and γ-tocopherol suppressed LDL receptor binding activity, protein and mRNA at all concentrations tested despite the cells incorporating similar amounts of the three homologues. In conclusion, α-tocopherol, exhibits a specific, concentration-dependent and biphasic "up then down" effect on the LDL receptor of HepG2 cells which appears to be at the level of gene transcription. Cholesterol synthesis appears to be similarly affected and the cell cholesterol concentration may mediate these effects.

Polyunsaturated fatty acids downregulate the low density lipoprotein receptor of human HepG2 cells

The aim of the study was to investigate the effect of different fatty acids on the low density lipoprotein (LDL) receptor of cultured human liver HepG2 cells. Previous studies investigating the effect of fatty acids on LDL expression have reported conflicting findings and are limited to measurements of LDL receptor binding activity. Therefore, this study is unique in that the relative effects of different fatty acids on the LDL receptor were investigated at three different stages of expression: 1) functional cellular LDL binding activity, 2) amount of LDL receptor protein and 3) LDL receptor mRNA level. The HepG2 cells were incubated for 24 hr with either 100 μM palmitic, oleic, linoleic or eicosapentaenoic acid (EPA). The measurement of LDL receptor binding activity was with colloidal gold-LDL conjugates, cellular LDL receptor protein was by western blotting and LDL receptor mRNA by Southern blotting of reverse-transcribed, polymerase chain reaction-amplified cDNA. The LDL receptor binding activity, protein and mRNA levels decreased as the degree of unsaturation of the fatty acids increased (palmitic acid ≥ oleic acid > linoleic acid > EPA) and the inverse relationship held whether or not cholesterol was included in the culture media. The relative differences were very similar for the three stages of expression indicating that modulation of the LDL receptor by the fatty acids occurred at the level of gene transcription. The increased susceptibility to oxidation of the polyunsaturated fatty acids was unlikely to be a factor in the effect because EPA and linoleic acid (250 μM) still downregulated the LDL receptor in the presence of the antioxidant vitamin E (50 μM). In conclusion, the polyunsaturates, linoleic acid and EPA, effectively downregulated the LDL receptor of HepG2 cells compared to palmitic acid. The effects of these fatty acids were observed at the level of LDL receptor binding activity, protein and mRNA, strongly suggesting that the fatty acid effects were at the level of gene transcription.

Green tea upregulates the low-density lipoprotein receptor through the sterol-regulated element binding Protein in HepG2 liver cells.

Green tea from Camellia sinensis lowers plasma cholesterol in animal models of hypercholesterolemia. The aim of this study was to determine the effects of green tea on the expression of the hepatic low-density lipoprotein (LDL) receptor, a cell surface protein involved in the control of plasma cholesterol. Incubating human HepG2 liver cells in culture with green tea increased both LDL receptor binding activity and protein. An ethyl acetate extract of green tea, containing 70% (w/w) catechins, also increased the LDL receptor binding activity, protein, and mRNA, indicating that (1) the effect was at the level of gene transcription and that (2) the catechins were the active constituents. The mechanism by which green tea up-regulated the LDL receptor was then investigated. Green tea decreased the cell cholesterol concentration (-30%) and increased the conversion of the sterol-regulated element binding protein (SREBP-1) from the inactive precursor form to the active transcription-factor form. Consistent with this, the mRNA of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis, was also increased by green tea. In conclusion, green tea up-regulated the LDL receptor in HepG2 cells. The effect was most likely mediated through SREBP-1 in response to a decrease in the intracellular cholesterol concentration. The LDL receptor may therefore play a role in the hypocholesterolemic effect of green tea in vivo.

Apolipoprotein E peptide stimulation of rat ovarian theca cell androgen synthesis is mediated by members of the low density lipoprotein receptor superfamily.

Ovarian androgen production is rate limiting for follicular maturation and can induce follicular atresia. Thus, it is important to define the actions of the intraovarian agents, such as apolipoprotein (apo) E, that modulate theca cell androgen production. Theca cell androgen production is stimulated at low concentrations and inhibited at higher concentrations of native apo E. The apo E peptide, acetyl-Y(LRKLRKRLLRDADDL)(2)C or acetyl-Y(141-155)(2)C, has low density lipoprotein (LDL) receptor and LDL receptor-related protein-binding activity, and it mimics the activity of native apo E in the theca-interstitial cell system. To define the role of members of the LDL receptor superfamily in the apo E peptide-mediated responses, we found that receptor-associated protein prevented the stimulation without altering the inhibition of androstenedione production. The apo E peptide (129-162), which has no LDL receptor-binding activity, did not stimulate androstenedione production. The apo E peptide acetyl-Y(141-155)(2)C did not stimulate androstenedione production when cell surface heparan sulfate proteoglycans were degraded with heparinase. The apo E peptide acetyl-Y(141-155)(2)C bound to heparin, a property of LDL receptor ligands, and in this complex the peptide had no effect on androstenedione production. These observations support the conclusion that apo E-mediated stimulation, but not inhibition, of ovarian theca cell androstenedione production was mediated by members of the LDL receptor superfamily.

In the Absence of Endogenous Mouse Apolipoprotein E, Apolipoprotein E*2(Arg-158 → Cys) Transgenic Mice Develop More Severe Hyperlipoproteinemia than Apolipoprotein E*3-Leiden Transgenic Mice

Apolipoprotein E*2(Arg-158 --> Cys) (APOE*2) transgenic mice were generated and compared to the previously generated apolipoprotein E*3-Leiden (APOE*3-Leiden) transgenic mice to study the variable expression of hyperlipoproteinemia associated with these two APOE variants. In the presence of the endogenous mouse Apoe gene, the expression of the APOE*3-Leiden gene resulted in slightly elevated levels of serum cholesterol as compared with control mice (2.7 +/- 0. 5 versus 2.1 +/- 0.2 mmol/liter, respectively), whereas the expression of the APOE*2(Arg-158 --> Cys) gene did not affect serum cholesterol levels, even after high/fat cholesterol feeding. The extreme cholesterol level usually found in apoE-deficient mice (Apoe-/- mice; 23.6 +/- 5.0 mmol/liter) could be rescued by introducing the APOE*3-Leiden gene (APOE*3-Leiden.Apoe-/-; 3.6 +/- 1. 5 mmol/liter), whereas the expression of the APOE*2(Arg-158 --> Cys) gene in Apoe-/- mice minimally reduced serum cholesterol levels (APOE*2.Apoe-/-; 16.6 +/- 2.9 mmol/liter). In vivo very low density lipoprotein (VLDL) turnover studies revealed that APOE*2.Apoe-/- VLDL and APOE*3-Leiden.Apoe-/- VLDL display strongly reduced fractional catabolic rates as compared with control mouse VLDL (4.0 and 6.1 versus 22.1 pools/h). In vitro low density lipoprotein (LDL) receptor binding studies using HepG2 and J774 cells showed that APOE*2. Apoe-/- VLDL is completely defective in binding to the LDL receptor, whereas APOE*3-Leiden.Apoe-/- VLDL still displayed a considerable binding activity to the LDL receptor. After transfection of APOE*2.Apoe-/- and APOE*3-Leiden.Apoe-/- mice with adenovirus carrying the gene for the receptor-associated protein (AdCMV-RAP), serum lipid levels strongly increased (15.3 to 42.8 and 1.4 to 15.3 mmol/liter for cholesterol and 5.0 to 35.7 and 0.3 to 20. 7 mmol/liter for triglycerides, respectively). This indicates that RAP-sensitive receptors, possibly the LDL receptor-related protein (LRP), mediate the plasma clearance of both APOE*2.Apoe-/- and APOE*3-Leiden. Apoe-/- VLDL. We conclude that in vivo the APOE*2 variant is completely defective in LDL receptor binding but not in binding to LRP, whereas for the APOE*3-Leiden mutant both LRP and LDL receptor binding activity are only mildly affected. As a consequence of this difference, APOE*2.Apoe-/- develop more severe hypercholesterolemia than APOE*3-Leiden.Apoe-/- mice.

Reduction of bovine plasma cholesterol concentration by partial interruption of enterohepatic circulation of bile salts: a novel hypocholesterolemic model

Interruption of enterohepatic circulation (EHC) of bile salts in several species is known to cause a significant decrease in plasma concentrations of low density lipoprotein (LDL) cholesterol, but to have little effect on high density lipoprotein (HDL) cholesterol. The present study, for the first time, demonstrates that partial interruption of EHC dramatically reduces both plasma LDL and HDL cholesterol concentrations in cattle. Five adult Holstein cows were surgically altered to allow controlled portions of bile flow to be diverted from the body. The animals were fed a low-fat, cholesterol-free diet. In two experiments, bile was diverted at 50% and 22% of total flow rates. By day 8 of diversion, both rates reduced mean plasma cholesterol from baseline (85 mg/dl) to about 8 and 18 mg/dl, respectively. Cholesterol was reduced in equal proportions in all lipoprotein fractions. In addition, plasma concentrations of triglycerides and phospholipids were also dramatically reduced. All of these plasma lipids returned to baseline within 1 week after restoration of bile flow. To determine the hepatic response to bile diversion, liver cholesterol concentrations, cholesterol synthesis rates, and LDL receptor-binding activities were determined in biopsy samples. In response to bile diversion, hepatic cholesteryl esters were markedly depleted while hepatic cholesterol synthesis rates were increased by more than 10-fold. Nevertheless, because the basal cholesterol synthesis rate was so low, it was estimated that the increase in synthesis would have supplied no more than 5% of the sterols depleted during bile diversion (1.2 vs. 25 mmol/day). LDL receptor-binding activity was significantly elevated, suggesting an increased uptake of plasma lipoprotein cholesterol by the liver. These results suggest that the unique sensitivity of bovine plasma cholesterol to enterohepatic circulation interruption might occur as a result of the inherently low rate of hepatic cholesterol synthesis in cattle. This hypocholesterolemic model might serve as an interesting tool for the study of factors regulating plasma HDL cholesterol.

Identification and characterization of a novel apolipoprotein E variant, apolipoprotein E3' (Arg136–>His): association with mild dyslipidemia and double pre-beta very low density lipoproteins

Apolipoprotein (apo) E mediates the removal of chylomicron and VLDL remnants from plasma. In a proband with mild hyperlipidemia and a family history of premature coronary artery disease, we have identified a new mutant of apoE with an isoelectric point close to but distinct from that of apoE3. Sequencing of the apoE gene from this subject (JB) revealed that the subject was heterozygous for a G to A substitution in codon 136, resulting in the substitution of histidine for arginine; therefore, we have designated this isoform apoE3' (Arg136-->His). Examination of the proband's kindred revealed that the nine carriers (all heterozygotes) of the variant isoform displayed a twofold elevation in the concentration of very low density lipoprotein (VLDL) cholesterol (40 +/- 8 mg/dl) and triglyceride (109 +/- 19) compared to the nine noncarriers (19 +/- 3 and 55 +/- 13, respectively). In all carriers, the VLDL displayed an abnormal double pre-beta pattern upon electrophoresis. The low density lipoprotein receptor-binding activity of purified apoE3' (Arg136-->His) when complexed with DMPC was slightly defective (80% of the activity of normal apoE). The mutant apoE also displayed a reduced affinity for heparin compared to apoE3. As both of these biochemical parameters are known to be important in VLDL clearance, the defects associated with this variant are likely responsible for the increase in VLDL observed in carriers. None of the carriers displayed clinical features of type III hyperlipoproteinemia, suggesting that the relatively mild dyslipoproteinemic phenotype associated with this variant might be associated with recessive expression of this disorder. However, the abnormal VLDL phenotype appears to be dominantly expressed.

Site-directed mutagenesis of an apolipoprotein E mutant, apo E5(Glu 3→Lys) and its binding to low density lipoprotein receptors

Apo E5(Glu 3→Lys) is a naturally occurring apolipoprotein E (apo E) mutant found in patients with hyperlipoproteinemia and atherosclerosis. It has been shown to have a high affinity for low density lipoprotein (LDL) receptors. In this study, mutant apo E5 was produced by Chinese hamster ovary cells by means of an in vitro site-directed mutagenesis technique, and its LDL receptor binding activity was assessed. The apo E5 obtained from gene expression bound more readily to the LDL receptor than did plasma apo E3. The concentrations required for 50% competitive binding of 125I-labeled LDL to the LDL receptors were 58.9 ng/ml for plasma apo E3 and 25.7 ng/ml for the expressed apo E5. The expressed apo E5 displayed 229% normal binding. This result is highly consistent with that obtained with plasma apo E5, which showed 217% normal binding. Although the experimental apo E isoproteins contained more sialic acid than plasma apo E, the extent of sialylation had no effect on the receptor binding of apo E.

Importance of growth hormone for the induction of hepatic low density lipoprotein receptors.

This investigation was undertaken to determine the possible role of growth hormone (GH) in the hormonal regulation of hepatic low density lipoprotein (LDL) receptor expression. Treatment of normal rats with estrogen (ethynylestradiol, 5 mg/kg per day) increased the number of hepatic LDL receptors, and the LDL receptor mRNA levels were increased 2.4-fold. However, when hypophysectomized rats were treated with estrogen, the hepatic LDL receptor number and the mRNA levels only increased slightly. Treatment with GH was important to restore the induction of hepatic LDL receptors in hypophysectomized estrogen-treated rats. Further, the hypocholesterolemic effect of estrogen was abolished in hypophysectomized rats, and GH reversed this effect. To assess the effect of GH in humans, hepatic LDL receptor binding activity was determined in liver biopsy specimens from gallstone patients pretreated with GH (12 international units/day) prior to operation. GH administration induced hepatic LDL receptors approximately 2-fold, and this was accompanied by a 25% decrease in serum cholesterol. The LDL receptor stimulation caused by GH treatment was of similar magnitude as that observed upon 3 weeks of treatment with an established hypolipidemic drug (pravastatin or simvastatin). The data show that GH has an important role in the regulation of hepatic LDL receptors and suggest that GH secretion may be important for the control of plasma LDL levels in humans.

Regulation of Hepatic Cholesterol Metabolism in Man

The liver is a key element in regulating the amount of low density lipoprotein (LDL) cholesterol in plasma. The interference of cholestyramine treatment in the enterohepatic circulation of bile acids stimulates the activity of the rate limiting enzymatic step in bile acid biosynthesis (cholesterol 7 alpha-hydroxylase). This increases demand for cholesterol which is met by enhanced cholesterol biosynthesis (through the enzyme 3-hydroxy-3-methylglutaryl coenzyme A [HMG CoA] reductase) and by an increased expression of LDL receptors. Inhibition of HMG CoA reductase activity by treatment with specific inhibitors such as pravastatin enhances LDL receptor binding activity. Combination of the two treatments results in a significant stimulation of LDL receptor expression and a drastic reduction in the concentration of plasma LDL cholesterol. Thus, selective interference with bile acid enterohepatic circulation and cholesterol biosynthesis may be utilised to regulate plasma lipoprotein metabolism.

Low density lipoprotein receptor-binding activity in human tissues: quantitative importance of hepatic receptors and evidence for regulation of their expression in vivo.

The heparin-sensitive binding of 125I-labeled low-density lipoprotein (LDL) to homogenates from 18 different normal human tissues and some solid tumors was determined. The binding to adrenal and liver homogenates fulfilled criteria established for the binding of LDL to its receptor--namely, (i) saturability, (ii) sensitivity to proteolytic destruction, (iii) inhibition by EDTA, and (iv) heat sensitivity. When the binding of 125I-labeled LDL was assayed at a constant concentration (50 micrograms/ml), the adrenal gland and the ovary had the highest binding of normal tissues. The highest binding per g of tissue overall was obtained in homogenates of a gastric carcinoma and a parotid adenoma. When the weights of the parenchymatous organs were considered, the major amount of LDL receptors was contained in the liver. To study the possible regulation of hepatic LDL-receptor expression, 11 patients were pretreated with cholestyramine (8 g twice a day for 3 weeks). Increased binding activity (+105%, P less than 0.001) was obtained in homogenates from liver biopsies from the cholestyramine-treated patients as compared with 12 untreated controls. It is concluded that the liver is the most important organ for LDL catabolism in humans and that the receptor activity in this organ can be regulated upon pharmacologic intervention. Further studies are needed to confirm the possibility that certain solid tumors can exhibit high numbers of LDL receptors.

Heparin-binding growth factor type one and platelet-derived growth factor are required for the optimal expression of cell surface low density lipoprotein receptor binding activity in human adult arterial smooth muscle cells

Purified heparin-binding growth factor-1 (HBGF-1) stimulated low density lipoprotein binding, internalization, and degradation in isolated human adult arterial smooth muscle cells. Exposure of quiescent cells to HBGF-1 in serum-free, defined medium increased both low density lipoprotein (LDL) receptor activity and de novo cholesterol biosynthesis. Both events preceded the onset of DNA synthesis by 6 to 9 h. HBGF-1 acted additively with platelet-derived growth factor (PDGF) to maximally stimulate cell surface LDL receptor binding activity and DNA synthesis in the smooth muscle cells. The presence of LDL was required for maximal mitogenic activity of HBGF-1 and PDGF. In the presence of LDL, growth factor-stimulated, proliferating human smooth muscle cells accumulated cholesterol ester and triglycerides. The results suggest that HBGF-1, PDGF, and LDL act together to promote the maximal proliferation of smooth muscle cells in culture. Chronic exposure to the three growth promoters may contribute to the smooth muscle cell hyperplasia and lipid accumulation observed in atherosclerotic lesions.