Increase In Low Density Lipoprotein Uptake Research Articles

Abstract 2046: Overexpression Of Endothelial Lipase Enhances The Cellular Uptake Of Low-density Lipoproteins In Cultured Hepatocytes In The Absence Of Low-density Lipoprotein Receptor.

Background: Inhibitors of angiopoietin-like3 (ANGPTL3) reduce low-density lipoprotein (LDL) cholesterol, even in Familial Hypercholesterolemia (FH) patients with a dysfunctional LDL receptor (LDLR). Although the exact mechanism remains elusive, it may depend upon endothelial lipase (EL). Early studies showed that EL mediates the cellular uptake of high-density lipoproteins in hepatocytes; however, whether EL similarly mediates LDL uptake is unknown. Aim: Investigate the role of EL in mediating the cellular uptake of LDL in an LDLR-deficient model. Methods: Cas9-expressing HepG2 cells were transfected with a scrambled or an LDLR -targeting guide RNA to produce control and LDLR -knockout (KO) cells. Control and KO cells were transfected by lipofection with an empty plasmid or a plasmid containing the human LIPG gene (EL). Cellular uptake of fluorescent human LDL was measured by FACS. In addition, we measured the uptake of LDL in cells with and without the pre-incubation with 5 U/ml heparin or a cocktail of heparinases I, II, and II to test the contribution of heparan sulfate proteoglycans (HSPG). Results: LDLR -KO HepG2 cells showed a 20-25% residual uptake of LDL compared to controls (p<0.001) and 10-fold higher levels of EL mRNA. Remarkably, LIPG -transfected LDLR -KO hepatocytes showed a 2-fold increase in LDL uptake compared to LDLR-KO cells non-overexpressing EL (p<0.001). The pre-incubation with either heparinases or heparin completely blocked the uptake of LDL not only in LIPG -transfected LDLR -KO cells but also in those LDLR -KO cells transfected with the empty plasmid, suggesting a crucial role of HSPG in the LDL uptake. Conversely, overexpression of EL decreased LDL uptake in control cells (-11%, p=0.02), while unchanged or slightly increased LDL uptake was observed after incubating with heparinases or heparin. Conclusions: Our findings demonstrate that EL facilitates the uptake of LDL through an LDLR-independent, HSPG-dependent pathway. This pathway may be responsible for the residual uptake of LDL observed in LDLR -KO hepatocytes and may represent a potential druggable target to treat FH. Our data provides an alternative mechanism to explain the reduction of LDL cholesterol induced by ANGPTL3 inhibitors.

Loss-of-function genomic variants highlight potential therapeutic targets for cardiovascular disease

Pharmaceutical drugs targeting dyslipidemia and cardiovascular disease (CVD) may increase the risk of fatty liver disease and other metabolic disorders. To identify potential novel CVD drug targets without these adverse effects, we perform genome-wide analyses of participants in the HUNT Study in Norway (n = 69,479) to search for protein-altering variants with beneficial impact on quantitative blood traits related to cardiovascular disease, but without detrimental impact on liver function. We identify 76 (11 previously unreported) presumed causal protein-altering variants associated with one or more CVD- or liver-related blood traits. Nine of the variants are predicted to result in loss-of-function of the protein. This includes ZNF529:p.K405X, which is associated with decreased low-density-lipoprotein (LDL) cholesterol (P = 1.3 × 10−8) without being associated with liver enzymes or non-fasting blood glucose. Silencing of ZNF529 in human hepatoma cells results in upregulation of LDL receptor and increased LDL uptake in the cells. This suggests that inhibition of ZNF529 or its gene product should be prioritized as a novel candidate drug target for treating dyslipidemia and associated CVD.

Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

The development of cardiovascular disease is intimately linked to elevated levels of low-density lipoprotein (LDL) cholesterol in the blood. Hepatic LDL receptor (LDLR) levels regulate the amount of plasma LDL. We identified the secreted zinc metalloproteinase, bone morphogenetic protein 1 (BMP1), as responsible for the cleavage of human LDLR within its extracellular ligand-binding repeats at Gly171↓Asp172. The resulting 120 kDa membrane-bound C-terminal fragment (CTF) of LDLR had reduced capacity to bind LDL and when expressed in LDLR null cells had compromised LDL uptake as compared to the full length receptor. Pharmacological inhibition of BMP1 or siRNA-mediated knockdown prevented the generation of the 120 kDa CTF and resulted in an increase in LDL uptake into cells. The 120 kDa CTF was detected in the livers from humans and mice expressing human LDLR. Collectively, these results identify that BMP1 regulates cellular LDL uptake and may provide a target to modulate plasma LDL cholesterol.

N-Glycosylation Defects in Humans Lower Low-Density Lipoprotein Cholesterol Through Increased Low-Density Lipoprotein Receptor Expression.

The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying patients with type I congenital disorders of glycosylation (CDGs) with defective N-glycosylation. We studied 29 patients with the 2 most prevalent types of type I CDG, ALG6 (asparagine-linked glycosylation protein 6)-deficiency CDG and PMM2 (phosphomannomutase 2)-deficiency CDG, and 23 first- and second-degree relatives with a heterozygous mutation and measured plasma cholesterol levels. Low-density lipoprotein (LDL) metabolism was studied in 3 cell models-gene silencing in HepG2 cells, patient fibroblasts, and patient hepatocyte-like cells derived from induced pluripotent stem cells-by measuring apolipoprotein B production and secretion, LDL receptor expression and membrane abundance, and LDL particle uptake. Furthermore, SREBP2 (sterol regulatory element-binding protein 2) protein expression and activation and endoplasmic reticulum stress markers were studied. We report hypobetalipoproteinemia (LDL cholesterol [LDL-C] and apolipoprotein B below the fifth percentile) in a large cohort of patients with type I CDG (mean age, 9 years), together with reduced LDL-C and apolipoprotein B in clinically unaffected heterozygous relatives (mean age, 46 years), compared with 2 separate sets of age- and sex-matched control subjects. ALG6 and PMM2 deficiency led to markedly increased LDL uptake as a result of increased cell surface LDL receptor abundance. Mechanistically, this outcome was driven by increased SREBP2 protein expression accompanied by amplified target gene expression, resulting in higher LDL receptor protein levels. Endoplasmic reticulum stress was not found to be a major mediator. Our study establishes N-glycosylation as an important regulator of LDL metabolism. Given that LDL-C was also reduced in a group of clinically unaffected heterozygotes, we propose that increasing LDL receptor-mediated cholesterol clearance by targeting N-glycosylation in the LDL pathway may represent a novel therapeutic strategy to reduce LDL-C and cardiovascular disease.

Blackcurrant anthocyanins stimulated cholesterol transport via post-transcriptional induction of LDL receptor in Caco-2 cells.

We previously showed that polyphenol-rich blackcurrant extract (BCE) showed a hypocholesterolemic effect in mice fed a high fat diet. As direct cholesterol removal from the body via the intestine has been recently appreciated, we investigated the effect of BCE on the modulation of genes involved in intestinal cholesterol transport using Caco-2 cells as an in vitro model. Caco-2 cells were treated with BCE to determine its effects on mRNA and protein expression of genes important for intestinal cholesterol transport, low-density lipoprotein (LDL) uptake, cellular cholesterol content, and cholesterol transport from basolateral to apical membrane of Caco-2 cell monolayers. Cells were also treated with anthocyanin-rich or -poor fraction of BCE to determine the role of anthocyanin on BCE effects. BCE significantly increased protein levels of LDL receptor (LDLR) without altering its mRNA, which consequently increased LDL uptake into Caco-2 cells. This post-transcriptional induction of LDLR by BCE was markedly attenuated in the presence of rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). In addition, BCE altered genes involved in cholesterol transport in the enterocytes, including apical and basolateral cholesterol transporters, in such a way that could enhance cholesterol flux from the basolateral to apical side of the enterocytes. Indeed, BCE significantly increased the flux of LDL-derived cholesterol from the basolateral to the apical chamber of Caco-2 monolayer. LDLR protein levels were markedly increased by anthocyanin-rich fraction, but not by anthocyanin-free fraction. mTORC1-dependent post-transcriptional induction of LDLR by BCE anthocyanins drove the transport of LDL-derived cholesterol to the apical side of the enterocytes. This may represent a potential mechanism for the hypocholesterolemic effect of BCE.

Abstract 614: 1,25-dihydroxyvitamin D Enhances Vegf-stimulated Porcine Adipose-derived Mesenchymal Stem Cells Toward the Endothelial Phenotype Involving Wnt/β-catenin Pathway

Background: Cell-based therapy using adipose-derived mesenchymal stem cells (AMSCs) is an attractive option for re-endothelialization post-angioplasty procedures. Vitamin D levels in circulating blood may regulate AMSC-based re-endothelialization of injured arteries. To our knowledge, the role of vitamin D and vascular endothelial growth factor (VEGF) in the differentiation of adipose-derived AMSCs into endothelial cells (ECs) has not been examined. In this study, we investigated the effect of vitamin D on VEGF-induced differentiation of MSCs into ECs. Methods and Results: MSCs isolated from porcine adipose tissue were CD11b-CD34-CD44+CD73+CD90+ and showed characteristics of MSCs. The MSCs were stimulated and differentiated into ECs with endothelial growth media (EGM+50ng/ml of VEGF) and EGM media containing 10nM of calcitriol (EGM+50ng/ml of VEGF +10nM calcitriol) for 10 days. The EC differentiation was assessed by mRNA expression by qPCR and protein expression by flow cytometry for endothelial cell markers. Calcitriol enhanced EGM+VEGF-induced differentiation of MSCs into ECs, as revealed by 2-fold increase in mRNA and 4-fold increase in protein expression of EC markers. Angiogenesis assay and acetylated low density lipoprotein (LDL) uptake assay were used to assess the endothelial functionality that showed significant increase in capillary tube sprouting, and an increase in LDL uptake by the differentiated cells in response to EGM +VEGF+ calcitriol. Examination of the findings from Wnt Pathway array revealed a 3-fold decrease in β-catenin and 3-fold increase in KREMEN1 protein in the cells treated with EGM + VEGF+calcitriol. Conclusion: Vitamin D significantly enhanced VEGF-induced differentiation of MSCs into endothelial cells. Thus, vitamin D status of the patients undergoing coronary intervention might regulate the development of thrombosis and intimal hyperplasia.

The role of HCA2 (GPR109A) in regulating macrophage function

We investigated the novel role of HCA2 (GPR109A) and its ligand nicotinic acid in regulating macrophage function. Hca2 expression in the RAW264.7 murine macrophage cell line is strongly induced by LPS treatment and correlates with the expression of TNF-α. Treatment with 300 μM nicotinic acid (reported EC50 3 μM, peak plasma concentration 50-300 μM), significantly inhibited TNF-α, IL-6, IL-12p40, and IL-1β production (P<0.05) in LPS (1 ng/ml)-stimulated wild-type murine bone marrow-derived macrophages (BMMs) but failed to do so in Hca2(-/-) BMMs. Treatment with nicotinic acid reduced nuclear factor κB (NF-κB) activation levels by 43% (P<0.03) in wild-type BMMs 6 h after LPS stimulation but not in Hca2(-/-) BMMs. Nicotinic acid significantly inhibited wild-type BMM chemotaxis (P<0.001), but had no effect on the chemotaxis of Hca2(-/-) BMMs. A significant increase in low-density lipoprotein uptake by both wild-type (P<0.006) and Hca2(-/-) BMMs (P<0.03) in response to LPS was observed, which was significantly suppressed by nicotinic acid in wild-type BMMs (P<0.04) but not in Hca2(-/-) BMMs. Our results suggest that the nicotinic acid-HCA2 axis is a novel negative regulator of macrophage activation.

Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis

IntroductionOsteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.MethodsLDL receptor deficient (LDLr−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.ResultsA cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr−/− mice). Synovial wash-outs of LDLr−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.ConclusionsLDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.

Glioma cell activation by Alzheimer's peptide Abeta1-42, alpha1-antichymotrypsin, and their mixture.

We compared the effects ofAlzheimer's peptide (Abeta1-42), a,-antichymotrypsin (ACT) and an ACT/Abeta1-42 mixture on human glioma DK-MG cells. The solution of Abeta (5 microM) formed by 2-h incubation at room temperature induced tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 levels by 55 and 45%, respectively, and increased gelatinase B activity by 67%, while exposure of cells to the ACT/Abeta1-42 mixture (1:10 molar ratio ACT: Abeta1-42) under the same experimental conditions showed no effect on IL-6 levels or gelatinase B activity, but strongly induced TNF-alpha (by 190%), compared to the controls. Stimulation of the cells with Abeta1-42 alone, but not with ACT, increased by about 20% low-density lipoprotein (LDL) uptake and mRNA levels for LDL receptor and HMG-CoA reductase, while the ACT/Abeta1-42 mixture significantly increased LDL uptake (by 50%), up-regulated mRNA levels for LDL receptor and HMG-CoA reductase by 48 and 63%, respectively, and increased lipid accumulation by about 20-fold. These data suggest a possible new role for Abeta in Alzheimer's disease through its interaction with the inflammatory reactant, ACT.

Lipoprotein cholesterol and atherosclerosis.

Progressive accumulation of cholesterol in the arterial wall causes atherosclerosis, the pathologic process underlying most heart attacks and strokes. Low density lipoprotein (LDL), the major carrier of blood cholesterol, has been implicated in the buildup of cholesterol in atherosclerotic plaques. Endothelial cells that line arteries function to transport LDL into the vessel wall. Models for the mechanism of cholesterol accumulation in atherosclerotic plaques emphasize increased LDL uptake into the vessel wall or increased retention of LDL that has entered the vessel wall. This article reviews the pathways of cholesterol entry and removal, the metabolism, and the physical changes of cholesterol in the vessel wall. How these processes are believed to contribute to cholesterol buildup in atherosclerotic plaques is discussed.

Dysregulation of LDL receptor under the influence of inflammatory cytokines: A new pathway for foam cell formation

Lipid-mediated renal injury is an important component of glomerulosclerosis and its similarity to atherosclerosis is well described. This study focused on the relationship between lipid-mediated injury and inflammation by examining the role of inflammatory cytokines in the regulation of human mesangial cell low-density lipoprotein (LDL) receptors. A human mesangial cell line (HMCL) was used to study the effects of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on the regulation of LDL receptor mRNA and protein in the presence of a high concentration of native LDL (250 microg/mL). Native LDL caused foam cell formation in HMCL in the presence of antioxidants, TNF-alpha and IL-1beta. Both cytokines overrode LDL receptor suppression induced by a high concentration of LDL and increased LDL uptake by enhancing receptor expression. These cytokines also caused increased expression of SCAP [sterol responsive element binding protein (SREBP) cleavage activation protein], and an increase in the nuclear translocation of SREBP, which induces LDL receptor expression. These observations demonstrate that inflammatory cytokines can modify cholesterol-mediated LDL receptor regulation in mesangial cells, permitting unregulated intracellular accumulation of unmodified LDL and causing foam cell formation. These findings suggest that inflammatory cytokines contribute to lipid-mediated renal damage, and also may have wider implications for the study of inflammation in the atherosclerotic process.

Annexin VI Stimulates Endocytosis and Is Involved in the Trafficking of Low Density Lipoprotein to the Prelysosomal Compartment

Annexins are calcium-binding proteins with a wide distribution in most polarized and nonpolarized cells that participate in a variety of membrane-membrane interactions. At the cell surface, annexin VI is thought to remodel the spectrin cytoskeleton to facilitate budding of coated pits. However, annexin VI is also found in late endocytic compartments in a number of cell types, indicating an additional important role at later stages of the endocytic pathway. Therefore overexpression of annexin VI in Chinese hamster ovary cells was used to investigate its possible role in endocytosis and intracellular trafficking of low density lipoprotein (LDL) and transferrin. While overexpression of annexin VI alone did not alter endocytosis and degradation of LDL, coexpression of annexin VI and LDL receptor resulted in an increase in LDL uptake with a concomitant increase of its degradation. Whereas annexin VI showed a wide intracellular distribution in resting Chinese hamster ovary cells, it was mainly found in the endocytic compartment and remained associated with LDL-containing vesicles even at later stages of the endocytic pathway. Thus, data presented in this study suggest that after stimulating endocytosis at the cell surface, annexin VI remains bound to endocytic vesicles to regulate entry of ligands into the prelysosomal compartment.

Reversal of cyclosporine-inhibited low-density lipoprotein receptor activity in HepG2 cells by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

Previously we have shown that cyclosporine inhibits low-density lipoprotein (LDL) catabolism in HepG2 cells. This inhibition mainly occurs through reduced LDL-receptor activity. 3-Hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase inhibitors up-regulate LDL receptor activity with a subsequent increase in LDL uptake and degradation. In this study, in HepG2 cells, we investigated the effects of HMG-CoA reductase inhibitors on cellular LDL catabolism in the presence of cyclosporine. Different concentrations of cyclosporine and HMG-CoA reductase inhibitors, which were within the range of therapeutic concentrations used in humans, were added to the culture medium and the cellular LDL receptor activity was then measured. The results show that HMG-CoA reductase inhibitors reverse the down-regulatory effect of cyclosporine on LDL receptor activity, thus further supporting our previous findings and also providing a rationale for the already established treatment in cyclosporine-induced hypercholesterolemia with HMG-CoA reductase inhibitors.

Low density lipoprotein catabolism is enhanced by the cleaved form of alpha-1-antitrypsin

The frequent occurrence of hypocholesterolaemia following inflammatory processes is well known but unexplained. Elevated plasma levels of serine proteinase inhibitors (serpins) and their complexes with target enzymes have been demonstrated in inflammatory, malignant and infectious diseases which are also often accompanied by low plasma cholesterol levels. Under inflammatory conditions, uncomplexed, but cleaved inactive serpins arising from slow deacylation of the serpin-proteinase complex may be present in the circulation. To determine the influence of native and cleaved forms of serpins, such as alpha-1-antitrypsin (AAT), on lipoprotein metabolism, we investigated the effect of these forms on low density lipoprotein (LDL) catabolism in human HepG2 cell line. We have found that the cleaved form of AAT in concentrations from 125 to 2000 nmol 1−1 stimulates LDL binding to the HepG2 cells, by up to 49% with a subsequent increase in LDL uptake and degradation of up to 79 and 65% respectively. Native AAT was also found to increase LDL binding and internalization by 20–25%, independently of the amount of AAT added, an effect most probably due to the cleaved form of AAT produced by local proteolysis of native AAT incubated in the cell culture. Moreover, we have shown that the cleaved form of AAT interacts with LDL in vitro, and that such an interaction abolishes AAT ability to stimulate LDL binding and internalization. This study for the first time describes the ability of the cleaved form of AAT to stimulate LDL binding and internalization in HepG2 cell culture, and provides evidence that hypocholesterolaemia occurring during inflammatory processes may be mediated by cleaved forms of serpins.

Regulation of human placental trophoblast low-density lipoprotein uptake in vitro by estrogen.

In the present study, human trophoblast cells were studied in culture to determine the effect of estrogen on low-density lipoprotein (LDL) uptake and progesterone formation. Cytotrophoblasts were obtained from term human placentas and incubated for 72 h with 10% FBS to stimulate formation of syncytia. Syncytiotrophoblasts were then incubated for an additional 48 h with estradiol and/or LDL protein. Estradiol plus LDL stimulated progesterone to a level that was 133% greater (P < 0.05) than control (314 +/- 69 ng/mg protein) or LDL alone, suggesting that estrogen stimulated progesterone formation via an increase in LDL uptake and utilization. To examine this possibility, trophoblast cells were cultured with estradiol for 48 h as above, then incubated for 12 h with 6-200 micrograms/mL [125I]LDL. Mean (+/- SE) specific uptake of [125I]LDL (ng/mg cell protein) was approximately 50% greater (P < 0.01) with estradiol (638 +/- 23) than with vehicle (429 +/- 54). Scatchard analysis demonstrated that the dissociation constant for LDL uptake was similar in the presence (2.9 +/- 0.4 x 10(-6)M) and absence (2.8 +/- 0.6 x 10(-8)M) of estradiol, indicating that estrogen increased LDL receptor number without affecting affinity. LDL uptake was increased (P < 0.05) by incubating trophoblast with as little as 0.10 ng/mL estradiol (approximately 10(-9) M). We conclude that estrogen regulates placental trophoblast cell uptake of LDL and, thus, the availability of cholesterol for progesterone bio-synthesis during human pregnancy.

Glycoprotein 330/Low Density Lipoprotein Receptor-related Protein-2 Mediates Endocytosis of Low Density Lipoproteins via Interaction with Apolipoprotein B100

The ability of glycoprotein 330/low density lipoprotein receptor-related protein-2 (LRP-2) to function as a lipoprotein receptor was investigated using cultured mouse F9 teratocarcinoma cells. Treatment with retinoic acid and dibutyryl cyclic AMP, which induces F9 cells to differentiate into endoderm-like cells, produced a 50-fold increase in the expression of LRP-2. Levels of the other members of the low density lipoprotein (LDL) receptor (LDLR) family, including LDLR, the very low density lipoprotein receptor, and LRP-1, were reduced. When LDL catabolism was examined in these cells, it was found that the treated cells endocytosed and degraded at 10-fold higher levels than untreated cells. The increased LDL uptake coincided with increased LRP-2 activity of the treated cells, as measured by uptake of both 125I-labeled monoclonal LRP-2 antibody and the LRP-2 ligand prourokinase. The ability of LDL to bind to LRP-2 was demonstrated by solid-phase binding assays. This binding was inhibitable by LRP-2 antibodies, receptor-associated protein (the antagonist of ligand binding for all members of the LDLR family), or antibodies to apoB100, the major apolipoprotein component of LDL. In cell assays, LRP-2 antibodies blocked the elevated 125I-LDL internalization and degradation observed in the retinoic acid/dibutyryl cyclic AMP-treated F9 cells. A low level of LDL endocytosis existed that was likely mediated by LDLR since it could not be inhibited by LRP-2 antibodies, but was inhibited by excess LDL, receptor-associated protein, or apoB100 antibody. The results indicate that LRP-2 can function to mediate cellular endocytosis of LDL, leading to its degradation. LRP-2 represents the second member of the LDLR family identified as functioning in the catabolism of LDL.

Stimulation of low-density lipoprotein uptake in HepG2 cells by epidermal growth factor via a tyrosine kinase-dependent, but protein kinase C-independent, mechanism

Epidermal growth factor (EGF), a potent mitogenic polypeptide, stimulated the uptake and degradation of [3H]sucrose-labelled low-density lipoprotein (LDL) by HepG2 cells. The increase in LDL uptake was prevented by the presence of the tyrosine kinase inhibitor genistein. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) also stimulated the uptake of [3H]LDL by HepG2 cells. When EGF and PMA were added together, PMA increased the response to EGF in an additive manner. The protein kinase C inhibitor Ro-31-8220 prevented the increase in LDL uptake caused by PMA, but did not affect EGF stimulation of LDL uptake. Similarly, down-regulation of protein kinase C activity by chronic treatment with PMA also did not affect the EGF stimulation of LDL uptake. These results suggest that the EGF stimulation of LDL uptake and degradation by HepG2 cells is mediated by a tyrosine kinase-dependent, but protein kinase C-independent, mechanism.

The effects of perhexiline maleate on low density lipoprotein processing and cholesterol metabolism in cultured human fibroblasts.

The effects of perhexiline maleate (PM, Pexid) on low density lipoprotein (LDL) processing and cholesterol metabolism were investigated after a 24 h pretreatment with the drug. Perhexiline maleate increased LDL uptake in the range 10(-6) to 10(-5) M (180% of control for 10(-5) M), whereas LDL binding and degradation were not affected. Sterol synthesis from sodium acetate was enhanced by perhexiline maleate (X 2.2 for 10(-5) M), while cholesterol esterification with oleic acid was decreased (40% of control for 10(-5) M). These effects of perhexiline on cholesterol metabolism are specific, since the synthesis of triacylglycerols from sodium acetate is not affected and since the incorporation of oleic acid into triacylglycerols is much less impaired. The decreased cholesterol esterification is accompanied by a reduction of acylcoenzyme A-cholesterol-O-acyltransferase (ACAT) activity measured in vitro on cell extracts.

Rates of low density lipoprotein uptake and cholesterol synthesis are regulated independently in the liver.

The relationship between rates of hepatic sterol synthesis and rates of hepatic low density lipoprotein (LDL) uptake (clearance) was studied in animals with high (rats), low (female hamsters), and very low (male hamsters) basal rates of hepatic sterol synthesis. In rats and female hamsters, rates of hepatic sterol synthesis were varied over a 110-fold range by feeding cholesterol or cholestyramine; nevertheless, rates of hepatic LDL clearance remained essentially unchanged as did plasma LDL-cholesterol concentrations. In contrast, in male hamsters, which have a very limited capacity to synthesize cholesterol in the liver, cholestyramine feeding increased rates of hepatic LDL uptake by 2.5-fold and this was associated with a 50% reduction in plasma LDL-cholesterol concentrations. The observed increase in LDL uptake was due to an increase in receptor-dependent LDL transport while receptor-independent lipoprotein uptake remained constant. These studies suggest that rates of hepatic cholesterol synthesis and receptor-dependent LDL uptake are regulated independently. Furthermore, the primary response of the liver to changes in cholesterol availability is regulation of sterol synthesis and only when the capacity of this compensatory mechanism is exceeded is the rate of LDL transport altered.