Receptor-mediated Uptake Of LDL Research Articles

LDL, LDL receptors, and PCSK9 as modulators of the risk for type 2 diabetes: a focus on white adipose tissue.

Type 2 diabetes (T2D) and cardiovascular disease (CVD) share many risk factors such as obesity, unhealthy lifestyle, and metabolic syndrome, whose accumulation over years leads to disease onset. However, while lowering plasma low-density lipoprotein cholesterol (LDLC) is cardio-protective, novel evidence have recognised a role for common LDLC-lowering variants (e.g. in HMGCR, PCSK9, and LDLR) and widely used hypocholesterolemic drugs that mimic the effects of some of these variants (statins) in higher risk for T2D. As these conditions decrease plasma LDLC by increasing tissue-uptake of LDL, a role for LDL receptor (LDLR) pathway was proposed. While underlying mechanisms remain to be fully elucidated, work from our lab reported that native LDL directly provoke the dysfunction of human white adipose tissue (WAT) and the activation of WAT NLRP3 (Nucleotide-binding domain and Leucine-rich repeat Receptor, containing a Pyrin domain 3) inflammasome, which play a major role in the etiology of T2D. However, while elevated plasma numbers of apolipoprotein B (apoB)-containing lipoproteins (measured as apoB, mostly as LDL) is associated with WAT dysfunction and related risk factors for T2D in our cohort, this relation was strengthened in regression analysis by lower plasma proprotein convertase subtilisin/kexin type 9 (PCSK9). This supports a central role for upregulated pathway of LDLR and/or other receptors regulated by PCSK9 such as cluster of differentiation 36 (CD36) in LDL-induced anomalies. Targeting receptor-mediated uptake of LDL into WAT may reduce WAT inflammation, WAT dysfunction, and related risk for T2D without increasing the risk for CVD.

Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection.

Niemann-Pick C1 (NPC1) is a lysosomal membrane protein that exports cholesterol derived from receptor-mediated uptake of LDL, and it also mediates cellular entry of Ebola virus. Cholesterol export is inhibited by nanomolar concentrations of U18666A, a cationic sterol. To identify the target of U18666A, we synthesized U-X, a U18666A derivative with a benzophenone that permits ultraviolet-induced crosslinking. When added to CHO cells, U-X crosslinked to NPC1. Crosslinking was blocked by U18666A derivatives that block cholesterol export, but not derivatives lacking blocking activity. Crosslinking was prevented by point mutation in the sterol-sensing domain (SSD) of NPC1, but not by point mutation in the N-terminal domain (NTD). These data suggest that the SSD contains a U18666A-inhibitable site required for cholesterol export distinct from the cholesterol-binding site in the NTD. Inasmuch as inhibition of Ebola requires 100-fold higher concentrations of U18666A, the high affinity U16888A-binding site is likely not required for virus entry.

Use of mutant 125 I-Perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells

Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study, we generate an (125)I-labeled mutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to (125)I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The (125)I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines, we show that this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.

Apolipoprotein B Reconstruction at Single Molecular Level

Low density lipoprotein-cholesterol (LDL-C) is a clinical significant marker of cardiovascular disease risk. Each particle of LDL contains only one protein, apolipoprotein B (apoB). In human, the liver secretes full-length apoB (apoB-100) which also serves as a ligand for receptor-mediated uptake of LDL by a variety of cell types, such as monocyte and A549. Many diseases’ progression may cause by the deficiency of the LDL assembly. Therefore, it is desired to reveal the folding/assembly process of apoB. However, it is challenge to refold the apoB, because of its high insolubility in solution. In this study, the native apoB had been purified from LDL by ice cheer method, and truncated mutants of various lengths of apoB were recombinant expressed from E. coli system. The apoB-100 and its truncated proteins then were dissolved in the denature buffer which contained additional detergents and were refolded via an over-critical refolding process. The folding intermediates of apoB-100 and its truncated mutants could be observed by immunofluorescence microscopy at the single molecular level and the lipidation processes and secondary structures of apoBs can be also analyzed by SRCD. Moreover, the confocal microscopy showed that the refolded and native LDL could be absorbed by THP-1 and A549 cell. According to our observation the LDL assembly process can be proposed. This is the first study to refold the structural and function of apoB in vitro. Meanwhile, this refolded lipoprotein can be used as carrier for hydrophobic particles delivery.

The Ile128Thr polymorphism influences stability and ligand binding properties of the microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTTP) is essential for the assembly of VLDLs. We recently observed that a polymorphism in the MTTP promoter (-493G>T), which is in allelic association with an isoleucine-to-theronine substitution at position 128 (Ile128Thr) in the expressed protein, confers an increased risk of coronary heart disease. Two variant proteins comprising amino acids 16-297 of intact MTTP, MTTP(N)-Ile128 and MTTP(N)-Thr128, had similar native secondary structure content, as judged by circular dichroism. However, the thermal stability of MTTP(N)-Thr128 was greatly reduced, and this protein was also more extensively cleaved in limited proteolysis experiments compared with MTTP(N)-Ile128; both of these findings support a less compact fold. On adding LDL, which includes natively folded apolipoprotein B (apoB), decreased stability of the MTTP(N)-Thr128-LDL complex was observed compared with that of the MTTP(N)-Ile128-LDL complex. In a refined model of the N-terminal domain of MTTP, residue 128 is located in a surface-exposed position, in the same region as an identified MTTP binding site in the homologous apoB protein. Thus, the Ile128Thr polymorphism confers reduced structural stability, leading to decreased binding of MTTP to LDL particles. Because the major MTTP binding target on LDL is apoB, the Ile128Thr polymorphism could target the MTTP-apoB interaction.

Control of lipid metabolism by phosphorylation-dependent degradation of the SREBP family of transcription factors by SCF Fbw7

The sterol regulatory element binding protein (SREBP) family of transcription factors controls cholesterol and lipid metabolism. The nuclear forms of these proteins are rapidly degraded by the ubiquitin-proteasome pathway, but the signals and factors required for this are unknown. Here, we identify a phosphodegron in SREBP1a that serves as a recognition motif for the SCF(Fbw7) ubiquitin ligase. Fbw7 interacts with nuclear SREBP1a and enhances its ubiquitination and degradation in a manner dependent on the phosphorylation of T426 and S430 by GSK-3. Fbw7 also degrades nuclear SREBP1c and SREBP2, and inactivation of endogenous Fbw7 results in stabilization of nuclear SREBP1 and -2, enhanced expression of SREBP target genes, enhanced synthesis of cholesterol and fatty acids, and enhanced receptor-mediated uptake of LDL. Thus, our results suggest that Fbw7 may be a major regulator of lipid metabolism through control of the phosphorylation-dependent degradation of the SREBP family of transcription factors.

Cholesterol-lowering effect of policosanol on rabbits with hypercholesterolaemia induced by a wheat starch-casein diet.

The effect of policosanol, a mixture of high-molecular-weight aliphatic alcohols isolated from sugarcane wax, on casein-induced hypercholesterolaemia in rabbits was studied. When policosanol was administered by the oral route once daily for 30 d (50 mg/kg) the increases in plasma total cholesterol and LDL-cholesterol (LDC-C) were significantly reduced when compared with the control group. The incorporation of 3H2O into sterols in the liver was significantly depressed, suggesting inhibition of hepatic cholesterol biosynthesis. The oral administration of policosanol raised the rate of removal of 125I-labelled LDL from serum. Kinetic parameters calculated following injection of [125I]LDL showed than in casein-fed rabbits, the terminal half-life (t1/2) was significantly decreased after policosanol treatment. The hepatic LDL-binding activity was increased after policosanol administration which suggested that the enhanced clearance was due, at least in part, to increased receptor-mediated uptake of LDL by the liver. Considered together, these results suggest that policosanol can significantly reduce the increase of plasma LDL-C in rabbits fed on a wheat starch-casein diet by reducing cholesterol biosynthesis in the liver. Such an effect could account for the enhancement of LDL catabolism through the receptor-mediated pathway.

Linoleic acid esterified in low density lipoprotein serves as substrate for increased arachidonic acid synthesis in differentiating monocytic cells.

The cellular metabolism of albumin- and lipoprotein-bound 18:2(n - 6) during monocytic differentiation was examined in the human premonocytic U937 and Mono Mac 6 cells. Differentiation for 72 h of U937 cells with retinoic acid (RA, 1 microM) or 1,25-(OH)2-vitamin D3 (1,25-D3, 10 nM) and of Mono Mac 6 cells with RA (1 microM) or lipopolysaccharide (LPS, 10 ng/ml) increased the desaturation and elongation of [1-14C]18:2(n - 6) to [1-14C]20:4(n - 6). In undifferentiated U937 and Mono Mac 6 cells, incubations with human LDL (100 micrograms/ml, 18 h) resulted in a 2.5-fold increase in 18:2(n - 6) levels in the cellular phospholipids. Differentiation of U937 cells with RA or or of Mono Mac 6 cells with LPS prior to LDL addition. Significantly reduced 18:2(n - 6) and elevated 20:4(n - 6) levels in cellular phospholipids. This increase in 20:4(n - 6) was likely not due to an increased incorporation of preformed 20:4(n - 6) esterified in LDL, as the receptor-specific degradation of [125I]LDL was reduced in both the RA-treated U937 and LPS-treated Mono Mac 6 cells. In U937 cells incubated with [1-14C]18:2(n - 6), the synthesis of TXB2, PGE2 and HHT could be detected after differentiation with RA. suggesting the availability of [1-14C]20:4(n - 6), derived from [1-14C]18:2(n - 6), for cyclooxygenase metabolism. Our results show that the conversion of 18:2(n - 6) to 20:4(n - 6) increases during monocyte differentiation. The 18:2(n - 6) supplied to the cells via the receptor-mediated uptake of LDL was utilized as substrate for the increased 20:4(n - 6) synthesis.

Actions of Placental and Fetal Adrenal Steroid Hormones in Primate Pregnancy*

It is clear that steroid hormones of placental and fetal adrenal origin have critically important roles in regulating key physiological events essential to the maintenance of pregnancy and development of the fetus for extrauterine life. Thus, progesterone has suppressive actions on lymphocyte proliferation and activity and on the immune system to prevent rejection of the developing fetus and placenta (see Fig. 9). Progesterone also suppresses the calcium-calmodulin-MLCK system and thus activity of uterine smooth muscle, thereby promoting myometrial quiescence to ensure the maintenance of pregnancy. Estrogen enhances uteroplacental blood flow and possibly placental neovascularization to provide optimal gas exchange and the nutrients required for the rapidly developing fetus and placenta. In turn, estrogen has specific stimulatory effects on the receptor-mediated uptake of LDL by, and P-450scc activity within, syncytiotrophoblasts, thus promoting the biosynthesis of progesterone. Moreover, there is an estrogen-dependent developmental regulation of expression of the LDL receptor and NAD-dependent 11 beta-HSD in the placenta, processes reflecting functional/biochemical differentiation of the trophoblast cells with advancing gestation. The increase in 11 beta-HSD causes a change in transplacental corticosteroid metabolism, which results in activation of the HPAA in the fetus. As a result of this cascade of events, there is an increase in expression of pituitary POMC/ACTH and key enzymes, e.g. 3 beta-HSD and P-450 17 alpha-hydroxylase, important for de novo cortisol formation by, and consequently maturation of, the fetal adrenal gland. In turn, cortisol has well defined actions on surfactant biosynthesis and consequently fetal lung maturation, as well as effects on placental CRH/POMC release, which may be important to the initiation of labor. At midgestation, estrogen also selectively feeds back on the fetal adrenal to suppress DHA and maintain physiologically normal levels of estrogen. Preparation of the breast for lactation and nourishment of the newborn appears to involve a multifactorial system of regulation that includes estrogen. It is apparent, therefore, that autocrine/paracrine, as well as endocrine, systems of regulation are operative within the fetoplacental unit during primate pregnancy. A major goal of this review has been to illustrate the critically close functional communication existing between the developing placenta and fetus in the biosynthesis and the actions of steroid hormones during primate pregnancy. The functional interaction of the human fetal adrenal and placenta with respect to the biosynthesis of estrogen was demonstrated many years ago. However, the recent studies presented in this review show that the endocrine interaction between the fetus and placenta is more extensive, involving complex physiological regulatory mechanisms. Thus, as illustrated in Fig. 9, estrogen, acting via its receptor within the placenta and other reproductive tissues, orchestrates the dynamic interchange between the placenta and fetus responsible for the developmental regulation of the biosynthesis of the various steroid and peptide hormones and their receptors necessary for the maintenance of pregnancy and development of a live newborn. It would appear, therefore, that the immediate and long range challenges in this area of reproductive endocrinology are to employ in vitro molecular and in vivo experimental approaches simultaneously to elucidate the nature of these complex interactions and define the cellular and molecular mechanisms underlying these important regulatory events.

Effects of different phenotypes of hyperlipoproteinemia and of treatment with fibric acid derivatives on the rates of cholesterol 7 alpha-hydroxylation in humans.

Little is known about the relationships between hyperlipidemia and bile acid metabolism. However, hypolipidemic treatment with fibric acid derivatives has been shown to increase biliary cholesterol secretion, presumably by reducing bile acid synthesis. To clarify such relationships, we investigated the effects of different hyperlipoproteinemic conditions and of treatment with fibric acid derivatives on the rates of cholesterol 7 alpha-hydroxylation (the limiting step of bile acid synthesis) in humans. We studied 10 patients (aged 36 to 68 years) with lipoprotein phenotype IIa and with a clinical diagnosis of heterozygous familial hypercholesterolemia, a condition of reduced activity of LDL receptors, and 11 patients (aged 48 to 70 years) with lipoprotein phenotype IIb or IV and clinical diagnosis of familial combined hyperlipidemia, a condition probably related to increased hepatic lipoprotein synthesis. Cholesterol 7 alpha-hydroxylation rates were assayed in vivo by tritium release assay after an intravenous injection of [7 alpha-3H]cholesterol. The results were compared by ANOVA to the values obtained in a group of 28 normolipidemic patients (aged 34 to 83 years), with age as the covariate. Six patients were also studied after treatment with gemfibrozil (900 to 1200 mg/d for 6 to 8 weeks) and 5 patients were studied after treatment with bezafibrate (400 mg/d for 6 to 8 weeks). Hydroxylation rates were 0.82 +/- 0.22 mmol/d in the familial hypercholesterolemia group and 1.30 +/- 0.47 mmol/d in the familial combined hyperlipidemia group (P < .05 between the two groups and between patients with familial combined hyperlipidemia and control subjects; P = NS between patients with familial hypercholesterolemia and control subjects, as determined by ANOVA).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of LDL receptor binding determinants in very low density lipoproteins.

We have used both proteolysis and reconstitution experiments to characterize the determinants for LDL receptor binding of HTG-VLDL. In these studies, we showed that the removal of approximately one mole of apo E per mole of HTG-VLDL (Sf 100-400) and HTG-VLDL (Sf 60-100) by thrombin-specific cleavage results in loss of receptor binding and concomitant loss of suppression of HMG-CoA reductase. This is in direct contrast to the lack of effect thrombin cleavage has on the receptor-mediated uptake of LDL, an apo B-mediated process. We were able to reconstitute receptor binding in thrombin-treated HTG-VLDL (Sf 100-400) by the specific reincorporation of one mole of apo E into the VLDL. The incorporation of one mole of apo E into normal non-suppressive VLDL (Sf 60-400) also enables this lipoprotein to bind to the receptor as effectively as LDL. Trypsin, which destroys apo E-mediated, but not apo B-mediated binding to the LDL receptor, abolishes binding of HTG-VLDL (Sf 100-400) and HTG-VLDL (Sf 60-100), but not that of HTG-VLDL (Sf 20-60), IDL, or LDL to the LDL receptor. Therefore, we conclude that apo E of the appropriate conformation is required for receptor-mediated uptake by the LDL receptor of large TG-rich lipoproteins (Sf greater than 60). This conformation of apo E is probably related to the surface on which it is found (i.e., size of the particle) and the mode of incorporation into the phospholipid surface (i.e., transferred from plasma HDL). In large TG-rich particles, it appears that the intact apo E is necessary for the proper orientation of the molecule on the surface, with the carboxy-terminal one-third needed to anchor the apoprotein to the phospholipid surface. We believe that the binding of apo E to the LDL receptor is a redundant system and is used as a backup system in abnormal pathological states such as hypertriglyceridemia, abetalipoproteinemia, and hypobetalipoproteinemia. In the case of hypertriglyceridemia, where the lipolysis mechanism is overloaded, the abnormal binding of HTG-VLDL (Sf greater than 60) provides an alternate catabolic route for their removal from plasma. In the cases of a beta- and hypobetalipoproteinemia, where the normal particles for cholesterol delivery are either absent or at low levels, apo E-containing particles can serve to deliver cholesterol to cells as has been recently observed in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Angiotensin II stimulates receptor-mediated uptake of LDL by bovine adrenal cortical cells in primary culture

Bovine adrenal cells were isolated from the subcapsular region of the gland to obtain cultures enriched in cells of the zona glomemlosa. The cells kept in primary cultures were shown to respond to angiotensin II and adrenocorticotropin (ACTH) by a significant increase in aldosterone production. These primary adrenal cultures were used to study the effect of angiotensin II on LDL metabolism. Addition of angiotensin II for 48 h to the culture medium resulted in a 200–300% increase in LDL metabolism and the lowest effective concentration was 10 −8−10 −9 M. The angiotensin II effect became evident after 12–16 h of incubation. To compare the metabolism of the 125I-labeled protein moiety to that of cholesteryl ester of LDL, the lipoprotein was labeled also with cholesteryl linoleyl ether, a nonhydrolyzable analog of cholesteryl ester. Under basal conditions and in the presence of angiotensin II or ACTH the ratio of [ 3H]cholesteryl linoleyl ether to 125I indicate some preferential uptake of the cholesteryl ester moiety. Stimulation of specific LDL binding at 4°C and LDL metabolism at 37°C by 10 −7 M angiotensin II occurred at all concentrations of LDL studied. Linearization of the kinetic data showed that angiotensin II increased the LDL receptor number significantly but not the affinity of the LDL receptor for its ligand. The present findings indicate that in analogy to ACTH, angiotensin II can influence receptor-mediated uptake of LDL by adrenal cortical cells. It remains to be shown whether the angiotensin II effect on LDL metabolism is limited to adrenal cells or will affect other cells which express the angiotensin II receptor.

Interaction between macrophages and mesenchymal cells Effect of LDL- or HDL-containing media, added to cholesteryl ester-loaded macrophages, on cholesterol esterification in mesenchymal cells

Mouse peritoneal macrophages were loaded with cholesteryl ester (200 ±17 μ g cholesteryl ester /mg cell protein) by incubation with acetylated LDL (50 μ g protein/ml) for 72 h. Next, the loaded cells were incubated for 48 h in Dulbecco-Vogt medium containing 1% bovine serum albumin alone or together with low or high density lipoproteins, 15 μg protein/ml, or acetylated LDL, 25 μg protein/ml. Bovine aortic smooth muscle cells and human skin fibroblasts were labeled for 72 h with medium containing free [ 3H]cholesterol. The labeled cells were then incubated for an additional 48 h with media conditioned in the presence of macrophages. The cholesteryl ester content of the labeled smooth muscle cells incubated with macrophageconditioned media containing LDL or acetylated LDL increased 5-fold and there was also a 10-fold increase in the amount of labeled cholesteryl ester over that recorded prior to exposure to the macrophage-conditioned medium. Similar results were obtained with macrophage-conditioned media containing HDL, but the effects were less prominent. No enhancement of cholesterol esterification was seen with macrophage-conditioned media containing albumin only. Increase in cellular cholesteryl ester content and in labeled cholesteryl ester after incubation with macrophage-conditioned media containing LDL or acetylated LDL was obtained also in human skin fibroblasts. Cells derived from control donors and from a patient with familial homozygous hypercholesterolemia responded in a similar manner. The enhanced cholesterol esterification in human skin fibroblasts, incubated with macrophage-conditioned medium containing acetylated LDL, was counteracted by addition of HDL to the human skin fibroblast medium. The present results offer a mechanism for enrichment in cholesteryl ester of smooth muscle cells and fibroblasts which bypasses the receptor-mediated uptake of LDL and, thus, is not susceptible to autoregulation.