Nascent High-density Lipoprotein Formation Research Articles

Adiponectin's mechanisms in high-density lipoprotein biogenesis and cholesterol efflux

AimAmong adiponectin's beneficial properties is its ability to promote cellular cholesterol efflux, thereby generating high-density lipoprotein (HDL) particles. However, adiponectin's role in the regulation of macrophage lipid metabolism, a crucial process in atherogenesis, remains poorly investigated. The aim of this study was to characterize the adiponectin's role in HDL biogenesis. Methods and resultsWe perform kinetics studies in baby hamster kidney (BHK) and Tamm-Horsfall protein 1 (THP-1) cell lines to elucidate adiponectin's role in HDL biogenesis. In cholesterol-enriched cells, specific molar doses of adiponectin stimulated cholesterol efflux with high efficiency to apoA-I. In the presence of adiponectin, BHK cells expressing ATP binding cassette transporter A1 (ABCA1) or ABCG1 generated lipidated particles having α electrophoretic mobility (α-HDL) and a molecular size of 7.5–20 nm. Interestingly, in THP-1 macrophages, cholesterol efflux was associated with more lipidated preβ1-HDL particles. Direct molecular interaction of adiponectin with apoA-I enhanced the affinity of apoA-I to free cholesterol and resulted in an increase in preβ1-HDL particles from plasma ex vivo. Adiponectin increased ABCA1 and ABCG1 protein expression and activated the formation of ABCA1-linked cholesterol oxidase sensitive plasma membrane domains. ConclusionAdiponectin upregulated ABCA1 and ABCG1 protein expression, reduced lipid accumulation, and efficiently promoted nascent HDL formation. These results highlight that these cellular processes are interconnected through adiponectin and ABCA1- and ABCG1-dependent. In this pathway, adiponectin increased the affinity of apoA-I to cholesterol and effectively accelerated cholesterol removal from the plasma membrane to HDL particles. Thus, by accelerating HDL biogenesis, adiponectin may have therapeutic potential for atherosclerotic cardiovascular disease prevention and management.

Temporary sequestration of cholesterol and phosphatidylcholine within extracellular domains of ABCA1 during nascent HDL generation

The quality and quantity of high-density lipoprotein (HDL) in blood plasma are important for preventing coronary artery disease. ATP-binding cassette protein A1 (ABCA1) and apolipoprotein A-I (apoA-I) play essential roles in nascent HDL formation, but controversy persists regarding the mechanism by which nascent HDL is generated. In the “direct loading model”, apoA-I acquires lipids directly from ABCA1 while it is bound to the transporter. By contrast, in the “indirect model”, apoA-I acquires lipids from the specific membrane domains created by ABCA1. In this study, we found that trypsin treatment causes rapid release of phosphatidylcholine (PC) and cholesterol from BHK/ABCA1 cells, and that the time course of lipid release coincides with those of trypsin digestion of extracellular domains (ECDs) of surface ABCA1 and of release of ECD fragments into the medium. This trypsin-dependent lipid release was dependent on ABCA1 ATPase activity, and did not occur in cells that express ABCG1, which exports lipids like ABCA1 but does not have large ECDs. These results suggest that the trypsin-sensitive sites on the cell surface are the large ECDs of ABCA1, and that lipids transported by ABCA1 are temporarily sequestered within the ECDs during nascent HDL formation.

ATP-Binding Cassette Transporter A1 Deficiency in Human Induced Pluripotent Stem Cell-Derived Hepatocytes Abrogates HDL Biogenesis and Enhances Triglyceride Secretion

Despite the recognized role of the ATP-binding Cassette Transporter A1 (ABCA1) in high-density lipoprotein (HDL) metabolism, our understanding of ABCA1 deficiency in human hepatocytes is limited. To define the functional effects of human hepatocyte ABCA1 deficiency, we generated induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) from Tangier disease (TD) and matched control subjects. Control HLCs exhibited robust cholesterol efflux to apolipoprotein A-I (apoA-I) and formed nascent HDL particles. ABCA1-deficient HLCs failed to mediate lipid efflux or nascent HDL formation, but had elevated triglyceride (TG) secretion. Global transcriptome analysis revealed significantly increased ANGPTL3 expression in ABCA1-deficient HLCs. Angiopoietin-related protein 3 (ANGPTL3) was enriched in plasma of TD relative to control subjects. These results highlight the required role of ABCA1 in cholesterol efflux and nascent HDL formation by hepatocytes. Furthermore, our results suggest that hepatic ABCA1 deficiency results in increased hepatic TG and ANGPTL3 secretion, potentially underlying the elevated plasma TG levels in TD patients.

ATP binding cassette A1 (ABCA1) mediates microparticle formation during high-density lipoprotein (HDL) biogenesis

Background and aimsMicro-particles (MP) are secreted by various cells. Their biological roles in health and in disease remain unknown. Here we describe formation of MP in the process of ABCA1-dependent cholesterol efflux in different cell types. MethodsThe ATP-binding cassette transporter, subfamily A, member 1 (ABCA1) is the rate-limiting step in the biogenesis of high-density lipoproteins (HDL). We have found that ABCA1 and apoA-I contribute to the formation of MP. Using cell-based systems with overexpression and selective inactivation of ABCA1, pharmacological blockade and modulation of membrane cholesterol content, we characterized MP release from various cell lines. We studied MP release in BHK cells stably expressing ABCA1 under mifepristone control, human THP-1 macrophages and HepG2 cells without, or with incubation with human apoA-I. ResultsABCA1 mediates the production of MPs containing cholesterol. This was also confirmed in primary human monocyte-derived macrophages (MDMs). Adding apoA-I markedly increases MP release from cells. Inhibition of ABCA1 with probucol or decreasing plasma membrane cholesterol with methyl-β cyclodextrin (CDX) markedly reduced MP release and nascent HDL formation. MPs do not contain apoA-I, but contain flotilin-2, a marker of plasma membrane, and CD63, an exosome marker. MPs exhibit considerable size heterogeneity (50–250 nm). ConclusionsWe show that MPs are lipoprotein-sized structures created by the ABCA1 transporter, and contribute approximately 30% of ABCA1-and apoA-I mediated cholesterol efflux. In addition, we found that MPs release from cells consists, in part, of exosomes and depends on the same pathway used for HDL biogenesis.

High insulin level promotes the degradation of high density lipoprotein generation-related functional protein ABCA1 through calpain and proteasome pathway in 3T3-L1 adipocytes

To explore effects and potential mechanisms of high insulin environment on high density lipoprotein (HDL) generation-related functional protein ABCA1. [(3)H] labeled cholesterol efflux from mature 3T3-L1 adipocytes was detected by liquid scintillation counting. ABCA1 mRNA and protein expression in mature 3T3-L1 adipocytes post stimulation with various concentrations of insulin was detected by real-time fluorescence-based quantitative techniques and Western blot, respectively, in the absence and presence of CHX (cycloheximide, CHX), calpeptin (calpain pathway inhibitor) or MG-132 (proteasome pathway inhibitor). Cholesterol efflux rates were reduced post insulin stimulation in a dose-dependent manner ((7.06 ± 0.27)%, (6.59 ± 0.30)%, (6.34 ± 0.24)%, (5.07 ± 0.40)%, and (4.71 ± 0.40)% at 0, 1, 10, 10², and 10³ nmol/L of insulin, P < 0.05). Cholesterol efflux rates decreased in a time-dependent manner post 10³ nmol/L insulin stimulation (6.52 ± 0.30)%, (5.59 ± 0.71)%, (5.44 ± 0.37)%, (4.52 ± 0.32)%, and (4.38 ± 0.33)% at 0, 2, 4, 6, 12 h, respectively). ABCA1mRNA levels were not affected by insulin (P > 0.05). ABCA1 protein level was significantly downregulated in 10³ nmol/L insulin group compared to 0 nmol/L insulin group (P < 0.01). Compared with the 0 h group, ABCA1 protein level was significantly reduced in 6 h group (P < 0.05) and further reduced in 12 h group (P < 0.01). Both calpeptin and MG-132 could partly reduce insulin-induced degradation of ABCA1. Compared with the negative control group, ABCA1 protein levels were significantly upregulated by cotreatment with calpeptin and MG-132, respectively (both P < 0.01). Our data suggest that high insulin level could promote the ABCA1 protein degradation and reduce cholesterol efflux from mature 3T3-L1 adipocytes through calpain and proteasome pathway, thus, produce a circumference not suitable for nascent HDL formation in 3T3-L1 adipocytes.

Impact of phospholipid transfer protein on nascent high-density lipoprotein formation and remodeling.

Phospholipid transfer protein (PLTP), which binds phospholipids and facilitates their transfer between lipoproteins in plasma, plays a key role in lipoprotein remodeling, but its influence on nascent high-density lipoprotein (HDL) formation is not known. The effect of PLTP overexpression on apolipoprotein A-I (apoA-I) lipidation by primary mouse hepatocytes was investigated. Overexpression of PLTP through an adenoviral vector markedly affected the amount and size of lipidated apoA-I species that were produced in hepatocytes in a dose-dependent manner, ultimately generating particles that were <7.1 nm but larger than lipid-free apoA-I. These <7.1-nm small particles generated in the presence of overexpressed PLTP were incorporated into mature HDL particles more rapidly than apoA-I both in vivo and in vitro and were less rapidly cleared from mouse plasma than lipid-free apoA-I. The <7.1-nm particles promoted both cellular cholesterol and phospholipid efflux in an ATP-binding cassette transporter A1-dependent manner, similar to apoA-I in the presence of PLTP. Lipid-free apoA-I had a greater efflux capacity in the presence of PLTP than in the absence of PLTP, suggesting that PLTP may promote ATP-binding cassette transporter A1-mediated cholesterol and phospholipid efflux. These results indicate that PLTP alters nascent HDL formation by modulating the lipidated species and by promoting the initial process of apoA-I lipidation. Our findings suggest that PLTP exerts significant effects on apoA-I lipidation and nascent HDL biogenesis in hepatocytes by promoting ATP-binding cassette transporter A1-mediated lipid efflux and the remodeling of nascent HDL particles.

Targeted Deletion of Hepatocyte ABCA1 Leads to Very Low Density Lipoprotein Triglyceride Overproduction and Low Density Lipoprotein Hypercatabolism

Loss of ABCA1 activity in Tangier disease (TD) is associated with abnormal apoB lipoprotein (Lp) metabolism in addition to the complete absence of high density lipoprotein (HDL). We used hepatocyte-specific ABCA1 knock-out (HSKO) mice to test the hypothesis that hepatic ABCA1 plays dual roles in regulating Lp metabolism and nascent HDL formation. HSKO mice recapitulated the TD lipid phenotype with postprandial hypertriglyceridemia, markedly decreased LDL, and near absence of HDL. Triglyceride (TG) secretion was 2-fold higher in HSKO compared with wild type mice, primarily due to secretion of larger TG-enriched VLDL secondary to reduced hepatic phosphatidylinositol 3-kinase signaling. HSKO mice also displayed delayed clearance of postprandial TG and reduced post-heparin plasma lipolytic activity. In addition, hepatic LDLr expression and plasma LDL catabolism were increased 2-fold in HSKO compared with wild type mice. Last, adenoviral repletion of hepatic ABCA1 in HSKO mice normalized plasma VLDL TG and hepatic phosphatidylinositol 3-kinase signaling, with a partial recovery of HDL cholesterol levels, providing evidence that hepatic ABCA1 is involved in the reciprocal regulation of apoB Lp production and HDL formation. These findings suggest that altered apoB Lp metabolism in TD subjects may result from hepatic VLDL TG overproduction and increased hepatic LDLr expression and highlight hepatic ABCA1 as an important regulatory factor for apoB-containing Lp metabolism.

Electron Paramagnetic Resonance Spectroscopy of Site-directed Spin Labels Reveals the Structural Heterogeneity in the N-terminal Domain of ApoA-I in Solution

Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipid-dependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other in-solution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14-98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.

Effects of Apolipoprotein A-I on ATP-binding Cassette Transporter A1-mediated Efflux of Macrophage Phospholipid and Cholesterol: FORMATION OF NASCENT HIGH DENSITY LIPOPROTEIN PARTICLES

The mechanism of formation of high density lipoprotein (HDL) particles by the action of ATP-binding cassette transporter A1 (ABCA1) is not defined completely. To address this issue, we monitored efflux to apoA-I of phosphatidylcholine (PC), sphingomyelin (SM), and unesterified (free) cholesterol (FC) from J774 macrophages, in which ABCA1 is up-regulated, and investigated the nature of the particles formed. The various apoA-I/lipid particles appearing in the extracellular medium were separated by gel filtration chromatography. The presence of apoA-I in the extracellular medium led to the simultaneous formation of more than one type of poorly lipidated apoA-I-containing particle: there were 9- and 12-nm diameter particles containing approximately 3:1 and 1:1 phospholipid/FC (mol/mol), respectively, which were present together with 6-nm monomeric apoA-I. Removal of the C-terminal alpha-helix (residues 223-243) of apoA-I reduced phospholipid and FC efflux and prevented formation of the 9- and 12-nm HDL particles; the apoA-I variant formed larger particles that eluted in the void volume. FC loading of the J774 cells also led to the formation of larger apoA-I-containing particles that were highly enriched in FC. Besides creating HDL particles, ABCA1 mediated release of larger (20-450-nm diameter) FC-rich particles that were not involved in HDL formation and that are probably membrane vesicles. These particles contained 1:1 PC/SM in contrast to the HDL particles, which contained 2:1 PC/SM. This is consistent with lipid raft and non-raft plasma membrane domains being involved primarily in ABCA1-mediated vesicle release and nascent HDL formation, respectively.