ABCA12 regulates insulin secretion from ß-cells.

Dysregulation of lipid homeostasis is intimately associated with defects in insulin secretion, a key feature of type 2 diabetes. Here, we explore the role of the putative lipid transporter ABCA12 in regulating insulin secretion from ß-cells. Mice with ß-cell-specific deletion of Abca12 display impaired glucose-stimulated insulin secretion and eventual islet inflammation and ß-cell death. ABCA12's action in the pancreas is independent of changes in the abundance of two other cholesterol transporters, ABCA1 and ABCG1, or of changes in cellular cholesterol or ceramide content. Instead, loss of ABCA12 results in defects in the genesis and fusion of insulin secretory granules and increases in the abundance of lipid rafts at the cell membrane. These changes are associated with dysregulation of the small GTPase CDC42 and with decreased actin polymerisation. Our findings establish a new, pleiotropic role for ABCA12 in regulating pancreatic lipid homeostasis and insulin secretion.

Modification of lipid rafts by extracellular vesicles carrying HIV-1 protein Nef induces redistribution of amyloid precursor protein and Tau, causing neuronal dysfunction.

HIV-associated neurocognitive disorders (HANDs) are a frequent outcome of HIV infection. Effective treatment of HIV infection has reduced the rate of progression and severity but not the overall prevalence of HANDs, suggesting ongoing pathological process even when viral replication is suppressed. In this study, we investigated how HIV-1 protein Nef secreted in extracellular vesicles (exNef) impairs neuronal functionality. ExNef were rapidly taken up by neural cells in vitro, reducing the abundance of ABC transporter A1 (ABCA1) and thus cholesterol efflux and increasing the abundance and modifying lipid rafts in neuronal plasma membranes. ExNef caused a redistribution of amyloid precursor protein (APP) and Tau to lipid rafts and increased the abundance of these proteins, as well as of Aß42 ExNef further potentiated phosphorylation of Tau and activation of inflammatory pathways. These changes were accompanied by neuronal functional impairment. Disruption of lipid rafts with cyclodextrin reversed the phenotype. Short-term treatment of C57BL/6 mice with either purified recombinant Nef or exNef similarly resulted in reduced abundance of ABCA1 and elevated abundance of APP in brain tissue. The abundance of ABCA1 in brain tissue of HIV-infected human subjects diagnosed with HAND was lower, and the abundance of lipid rafts was higher compared with HIV-negative individuals. Levels of APP and Tau in brain tissue correlated with the abundance of Nef. Thus, modification of neuronal cholesterol trafficking and of lipid rafts by Nef may contribute to early stages of neurodegeneration and pathogenesis in HAND.

Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.

HIV infection has a profound effect on "bystander" cells causing metabolic co-morbidities. This may be mediated by exosomes secreted by HIV-infected cells and containing viral factors. Here we show that exosomes containing HIV-1 protein Nef (exNef) are rapidly taken up by macrophages releasing Nef into the cell interior. This caused down-regulation of ABCA1, reduction of cholesterol efflux and sharp elevation of the abundance of lipid rafts through reduced activation of small GTPase Cdc42 and decreased actin polymerization. Changes in rafts led to re-localization of TLR4 and TREM-1 to rafts, phosphorylation of ERK1/2, activation of NLRP3 inflammasome, and increased secretion of pro-inflammatory cytokines. The effects of exNef on lipid rafts and on inflammation were reversed by overexpression of a constitutively active mutant of Cdc42. Similar effects were observed in macrophages treated with exosomes produced by HIV-infected cells or isolated from plasma of HIV-infected subjects, but not with exosomes from cells and subjects infected with ΔNef-HIV or uninfected subjects. Mice injected with exNef exhibited monocytosis, reduced ABCA1 in macrophages, increased raft abundance in monocytes and augmented inflammation. Thus, Nef-containing exosomes potentiated pro-inflammatory response by inducing changes in cholesterol metabolism and reorganizing lipid rafts. These mechanisms may contribute to HIV-associated metabolic co-morbidities.

Cholesterol Efflux-Independent Modification of Lipid Rafts by AIBP (Apolipoprotein A-I Binding Protein).

OBJECTIVE: AIBP (apolipoprotein A-I binding protein) is an effective and selective regulator of lipid rafts modulating many metabolic pathways originating from the rafts, including inflammation. The mechanism of action was suggested to involve stimulation by AIBP of cholesterol efflux, depleting rafts of cholesterol, which is essential for lipid raft integrity. Here we describe a different mechanism contributing to the regulation of lipid rafts by AIBP. Approach and Results: We demonstrate that modulation of rafts by AIBP may not exclusively depend on the rate of cholesterol efflux or presence of the key regulator of the efflux, ABCA1 (ATP-binding cassette transporter A-I). AIBP interacted with phosphatidylinositol 3-phosphate, which was associated with increased abundance and activation of Cdc42 and rearrangement of the actin cytoskeleton. Cytoskeleton rearrangement was accompanied with reduction of the abundance of lipid rafts, without significant changes in the lipid composition of the rafts. The interaction of AIBP with phosphatidylinositol 3-phosphate was blocked by AIBP substrate, NADPH (nicotinamide adenine dinucleotide phosphate), and both NADPH and silencing of Cdc42 interfered with the ability of AIBP to regulate lipid rafts and cholesterol efflux. CONCLUSIONS: Our findings indicate that an underlying mechanism of regulation of lipid rafts by AIBP involves PIP-dependent rearrangement of the cytoskeleton.

Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood.

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1+/+ and Abca1-/- mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.

TRAK2, a novel regulator of ABCA1 expression, cholesterol efflux and HDL biogenesis.

Aims: The recent failures of HDL-raising therapies have underscored our incomplete understanding of HDL biology. Therefore there is an urgent need to comprehensively investigate HDL metabolism to enable the development of effective HDL-centric therapies. To identify novel regulators of HDL metabolism, we performed a joint analysis of human genetic, transcriptomic, and plasma HDL-cholesterol (HDL-C) concentration data and identified a novel association between trafficking protein, kinesin binding 2 (TRAK2) and HDL-C concentration. Here we characterize the molecular basis of the novel association between TRAK2 and HDL-cholesterol concentration. Methods and results: Analysis of lymphocyte transcriptomic data together with plasma HDL from the San Antonio Family Heart Study (n = 1240) revealed a significant negative correlation between TRAK2 mRNA levels and HDL-C concentration, HDL particle diameter and HDL subspecies heterogeneity. TRAK2 siRNA-mediated knockdown significantly increased cholesterol efflux to apolipoprotein A-I and isolated HDL from human macrophage (THP-1) and liver (HepG2) cells by increasing the mRNA and protein expression of the cholesterol transporter ATP-binding cassette, sub-family A member 1 (ABCA1). The effect of TRAK2 knockdown on cholesterol efflux was abolished in the absence of ABCA1, indicating that TRAK2 functions in an ABCA1-dependent efflux pathway. TRAK2 knockdown significantly increased liver X receptor (LXR) binding at the ABCA1 promoter, establishing TRAK2 as a regulator of LXR-mediated transcription of ABCA1. Conclusion: We show, for the first time, that TRAK2 is a novel regulator of LXR-mediated ABCA1 expression, cholesterol efflux, and HDL biogenesis. TRAK2 may therefore be an important target in the development of anti-atherosclerotic therapies.

Innate immune receptor NOD2 promotes vascular inflammation and formation of lipid-rich necrotic cores in hypercholesterolemic mice.

Atherosclerosis is an inflammatory disease associated with the activation of innate immune TLRs and nucleotide-binding oligomerization domain-containing protein (NOD)-like receptor pathways. However, the function of most innate immune receptors in atherosclerosis remains unclear. Here, we show that NOD2 is a crucial innate immune receptor influencing vascular inflammation and atherosclerosis severity. 10-week stimulation with muramyl dipeptide (MDP), the NOD2 cognate ligand, aggravated atherosclerosis, as indicated by the augmented lesion burden, increased vascular inflammation and enlarged lipid-rich necrotic cores in Ldlr(-/-) mice. Myeloid-specific ablation of NOD2, but not its downstream kinase, receptor-interacting serine/threonine-protein kinase 2, restrained the expansion of the lipid-rich necrotic core in Ldlr(-/-) chimeric mice. In vitro stimulation of macrophages with MDP enhanced the uptake of oxidized low-density lipoprotein and impaired cholesterol efflux in concordance with upregulation of scavenger receptor A1/2 and downregulation of ATP-binding cassette transporter A1. Ex vivo stimulation of human carotid plaques with MDP led to increased activation of inflammatory signaling pathways p38 MAPK and NF-κB-mediated release of proinflammatory cytokines. Altogether, this study suggests that NOD2 contributes to the expansion of the lipid-rich necrotic core and promotes vascular inflammation in atherosclerosis.

Mechanism of cholesterol efflux in humans after infusion of reconstituted high-density lipoprotein.

OBJECTIVES: Infusion of reconstituted HDL (rHDL) leads to changes in HDL metabolism as well as to an increased capacity of plasma to support cholesterol efflux providing an opportunity to investigate mechanisms linking cholesterol efflux to changes in plasma HDL. METHODS AND RESULTS: Patient plasmas after infusion of rHDL were tested ex vivo for their capacity to stimulate cholesterol efflux. Reconstituted HDL enhanced mobilization of cholesterol from tissues in vivo as shown by rising HDL cholesterol concentrations over the infusion period. Infusion of rHDL in vivo led to increased cholesterol efflux ex vivo; surprisingly, removing apoB-containing lipoproteins while preserving all HDL subfractions eliminated this increase. Infusion of rHDL led to the remodelling of plasma HDL; however, the capacity of plasma to support cholesterol efflux did not correlate with changes in the concentrations of any of HDL subfractions. Unmodified rHDL accounted for only a proportion of the increment in cholesterol efflux capacity. Furthermore, studies using HeLa and BHK cells overexpressing ABCA1, ABCG1, and SR-B1 showed that the contribution of these cellular mediators of cholesterol efflux to the enhanced capacity of plasma for the efflux was minimal. CONCLUSION: Enhanced cholesterol efflux from tissues requires the presence of apoB-containing lipoproteins and may involve enhanced flow of cholesterol through multiple components of the reverse cholesterol transport pathway rather than being determined by a specific HDL subfraction.

Global functional knockdown of ATP binding cassette transporter A1 stimulates development of atherosclerosis in apoE K/O mice.

ABCA1 is a key element of cellular cholesterol homeostasis. ApoE K/O mice fed with high-fat diet were infused with anti-ABCA1 antibody or control IgM. Infusion of anti-ABCA1 antibody led to 72% increase in the area of atherosclerotic plaque in aorta. After 16 weeks on high-fat diet plasma level of high density lipoprotein cholesterol (HDL-C) was reduced in control group, but was unchanged in mice infused with anti-ABCA1 antibody. Total plasma cholesterol level was elevated while the capacity of plasma to support cholesterol efflux ex vivo was reduced after 16 weeks on high-fat diet; the effects were similar in the two groups. We conclude that functional blocking of ABCA1-dependent cholesterol efflux stimulates development of atherosclerosis in apoE K/O mice independently from HDL-C levels.

Isolation of Lipid Rafts from Cultured Mammalian Cells and Their Lipidomics Analysis.

Lipid rafts are distinct liquid-ordered domains of plasma membranes of most eukaryotic cells providing platform for signaling pathways. Lipid composition of rafts is critical for their structural integrity and for regulation of signaling pathways originating from rafts. Here we provide a protocol to isolate lipid rafts from cultured human and animal cells and comprehensively analyse their lipid composition.

Lipids, biomarkers, and subclinical atherosclerosis in treatment-naive HIV patients starting or not starting antiretroviral therapy: Comparison with a healthy control group in a 2-year prospective study.

OBJECTIVE: To assess the effect of HIV infection and combined antiretroviral therapy (c-ART) on various proatherogenic biomarkers and lipids and to investigate their relationship with subclinical atherosclerosis in a cohort of treatment-naive HIV-infected patients. METHODS: We performed a prospective, comparative, multicenter study of 2 groups of treatment-naive HIV-infected patients (group A, CD4>500 cells/µL, not starting c-ART; and group B, CD4<500 cells/µL, starting c-ART at baseline) and a healthy control group. Laboratory analyses and carotid ultrasound were performed at baseline and at months 12 and 24. The parameters measured were low-density lipoprotein (LDL) particle phenotype, lipoprotein-associated phospholipase A2 (Lp-PLA2), interleukin-6 (IL-6), high-sensitivity C-reactive protein (hs-CRP), sCD14, sCD163, monocyte chemoattractant protein-1(MCP-1), and asymmetric dimethylarginine (ADMA). A linear mixed model based on patient clusters was used to assess differences in biomarkers between the study groups and over time. RESULTS: The study population comprised 62 HIV-infected patients (group A, n = 31; group B, n = 31) and 22 controls. Age was 37 (30-43) years, and 81% were men. At baseline, the HIV-infected patients had a worse LDL particle phenotype and higher plasma concentration of sCD14, sCD163, hs-CRP, and LDL-Lp-PLA2 than the controls. At month 12, there was an increase in total cholesterol (p = 0.002), HDL-c (p = 0.003), and Apo A-I (p = 0.049) and a decrease in sCD14 (p = <0.001) and sCD163 (p<0.001), although only in group B. LDL particle size increased in group B at month 24 (p = 0.038). No changes were observed in group A or in the healthy controls. Common carotid intima-media thickness increased in HIV-infected patients at month 24 (Group A p = 0.053; group B p = 0.048). Plasma levels of sCD14, sCD163, and hs-CRP correlated with lipid values. CONCLUSIONS: In treatment-naive HIV-infected patients, initiation of c-ART was associated with an improvement in LDL particle phenotype and inflammatory/immune biomarkers, reaching values similar to those of the controls. HIV infection was associated with progression of carotid intima-media thickness.

Serum amyloid A does not affect high-density lipoprotein cholesterol measurement by a homogeneous assay.

BACKGROUND: Serum amyloid A (SAA), which is one of the acute phase proteins, alters the structure of HDL by associating with it during circulation. We focused on whether SAA influences the values of HDL-cholesterol (HDL-C) measurements when using a homogeneous assay. METHODS: HDLs were isolated by ultracentrifugation from serum samples of 248 patients that were stratified into three groups based on their serum SAA concentrations (low: SAA ≤ 8 µg/mL; middle: 8 < SAA ≤ 100 µg/mL; and high: SAA > 100 µg/mL). HDL-C concentrations of the serum samples measured by the homogeneous assay were compared with the total cholesterol concentrations of HDL fractions isolated by ultracentrifugation. RESULTS: HDLs obtained from patients with low SAA concentrations were separated into their general particle sizes and classified as HDL2 and HDL3 by native-gel electrophoresis. On the other hand, HDLs obtained from patients with high SAA concentrations occasionally showed distributions different from the typical sizes of HDL2 and HDL3, such as extremely small or large particles. Nevertheless, HDL-C concentrations measured using the homogeneous assay were strongly correlated with those measured using the ultracentrifugation method, regardless of the SAA concentrations. However, the ratios of HDL-C concentrations obtained by the homogeneous assay to those obtained by the ultracentrifugation method for patients with high SAA concentrations were significantly lower than those of patients with low SAA concentrations. CONCLUSIONS: A large amount of SAA attached to HDL altered the HDL particle size but did not essentially affect HDL-C measurement by homogeneous assay.

HIV disease, metabolic dysfunction and atherosclerosis: A three year prospective study.

HIV infection is known to be associated with cardiometabolic abnormalities; here we investigated the progression and causes of these abnormalities. Three groups of participants were recruited: HIV-negative subjects and two groups of treatment-naïve HIV-positive subjects, one group initiating antiretroviral treatment, the other remaining untreated. Intima-media thickness (cIMT) increased in HIV-positive untreated group compared to HIV-negative group, but treatment mitigated the difference. We found no increase in diabetes-related metabolic markers or in the level of inflammation in any of the groups. Total cholesterol, low density lipoprotein cholesterol and apoB levels were lower in HIV-positive groups, while triglyceride and Lp(a) levels did not differ between the groups. We found a statistically significant negative association between viral load and plasma levels of total cholesterol, LDL cholesterol, HDL cholesterol, apoA-I and apoB. HIV-positive patients had hypoalphalipoproteinemia at baseline, and we found a redistribution of sub-populations of high density lipoprotein (HDL) particles with increased proportion of smaller HDL in HIV-positive untreated patients, which may result from increased levels of plasma cholesteryl ester transfer protein in this group. HDL functionality declined in the HIV-negative and HIV-positive untreated groups, but not in HIV-positive treated group. We also found differences between HIV-positive and negative groups in plasma abundance of several microRNAs involved in lipid metabolism. Our data support a hypothesis that cardiometabolic abnormalities in HIV infection are caused by HIV and that antiretroviral treatment itself does not influence key cardiometabolic parameters, but mitigates those affected by HIV.

Inhibition of Neuroinflammation by AIBP: Spinal Effects upon Facilitated Pain States.

Apolipoprotein A-I binding protein (AIBP) reduces lipid raft abundance by augmenting the removal of excess cholesterol from the plasma membrane. Here, we report that AIBP prevents and reverses processes associated with neuroinflammatory-mediated spinal nociceptive processing. The mechanism involves AIBP binding to Toll-like receptor-4 (TLR4) and increased binding of AIBP to activated microglia, which mediates selective regulation of lipid rafts in inflammatory cells. AIBP-mediated lipid raft reductions downregulate LPS-induced TLR4 dimerization, inflammatory signaling, and expression of cytokines in microglia. In mice, intrathecal injections of AIBP reduce spinal myeloid cell lipid rafts, TLR4 dimerization, neuroinflammation, and glial activation. Intrathecal AIBP reverses established allodynia in mice in which pain states were induced by the chemotherapeutic cisplatin, intraplantar formalin, or intrathecal LPS, all of which are pro-nociceptive interventions known to be regulated by TLR4 signaling. These findings demonstrate a mechanism by which AIBP regulates neuroinflammation and suggest the therapeutic potential of AIBP in treating preexisting pain states.

Lipidomic dataset of plasma from patients infected with wild type and nef-deficient HIV-1 strain.

Previous in vitro and in vivo studies demonstrated that HIV protein nef plays a key role in impairing cellular and systemic cholesterol metabolism in HIV disease, but clinical support for these findings is lacking. Here we present the data of comparative lipidomic analysis (330 lipid species) of plasma samples from HIV-negative subjects, patients infected with WT HIV-1 strain and patients infected with nef-deficient strain of HIV-1. We determine which effects of HIV on plasma lipidome are explained by the presence of nef. The data can be used to evaluate cardiovascular risk in HIV disease and to assess the role of nef in HIV-induced disturbances in systemic lipid metabolism. The full impact of nef deficiency on lipid and lipoprotein metabolism in HIV-infected patients is presented in the accompanying study "Lipid Metabolism in Patients Infected with Nef-deficient HIV-1 Strain" [1].

Lipid metabolism in patients infected with Nef-deficient HIV-1 strain.

BACKGROUND: HIV protein Nef plays a key role in impairing cholesterol metabolism in both HIV infected and bystander cells. The existence of a small cohort of patients infected with Nef-deficient strain of HIV presented a unique opportunity to test the effect of Nef on lipid metabolism in a clinical setting. METHODS: Here we report the results of a study comparing six patients infected with Nef-deficient strain of HIV (ΔNefHIV) with six treatment-naïve patients infected with wild-type HIV (WT HIV). Lipoprotein profile, size and functionality of high density lipoprotein (HDL) particles as well as lipidomic and microRNA profiles of patient plasma were analyzed. RESULTS: We found that patients infected with ΔNefHIV had lower proportion of subjects with plasma HDL-C levels <1 mmol/l compared to patients infected with WT HIV. Furthermore, compared to a reference group of HIV-negative subjects, there was higher abundance of smaller under-lipidated HDL particles in plasma of patients infected with WT HIV, but not in those infected with ΔNefHIV. Lipidomic analysis of plasma revealed differences in abundance of phosphatidylserine and sphingolipids between patients infected with ΔNefHIV and WT HIV. MicroRNA profiling revealed that plasma abundance of 24 miRNAs, many of those involved in regulation of lipid metabolism, was differentially regulated by WT HIV and ΔNefHIV. CONCLUSION: Our findings are consistent with HIV protein Nef playing a significant role in pathogenesis of lipid-related metabolic complications of HIV disease.

Cytomegalovirus Restructures Lipid Rafts via a US28/CDC42-Mediated Pathway, Enhancing Cholesterol Efflux from Host Cells.

Cytomegalovirus (HCMV) contains cholesterol, but how HCMV interacts with host cholesterol metabolism is unknown. We found that, in human fibroblasts, HCMV infection increased the efflux of cellular cholesterol, despite reducing the abundance of ABCA1. Mechanistically, viral protein US28 was acting through CDC42, rearranging actin microfilaments, causing association of actin with lipid rafts, and leading to a dramatic change in the abundance and/or structure of lipid rafts. These changes displaced ABCA1 from the cell surface but created new binding sites for apolipoprotein A-I, resulting in enhanced cholesterol efflux. The changes also reduced the inflammatory response in macrophages. HCMV infection modified the host lipidome profile and expression of several genes and microRNAs involved in cholesterol metabolism. In mice, murine CMV infection elevated plasma triglycerides but did not affect the level and functionality of high-density lipoprotein. Thus, HCMV, through its protein US28, reorganizes lipid rafts and disturbs cell cholesterol metabolism.

The effect of HIV infection on atherosclerosis and lipoprotein metabolism: a one year prospective study.

OBJECTIVES: HIV infection is associated with dyslipidaemia and increased risk of cardiovascular disease. The effects of HIV infection and antiretroviral treatment on surrogate markers of atherosclerosis, and lipoprotein metabolism were evaluated in a 12 month prospective study. METHODS AND RESULTS: Treatment-naive HIV patients were recruited into one of three groups: untreated HIV infection not likely to require initiation of antiretroviral therapy (ART) for at least 12 months; initiating treatment with non nucleoside reverse transcriptase inhibitor-containing ART regimen and initiating treatment with protease inhibitor-containing ART regimen. The patients underwent assessment of carotid intima-media thickness (cIMT), pulse wave velocity (PWV), brachial flow-mediated dilation (FMD) and variables of plasma lipoprotein metabolism at baseline and 12 months. The findings were compared with published values for age and sex matched HIV-negative healthy subjects in a cross-sectional fashion. cIMT and FMD were lower while PWV was higher in HIV-patients compared with HIV-negative individuals; none of the markers changed significantly during 12 months follow up. HIV patients had hypoalphalipoproteinemia and elevated plasma levels of lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein. The only significant changes in lipid-related variables were elevation of total cholesterol and triglycerides in patients treated with PI-containing regimen and elevation of plasma LCAT levels in patients treated with NNRTI-containing regimen. The ability of whole and apoB-depleted plasma to effect cholesterol efflux was not impaired in all three groups. CONCLUSIONS: This study did not find evidence for rapid progression of subclinical atherosclerosis and deterioration of dyslipidaemia in HIV patients within 1 year.

Cholesterol efflux assay.

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare. Excessive cholesterol is more common problem. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required. The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.