Abundance Of Cholesterol Research Articles

Endometrial responsiveness to interferon-tau and its association with subsequent reproductive performance in dairy heifers

Our objectives were to evaluate the endometrial responsiveness of dairy heifers to an intrauterine infusion of recombinant bovine interferon-tau (rbIFN-τ) and to associate endometrial responses to rbIFN-τ with subsequent reproductive performance. In experiments 1 and 2, cyclic heifers were enrolled in a program for synchronization of the estrous cycle, and blood sampling and ultrasonography examinations were performed on d 0, 4, 7, 11, and 14 of the estrous cycle. In experiment 1, heifers were randomly assigned to receive an intrauterine infusion containing 2 µg of rbIFN-τ (rbIFN-τ = 19) or saline control (CTRL = 19) into the uterine horn ipsilateral to the corpus luteum (CL) on d 14 of the estrous cycle. Then, 6 hours after the infusion, the infused uterine horn was flushed for sampling of the uterine luminal fluid (ULF) for composition analysis, and the endometrium was biopsied for transcriptomics. In experiment 2, 100 heifers received an intrauterine infusion of rbIFN-τ, and the same procedures for uterine sample collection were performed as described in experiment 1. After the intrauterine test, heifers were enrolled in a breeding program and classified as highly fertile (HF; pregnant at first AI) or subfertile (SF; not pregnant at first AI). Statistical analyses were performed using regression models, which included the effects of treatment (experiment 1: CTRL vs. rbIFN-τ) or fertility group (experiment 2: HF vs. SF) and block of samples. Intrauterine infusion of rbIFN-τ increased the expression of classical interferon-stimulated genes in the endometrium (e.g., ISG15, MX1, OAS2, IRF9, and USP18), and an antiviral response was predicted to be the main downstream effect of the transcriptome changes. In addition, rbIFN-τ increased the abundance of cholesterol, glycerol, and the overall concentration of oxylipins in the ULF. Analysis of endometrial transcriptome between HF and SF heifers revealed important differences in the expression of genes associated with cell signaling, metabolism, attachment, and migration, with a large representation of genes encoding extracellular matrix proteins. In general, differentially expressed genes were expected to be downregulated by IFN-τ but seemed to fail to be downregulated in SF heifers, resulting in higher expression in SF compared with HF heifers. Subfertile heifers had lower concentrations of glycerol and an altered profile of oxylipins in the ULF, with a lower abundance of oxylipins derived from arachidonic acid and dihomo-γ-linolenic acid, and a greater abundance of oxylipins derived from linoleic acid. Measurements of ovarian function did not differ between groups and, therefore, did not influence the observed results in uterine biology. Overall, the endometrial responsiveness to IFN-τ is variable among individuals and associated with subsequent fertility of heifers, indicating that communication between conceptus and endometrium is critical for the uterine receptivity and survival of pregnancy.

Polyethylene microplastics induced lipidomic responses in Chironomus tepperi: A two-generational exploration

Microplastics (MPs) accumulating in freshwater sediment have raised concerns about potential risks to benthic dwelling organisms, yet few studies have examined the long-term impacts caused by MP exposure. This study investigated alterations to lipid profiles in an Australian freshwater invertebrate, Chironomus tepperi, induced by polyethylene MP fragments (1–45 μm) at environmentally relevant concentrations (125, 250, 500 and 1000 MPs/kg sediment), using a two-generational experimental design. In the parental generation, the relative abundance of triacylglycerols, total fatty acids and unsaturated fatty acids exhibited apparent hormetic patterns, with low-concentration stimulation and high-concentration inhibition observed. The overall trend in these lipid classes is consistent with previously observed changes to polar metabolite profiles, indicating that ingestion of MPs could inhibit nutrient assimilation from food leading to disruption of energy availability. In the first filial generation continuously exposed to MPs, however, abundance of cholesterol and total fatty acids increased with increasing exposure concentrations, suggesting different effects on energy metabolism between the parental generation and offspring. No differences in the lipidome were observed in first filial larvae that were not exposed, implying that MPs pose negligible carry-over effects. Overall, the combined results of this study together with a preceding metabolomics study provide evidence of a physical effect of MPs with subsequent impacts to bioenergetics. Nevertheless, future research is required to explore the potential long-term impacts caused by MPs, and to unravel the impacts of the surfactant control as a potential contributor to the observed hormetic response, particularly for studies exploring sub-lethal effects of MP exposure using sensitive omics techniques.

Dietary Cholesterol Differentially Regulates the Muscle Lipidomics of Farmed Turbot and Tiger Puffer.

Exogenous cholesterol has been supplemented into aqua-feeds due to the reduced proportions of fishmeal and fish oil. This study aimed to investigate the effects of dietary cholesterol supplementation on the muscle lipidomics of two marine fish species, turbot and tiger puffer. A 70-day feeding trial was conducted, where two low-fishmeal diets supplemented with 0 or 1% cholesterol were used. The lipidomic analysis with targeted tandem mass spectrometry showed that, in turbot, a total of 49 individual lipids exhibited significant differences in their abundance in response to dietary cholesterol, whereas the number was 30 for tiger puffer. Dietary cholesterol up-regulated the abundance of cholesterol and cholesterol ester in both species. In turbot, the dietary cholesterol also increased the abundance of triacylglycerol and acylcarnitine, whereas in tiger puffer, it primarily regulated the abundance of phospholipids and BMP. This was the first time the responses of marine fish muscle lipidomics to dietary cholesterol supplementation have been investigated.

Clinical strains of Mycobacterium tuberculosis exhibit differential lipid-metabolism associated transcriptome changes in in vitro cholesterol and infection models.

Many studies have identified host-derived lipids, characterized by the abundance of cholesterol as a major source of carbon nutrition for Mycobacterium tuberculosis (M. tb) during infection. Members of the Mycobacterium tuberculosis complex (MTBC) are biologically different with regards to degree of disease, host range, pathogenicity, and transmission. Therefore, the current study was aimed at elucidating transcriptome changes during early infection of pulmonary epithelial cells and on an in vitro cholesterol rich minimal media, in M. tb clinical strains F15/LAM4/KZN and Beijing, and the laboratory H37Rv strain. Infection of pulmonary epithelial cells elicited the upregulation fadD28 and hsaC in both the F15/LAM4/KZN and Beijing strains and the downregulation of several other lipid-associated genes. Growth curve analysis revealed F15/LAM4/KZN and Beijing to be slow growers in 7H9 medium and cholesterol-supplemented media. RNA-Seq analysis revealed strain-specific transcriptomic changes, thereby affecting different metabolic processes in an in vitro cholesterol model. The differential expression of these genes suggests that the genetically diverse M. tb clinical strains exhibit strain-specific behaviour that may influence their ability to metabolize lipids, specifically cholesterol, which may account for phenotypic differences observed during infection.

Relative Abundance of Lipid Metabolites in Spermatozoa across Three Compartments.

Male fertility, as manifest by the quantity and progressive motility of spermatozoa, is negatively impacted by obesity, dyslipidaemia and metabolic disease. However, the relative distribution of lipids in spermatozoa and the two compartments which supply lipids for spermatogenesis (seminal fluid and blood serum) has not been studied. We hypothesised that altered availability of lipids in blood serum and seminal fluid may affect the lipid composition and progressive motility of sperm. 60 men of age 35 years (median (range 20–45) and BMI 30.4 kg/m2 (24–36.5) under preliminary investigation for subfertility were recruited at an NHS clinic. Men provided samples of serum and semen, subject to strict acceptance criteria, for analysis of spermatozoa count and motility. Blood serum (n = 60), spermatozoa (n = 26) and seminal fluid (n = 60) were frozen for batch lipidomics analysis. Spermatozoa and seminal fluid had comparable lipid composition but showed marked differences with the serum lipidome. Spermatozoa demonstrated high abundance of ceramides, very-long-chain fatty acids (C20-22), and certain phospholipids (sphingomyelins, plasmalogens, phosphatidylethanolamines) with low abundance of phosphatidylcholines, cholesterol and triglycerides. Men with spermatozoa of low progressive motility had evidence of fewer concentration gradients for many lipid species between blood serum and spermatozoa compartments. Spermatozoa are abundant in multiple lipid species which are likely to contribute to key cellular functions. Lipid metabolism shows reduced regulation between compartments in men with spermatozoa with reduced progressive motility.

Cholesterol as a Second Messenger in the Hedgehog Signaling Pathway

The Hedgehog (HH) pathway is a cell-cell communication system essential for embryonic development; loss of proper HH signaling results in birth defects and cancer. Signaling is initiated when HH ligands are received on target cells by their receptor, Patched-1 (PTCH1). This relieves the inhibitory effect of PTCH1 on the G protein-coupled receptor Smoothened (SMO), allowing the HH signal to be transmitted across the membrane. The question of how PTCH1 inhibits SMO has remained a central mystery in developmental signaling for 25 years. Prior work implicated cholesterol as an endogenous lipid regulator of SMO. However, a major conundrum is presented by the abundance of cholesterol: how can a lipid that makes up 30%-50% of the plasma membrane be used to regulate a key signaling pathway? Using a custom CRISPR library focused on genes involved in lipid regulation, we asked the more general question of which lipids regulate HH signaling in target cells. We found that Sphingomyelin (SM) levels negatively regulate HH signaling. We show that SM exerts its effect by sequestering cholesterol into complexes, altering the chemical activity (or “accessibility”) of cholesterol. This highlights a key concept in membrane biology: only the accessible pool of cholesterol is available to interact with proteins and engage in signaling reactions. Using toxin-based sensors to measure accessible and sequestered cholesterol in cells, we find that HH ligands change cholesterol accessibility selectively in the membrane of the primary cilium. Primary cilia are antenna-like organelles required for HH signaling in all vertebrates. By compartmentalizing HH signaling in cilia, cells can use accessible cholesterol to communicate between PTCH1 and SMO without interfering with overall cellular cholesterol homeostasis. Our work suggests that the second messenger that communicates the signal between PTCH1 and SMO is the accessibility of cholesterol in a membrane compartment.

Nanosims Tools and Probes for Imaging Intracellular Structures

In eukaryotic cells, intracellular lipid membranes act as barriers that segregate specific chemical reactions into organelles for increased biochemical efficiency. Organelle efficiency is maintained by the regulation of cholesterol and different lipid distributions within these organelles and is tightly controlled by intracellular transport and lipid metabolism. Disturbances in intracellular lipid trafficking, biosynthesis and degradation are hallmarks of fatal diseases involving an accumulation of cholesterol or lipids in endosomes and lysosomes, such as Niemann Pick Type II disease. Efforts to elucidate the mechanisms for lipid-mediated cellular (dys)function would benefit from an imaging technique that enables assessing the relative abundances of cholesterol and lipid species of interest between organelles. Towards this aim, we developed a secondary ion mass spectrometry (SIMS) approach to image the sphingolipid and cholesterol distributions within intact cells in three dimensions with a NanoSIMS instrument. Because the components of interest must produce secondary ions that contain distinctive elements or isotopes to permit their detection with a NanoSIMS instrument, organelle-specific labels must be incorporated into the cells to enable identifying specific organelles. This work describes a strategy that utilizes small protein tags and functionalized substrates that permit identify organelles of interest with NanoSIMS, and the development of data processing tools that produce images of these organelles at the proper depth in the 3D SIMS images. We created a transfected cell line that stably expresses enzymatic proteins fused to organelle-specific proteins. These proteins covalently attach to functionalized small-molecule substrates which can be detected with NanoSIMS and fluorescence microscopy. By combining these tools for organelle detection with lipid-specific metabolic isotope incorporation, semi-quantitative information concerning the abundance of cholesterol or specific lipids within organelles of interest may be obtained.

Development of a Topographic Z-Correction Algorithm for 3D Depth Profiling Nanosims Images

Seasonal epidemics caused by the influenza virus, are responsible for thousands of deaths each year throughout the world. Anti-viral therapeutics that target the host cell components critical for its infectivity could reduce antiviral resistance. Cellular lipids such as sphingolipids and cholesterol are attractive targets because influenza a virus infectivity is sensitive to their abundance. Studies indicate the influenza a viral envelope is enriched with cholesterol and sphingolipids, and depletion of these components is detrimental to virus infectivity. Interestingly, influenza virus infection is also correlated with an accumulation of cholesterol within certain organelles in cells. The ability to produce intracellular three-dimensional images that show the abundance of cholesterol within distinct organelles could improve understanding of the role of intracellular cholesterol in influenza virus infectivity. Metabolically incorporated, rare stable isotope-labeled cholesterol and sphingolipids within intracellular compartments can be visualized by performing NanoSIMS imaging in a depth profiling mode. This depth profiling produces a series of NanoSIMS images that show the same location on the cell, but at increasing depth from its surface. Unfortunately, stacking the NanoSIMS images creates a three-dimensional (3D) image in which all the component-specific signals detected in a single scan are positioned at the same height. In contrast, the components that produced these ions were located at different distances from the substrate due to the cell's nonplanar topography. This poster describes an algorithm we developed that enables reshaping the 3D NanoSIMS images stacks to correct for sample topography in the absence of atomic force microscopy (AFM) data.

Cholesterol Pushes Vitamin E and Polyunsaturated Phospholipids Together

Vitamin E (α-tocopherol) is an essential micronutrient that protects membrane lipids from oxidative damage. Whether this lipid-soluble vitamin preferentially locates in the vicinity of polyunsaturated phospholipids that are most vulnerable to oxidation continues to be debated. We apply MD simulations to address this issue. A key role for cholesterol, ubiquitous in mammalian membranes, is revealed. Binding energies measured for vitamin E in all-atom (AA) umbrella sampling simulations are lower for polyunsaturated phospholipids than less unsaturated phospholipids - they do not indicate preferential interaction with polyunsaturated phospholipids. There is a reduction in the binding energy following the introduction of cholesterol, which we suggest drives vitamin E and polyunsaturated phospholipids together. Support is provided by largescale simulations performed over an extended timescale on vitamin E in mixtures of PDPC (1-palmitoyl-2-docosahexaenoylphosphatidylcholine) with SM (sphingomyelin) and cholesterol that separate into a (raft-like) domain enriched in SM and cholesterol (raft-like) and a (non-raft ) region composed primarily of PDPC (non-raft) but depleted in cholesterol. In coarse-grained (CG) and AA simulations vitamin E is excluded from the raft-like domain and thereby placed in closer proximity to polyunsaturated phospholipids. We surmise that the much greater abundance of cholesterol, which has high affinity for saturated SM, relative to vitamin E is responsible.

Glucosylated cholesterol in skin: Synthetic role of extracellular glucocerebrosidase

The existence of glucosylated cholesterol (GlcChol) in tissue has recently been recognized. GlcChol is generated from glucosylceramide (GlcCer) and cholesterol through transglucosylation by two retaining β-glucosidases, GBA and GBA2. Given the abundance of GBA, GlcCer and cholesterol in the skin’s stratum corneum (SC), we studied the occurrence of GlcChol.A significant amount of GlcChol was detected in SC (6 pmol/mg weight). The ratio GlcChol/GlcCer is higher in SC than epidermis, 0.083 and 0.011, respectively. Examination of GlcChol in patients with Netherton syndrome revealed comparable levels (11 pmol/mg).Concluding, GlcChol was identified as a novel component in SC and is likely locally metabolized by GBA. The physiological function of GlcChol in the SC warrants future investigation.

Cholesterol Acceptors Regulate the Lipidome of Macrophage Foam Cells.

Arterial foam cells are central players of atherogenesis. Cholesterol acceptors, apolipoprotein A-I (apoA-I) and high-density lipoprotein (HDL), take up cholesterol and phospholipids effluxed from foam cells into the circulation. Due to the high abundance of cholesterol in foam cells, most previous studies focused on apoA-I/HDL-mediated free cholesterol (FC) transport. However, recent lipidomics of human atherosclerotic plaques also identified that oxidized sterols (oxysterols) and non-sterol lipid species accumulate as atherogenesis progresses. While it is known that these lipids regulate expression of pro-inflammatory genes linked to plaque instability, how cholesterol acceptors impact the foam cell lipidome, particularly oxysterols and non-sterol lipids, remains unexplored. Using lipidomics analyses, we found cholesterol acceptors remodel foam cell lipidomes. Lipid subclass analyses revealed various oxysterols, sphingomyelins, and ceramides, species uniquely enriched in human plaques were significantly reduced by cholesterol acceptors, especially by apoA-I. These results indicate that the function of lipid-poor apoA-I is not limited to the efflux of cholesterol and phospholipids but suggest that apoA-I serves as a major regulator of the foam cell lipidome and might play an important role in reducing multiple lipid species involved in the pathogenesis of atherosclerosis.

Analysis of serum changes in response to a high fat high cholesterol diet challenge reveals metabolic biomarkers of atherosclerosis.

Atherosclerotic plaques are characterized by an accumulation of macrophages, lipids, smooth muscle cells, and fibroblasts, and, in advanced stages, necrotic debris within the arterial walls. Dietary habits such as high fat and high cholesterol (HFHC) consumption are known risk factors for atherosclerosis. However, the key metabolic contributors to diet-induced atherosclerosis are far from established. Herein, we investigate the role of a 2-year HFHC diet challenge in the metabolic changes of development and progression of atherosclerosis. We used a non-human primate (NHP) model (baboons, n = 60) fed a HFHC diet for two years and compared metabolomic profiles in serum from animals on baseline chow with serum collected after the challenge diet using two-dimensional gas chromatography time-of-flight mass-spectrometry (2D GC-ToF-MS) for untargeted metabolomic analysis, to quantify metabolites that contribute to atherosclerotic lesion formation. Further, clinical biomarkers associated with atherosclerosis, lipoprotein measures, fat indices, and arterial plaque formation (lesions) were quantified. Using two chemical derivatization (i.e., silylation) approaches, we quantified 321 metabolites belonging to 66 different metabolic pathways, which revealed significantly different metabolic profiles of HFHC diet and chow diet fed baboon sera. We found heritability of two important metabolites, lactic acid and asparagine, in the context of diet-induced metabolic changes. In addition, abundance of cholesterol, lactic acid, and asparagine were sex-dependent. Finally, 35 metabolites correlated (R2, 0.068–0.271, P < 0.05) with total lesion burden assessed in three arteries (aortic arch, common iliac artery, and descending aorta) which could serve as potential biomarkers pending further validation. This study demonstrates the feasibility of detecting sex-specific and heritable metabolites in NHPs with diet-induced atherosclerosis using untargeted metabolomics allowing understanding of atherosclerotic disease progression in humans.

Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane.

Cholesterol is an essential constituent of membranes in mammalian cells. The plasma membrane and the endocytic recycling compartment (ERC) are both highly enriched in cholesterol. The abundance and distribution of cholesterol among organelles are tightly controlled by a combination of mechanisms involving vesicular and nonvesicular sterol transport processes. Using the fluorescent cholesterol analogue dehydroergosterol, we examined sterol transport between the plasma membrane and the ERC using fluorescence recovery after photobleaching and a novel sterol efflux assay. We found that sterol transport between these organelles in a U2OS cell line has a t1/2 =12-15 min. Approximately 70% of sterol transport is ATP independent and therefore is nonvesicular. Increasing cellular cholesterol levels dramatically increases bidirectional transport rate constants, but decreases in cholesterol levels have only a modest effect. A soluble sterol transport protein, STARD4, accounts for ∼25% of total sterol transport and ∼33% of nonvesicular sterol transport between the plasma membrane and ERC. This study shows that nonvesicular sterol transport mechanisms and STARD4 in particular account for a large fraction of sterol transport between the plasma membrane and the ERC.

Biological Systems of Vitamin K: A Plasma Nutriproteomics Study of Subclinical Vitamin K Deficiency in 500 Nepalese Children.

Vitamin K (VK) is a fat-soluble vitamin whose deficiency disrupts coagulation and may disturb bone and cardiovascular health. However, the scale and systems affected by VK deficiency in pediatric populations remains unclear. We conducted a study of the plasma proteome of 500 Nepalese children 6–8 years of age (male/female ratio = 0.99) to identify proteins associated with VK status. We measured the concentrations of plasma lipids and protein induced by VK absence-II (PIVKA-II) and correlated relative abundance of proteins quantified by mass spectrometry with PIVKA-II. VK deficiency (PIVKA-II >2 μg/L) was associated with a higher abundance of low-density lipoproteins, total cholesterol, and triglyceride concentrations (p < 0.01). Among 978 proteins observed in >10% of the children, five proteins were associated with PIVKA-II and seven proteins were differentially abundant between VK deficient versus sufficient children, including coagulation factor-II, hemoglobin, and vascular endothelial cadherin, passing a false discovery rate (FDR) threshold of 10% (q < 0.10). Among 27 proteins associated with PIVKA-II or VK deficiency at a less stringent FDR (q < 0.20), a network comprised of hemoglobin subunits and erythrocyte anti-oxidative enzymes were highly and positively correlated each other (all r > 0.7). Untargeted proteomics offers a novel systems approach to elucidating biological processes of coagulation, vascularization, and erythrocyte oxidative stress related to VK status. The results may help elucidate subclinical metabolic disturbances related to VK deficiency in populations.

CYP46A1 inhibition, brain cholesterol accumulation and neurodegeneration pave the way for Alzheimer's disease.

Abnormalities in neuronal cholesterol homeostasis have been suspected or observed in several neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and Huntington's disease. However, it has not been demonstrated whether an increased abundance of cholesterol in neurons in vivo contributes to neurodegeneration. To address this issue, we used RNA interference methodology to inhibit the expression of cholesterol 24-hydroxylase, encoded by the Cyp46a1 gene, in the hippocampus of normal mice. Cholesterol 24-hydroxylase controls cholesterol efflux from the brain and thereby plays a major role in regulating brain cholesterol homeostasis. We used an adeno-associated virus vector encoding short hairpin RNA directed against the mouse Cyp46a1 mRNA to decrease the expression of the Cyp46a1 gene in hippocampal neurons of normal mice. This increased the cholesterol concentration in neurons, followed by cognitive deficits and hippocampal atrophy due to apoptotic neuronal death. Prior to neuronal death, the recruitment of the amyloid protein precursor to lipid rafts was enhanced leading to the production of β-C-terminal fragment and amyloid-β peptides. Abnormal phosphorylation of tau and endoplasmic reticulum stress were also observed. In the APP23 mouse model of Alzheimer's disease, the abundance of amyloid-β peptides increased following inhibition of Cyp46a1 expression, and neuronal death was more widespread than in normal mice. Altogether, these results suggest that increased amounts of neuronal cholesterol within the brain may contribute to inducing and/or aggravating Alzheimer's disease.

Colorimetric polymer assay for the diagnosis of plasma lipids atherogenic quality in hypercholesterolemic patients.

Hypercholesterolemia (increased blood cholesterol level) is considered a major risk factor for developing atherosclerotic diseases. As such, alerting individuals on hypercholesterolemic conditions is a crucial component in averting onset of atherosclerosis and its outcome-cardiovascular diseases. While common diagnostic tools such as cholesterol and lipoproteins determination are widely employed for hypercholesterolemia screening, their effectiveness has been questioned since they do not shed light on critical physiological factors like lipid oxidation and inflammation levels, which constitute prominent determinants for development of atherosclerotic diseases. The objective of this study is to develop a simple assay for identifying hypercholesterolemia, and assessing the impact of therapeutic treatments. We developed a diagnostic assay based upon color transformations of polydiacetylene, a unique conjugated polymer, upon interactions with blood plasma obtained from healthy individuals, hypercholesterolemic patients, hypercholesterolemic patients treated with statin, and hypercholesterolemic patients treated with statin together with pomegranate extracts. The color transformations of the polymer were monitored through desktop color scanning combined with colorimetric image analysis. We show that the colorimetric assay was able to distinguish among plasma. Bio-analytical characterization reveals that the distinct colorimetric responses likely arise from interactions with plasma lipoproteins. Importantly, the colorimetric changes are not simply correlated with the relative abundance of cholesterol (or other lipids) in the plasma of hypercholesterolemic or healthy patients, but also reflect the presence of oxidized and inflamed species. This paper introduces a simple color assay for detection of hypercholesterolemia and monitoring the effect of therapies directed at mitigating this physiological condition. The colorimetric system might constitute a novel platform for assessing patient vulnerability towards the development of atherosclerosis.

The lipid raft-bound alkaline phosphatase activity increases and the level of transcripts remains unaffected in liver of merosin-deficient LAMA2dy mouse

Alkaline phosphatase (AP) and other proteins add glycosylphosphatidylinositol (GPI) before addressing to raft domains of the cell membrane. Our previous report showing an increased density of lipid rafts in muscle of dystrophic Lama2dy mice prompted us to compare livers of normal (NL) and dystrophic mice (DL) for their levels of rafts. With this aim, hepatic rafts were isolated as Triton X-100 resistant membranes, and identified by their abundance of flotillin-2, alkaline phosphatase (AP) and other raft markers. The comparable abundance of cholesterol and flotillin-2 in rafts of NL and DL contrasted with the double AP activity both in rafts of DL and whole DL. The AP mRNA level was the same in NL and DL. Sedimentation analysis profiles revealed AP activity of NL distributed between dimeric (dAP) and monomeric AP (mAP), whose proportions and lectin-binding extent changed in DL. The increased AP activity and changed AP glycosylation in DL, the prevalence of mAP in NL and the enhanced stability of dAP in DL demonstrated the critical role that glycosylation and oligomerization play for AP catalysis. The higher AP activity of DL probably arises from dystrophy-associated changes in glycosyl transferases, which alter AP glycosylation and subunit folding with profitable effects for AP stability and catalysis.

Proteomic Analysis of Detergent-resistant Membrane Microdomains in Trophozoite Blood Stage of the Human Malaria Parasite Plasmodium falciparum

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum.

Emerging role of lymphatic vessels in reverse cholesterol transport

As a major constituent of cell membrane, cholesterol is essential for cellular homeostasis through the control of membrane fluidity and permeability. Abnormal levels of cholesterol can have important cellular consequences and may lead to diseases such as atherosclerosis and type II diabetes. Therefore, cells have developed complex mechanisms to regulate the abundance of intracellular cholesterol. Among these mechanisms, the process referred to as reverse cholesterol transport (RCT) has been shown to be critical for the removal of excess cholesterol from peripheral tissues and its transport to the liver via plasma for excretion [1]. RCT is initiated when cells including macrophages efflux out the excess of cholesterol to prevent intracellular over-accumulation. This step is tightly regulated through the action of ATP-binding cassette transporters ABCA1, ABCG1 and scavenger receptor class BI (SR-BI) and high density lipoprotein (HDL) which serves as the major acceptor for cellular cholesterol released in extrahepatic tissues [2]. Following cholesterol efflux, HDL-cholesterol is transported from the tissue back into the blood circulation. In the final step of RCT, the majority of HDL-derived cholesterol is removed from the liver by secretion into the bile [1, 2]. In contrast to the knowledge on the mechanisms by which cholesterol is effluxed out of the cells and its fate after reaching the liver, little information is available on how HDL-cholesterol travels from the peripheral tissues back to the blood circulation. Since one of the major roles of lymphatic vessels is to drain macromolecules from the interstitial space back to the circulation and lymph is rich in cholesterol and HDL [3], the implication of lymphatics in the transport of lipoproteins from interstitium to blood has been suspected. The recent studies by Martel et al., and Lim et al., now provide convincing evidence for a role of lymphatic vessels in RCT [4, 5]. Indeed, impairment of lymphatic drainage induced by surgical excision in wild-type mice [4, 5] or naturally occurring in mice lacking the expression of one allele of vascular endothelial growth factor-C receptor [4] significantly decreased the efficiency of RCT. Furthermore, Lim et al., showed that the transport of effluxed cholesterol from peripheral tissue to the blood circulation via lymphatics is not passive as anticipated but an active process dependent on HDL and SR-BI [5]. Therefore, these new findings establish that lymphatic vessels tightly regulate RCT through the active transport of HDL-cholesterol. Accumulation of cholesterol in tissues including arteries and skin is a hallmark of atherosclerosis and xanthomas, respectively, and these features are recapitulated in mouse models lacking apoE (apoE−/−) or LDL receptor which develop hypercholesterolemia. Interestingly, we showed previously that skin lymphatic vessels in these hypercholesterolemic mice exhibit structural defects which severely compromise their function [6]. These observations suggest that the accumulation of cholesterol in arteries and skin from hyper-cholesterolemic mice may result from poor lymphatic drainage. We now provide definitive evidence that restoring lymphatic function in apoE−/− mice ameliorates the clearance of cholesterol from skin by supporting RCT [5]. In strong agreement with these findings in skin, Martel et al., showed in an elegant surgical model of aorta transplantation that impeding lymphatic vessel growth interferes with the removal of cholesterol from the transplanted aorta [4]. Altogether, these findings reveal the potential atheroprotective role of lymphatic drainage by actively contributing to RCT. The relevance of these findings in humans is supported by clinical studies reporting a possible interconnection between lymphatic function and lipid accumulation in tissues (Figure ​(Figure1).1). Patients with familial combined hyperlipidemia, a genetic dyslipidemia, which share similar clinical and biochemical features with type II diabetes mellitus, obesity and the metabolic syndrome showed decreased expression of FOXC2 and Prox-1 genes in their adipose tissue [7]. Since these two genes regulate the structure and function of lymphatic vessels these findings support the hypothesis that defective lymphatic drainage may contribute to this lipid disorder. Massive lipid deposits are evident in the edematous tissues of patients with lymphedema and poor lymphatic drainage has been associated with the formation of xanthomas in humans [8]. However, it remains to be determined whether RCT is affected in patients with lymphedema. Interestingly, the use of compression garments to resolve lymphedema ameliorate xanthomatous eruptions in patients [8]. Therefore, promoting efficient lymphatic drainage in lipid-related diseases including atherosclerosis and diabetes whose prevalence will continue to augment as a result of increase in aging population may be an attractive therapeutic intervention that deserves future investigations. Figure 1 Lymphatic vessels are important for peripheral lipid clearance. Under normocholesterolemic conditions, functional lymphatic vessels regulate cholesterol transport from extra-hepatic tissues by contributing to RCT. Hypercholesterolemic conditions results ...

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging reveals cholesterol overload in the cerebral cortex of Alzheimer disease patients

Although cholesterol has been involved in the pathophysiology of Alzheimer disease (AD), its distribution in the cerebral cortex over the course of AD is unknown. We describe an original method to quantify cholesterol distribution using time-of-flight secondary ion mass spectrometry imaging. Cholesterol was unevenly distributed along the cortical thickness, being more abundant close to the white matter, in both control and AD cases. However, the mean cholesterol signal was significantly higher in the lower half of the cortex in AD samples compared to controls. This increase, when converted into cortical layers, was statistically significant for layers III and IV and did not reach significance in layers V + VI, the variability being too high at the interface between grey and white matter. The density of neurofibrillary tangles and of senile plaques was not statistically linked to the abundance of cholesterol. Cholesterol overload thus appears a new and independent alteration of AD cerebral cortex. The structure in which cholesterol accumulates and the mechanism of this accumulation remain to be elucidated.