Human Lipoprotein Research Articles

Abstract 229: Mucosal Immunity Shapes the Lipoprotein Small-RNA Fingerprint from Commensal, Dietary and Environmental Microbiota

Cardiovascular disease (CVD) is a leading cause of mortality in developed countries and is a frequent comorbidity of numerous metabolic and inflammatory diseases that demands more effective therapies. Dyslipidemias are a classical risk factor for CVD, but emerging alternative functions of lipoproteins have implicated them in novel narratives for the pathophysiology of many diseases that warrant further study. Our lab has identified functional, intercellular gene regulatory networks mediated by extracellular transport of microRNAs (miRNA) by lipoproteins. Here, we quantified the landscape of small RNAs (sRNA) on human and animal lipoproteins and discovered that most lipoprotein-sRNAs are derived from microorganisms of multiple kingdoms, primarily bacteria. Based on these observations, our over-arching hypothesis is that lipoprotein-sRNA signatures are shaped by the interface of host tissues with resident, environmental and dietary microbiota, and likely participate in unique gene regulation networks that contribute to complex (patho)physiological traits. To investigate this hypothesis, we developed a sRNA-sequencing analysis pipeline that identifies and quantifies both host and non-host sRNAs. Using this bioinformatic tool, we identified and validated a number of lipoprotein-sRNAs derived from bacteria that are similar in size to miRNAs with identical seed regions, termed Doppelganger (Dopl)-miRNAs. We hypothesized that mucosal immunity contributes to non-host sRNAs on lipoproteins, as mucosal linings are a primary interface between host tissues and microorganisms. To this end, we investigated the lipoprotein-sRNAs of mice lacking the polymeric immunoglobulin receptor (pIgR -/- ), which is devoid of polymeric IgA and IgM at mucosal linings and models human respiratory and intestinal diseases. We report a global increase in lipoprotein-miRNAs juxtaposed with a profound decrease in bacterial sRNA and Dopl-miRNAs from specific taxa. This work unfurls novel links between microbiota, mucosal immunity, and lipoprotein-sRNA gene networks and emphasizes the potential for novel nucleic-acid based therapeutics.

Abstract 556: Lipoprotein Remodeling and Cholesterol Exchange Monitored Using Fluorescent Lipids and Proteins

We developed a sensitive and robust in vitro method to monitor lipoprotein cholesterol and protein exchange and lipoprotein remodeling, using non-exchangeable fluorescent phosphatidylethanolamine (PE) as a lipoprotein marker. We applied this method to monitor the exchange of unesterified cholesterol (FC) and apoA-I among isolated human lipoproteins and synthetic lipoprotein-X (LpX). Fluorescent FC, but not PE, rapidly equilibrated between VLDL and HDL, and transferred almost entirely from VLDL or HDL to LDL. Fluorescent apoA-I bound specifically to HDL and remodeled fluorescent PE and FC-labeled LpX into a new lipoprotein particle that contained both fluorescent lipids and apoA-I. LpX-derived fluorescent PE incorporated into plasma HDL only. The incorporation of LpX-derived fluorescent FC into plasma lipoproteins was similar to fluorescent FC alone, consistent with remodeling of LpX to HDL with concomitant exchange of FC between lipoproteins. LPL remodeled fluorescent PE and FC-tagged VLDL into a new particle containing both fluorescent lipids and apoA-I. We also developed a model system to study lipid transfer in vitro and in vivo by depositing lipids on calcium silicate hydrate crystals to form dense lipid coated donor particles that are readily separated from acceptor membranes and can be used as a surrogate for cell-dependent cholesterol efflux. These methodologies can readily be applied to study the other members of the vast lipoprotein proteome and the wide variety of remodeling events involved in lipoprotein-mediated lipid homeostasis in health and disease.

Abstract 26: Identification of a Novel Gene, DENND5B, Associated With Plasma High Density Lipoprotein Levels and Intestinal Fat Absorption

In an effort to identify novel high density lipoprotein (HDL) receptors expressed by the liver, a bacteriophage library expressing liver cDNA fragments was used to screen for clones that bind to HDL. The gene DENND5B showed significant enrichment for HDL binding over 3 cycles of biopanning, with negative selection against low density lipoprotein binding. Although little is known about the recently described DENN-domain containing family of proteins, some members act as guanine nucleotide exchange factors involved in the activation of Rab proteins and membrane trafficking. To examine the influence of DENND5B on HDL physiology, a DENND5B knockout mouse was generated using a custom zinc finger nuclease. DENND5B -/- mice had decreased plasma total cholesterol (-30.9%, p < 0.0001) and phosphatidylcholine (-31.4%, p < 0.0001). When plasma was analyzed by size-exclusion chromatography, the lipid effects were attributed entirely to decreased HDL. When fed western diet for 4 months, DENND5B -/- mice showed significantly smaller increases in plasma lipids and body weight compared to wild type mice. After sacrifice, DENND5B -/- mice were found to have a distended small intestine that was whitish in color. To evaluate fat absorption, mice were given an oral gavage of vegetable oil (10 uL/gram body weight) and plasma lipids were measured at 0, 2, and 4 hours post-gavage. DENND5B -/- mice had significantly reduced plasma triglyceride (p < 0.05) and free fatty acids (p < 0.01) in response to oil gavage compared to wild type mice. To identify a possible role for this gene in human lipoprotein metabolism, we examined gene expression data from 5,458 participants in the Framingham Heart Study. Consistent with the mouse data, a significant negative relationship exists between DENND5B expression and plasma HDL-C (p = 4.82 x 10 -5 ). In summary, using a phage display screening approach, we have identified a novel gene associated with circulating HDL levels in mice and humans. Additionally, in a homozygous knockout mouse model, a defect in intestinal fat absorption is present. Future studies will be aimed at evaluating the influence of this gene on HDL function, fat absorption, and atherosclerotic disease burden.

Probing the Interactions of Cytotoxic [Pt(1S,2S-DACH)(5,6-dimethyl-1,10-phenanthroline)] and Its PtIV Derivatives with Human Serum.

The discrepancy between the in vitro cytotoxic results and the in vivo performance of Pt56MeSS prompted us to look into its interactions and those of its PtIV derivatives with human serum (HS), human serum albumin (HSA), lipoproteins, and serum-supplemented cell culture media. The PtII complex, Pt56MeSS, binds noncovalently and reversibly to slow-tumbling proteins in HS and in cell culture media and interacts through the phenanthroline group with HSA, with a Kd value of ∼1.5×10-6 m. All PtIV complexes were found to be stable toward reduction in HS, but those with axial carboxylate ligands, cct-[Pt(1S,2S-DACH)(5,6-dimethyl-1,10-phenantroline)(acetato)2 ](TFA)2 (Pt56MeSS(OAc)2 ) and cct-[Pt(1S,2S-DACH)(5,6-dimehtyl-1,10-phenantroline)(phenylbutyrato)2 ](TFA)2 (Pt56MeSS(PhB)2 ), were spontaneously reduced at pH 7 or higher in phosphate buffer, but not in Tris buffer (pH 8). HS also decreased the rate of reduction by ascorbate of the PtIV complexes relative to the reduction rates in phosphate buffer, suggesting that for this compound class, phosphate buffer is not a good model for HS.

Regulation of the human lipoprotein lipase gene by the forkhead box transcription factor FOXA2/HNF-3β in hepatic cells

Lipoprotein lipase (LPL) plays a major role in the hydrolysis of triglycerides (TG) from circulating TG-rich lipoproteins. The role of LPL in the liver has been controversial but recent studies in mice with liver LPL overexpression or deficiency have revealed important new roles of the enzyme in glucose and lipid metabolism. The objective of this study was to identify regulatory elements and factors that control the transcription of the human LPL gene in hepatocytes. Deletion analysis of the human LPL promoter revealed that the proximal region which harbors a binding site for the forkhead box transcription factor FOXA2/HNF-3β at position −47/−40 is important for its hepatic cell activity. Silencing of FOXA2 in HepG2 cells reduced the LPL mRNA and protein levels. Direct binding of FOXA2 to the novel binding site was established in vitro and ex vivo. Mutagenesis of the FOXA2 site reduced the basal activity and abolished the FOXA2-mediated transactivation of the LPL promoter. Ιnsulin decreased LPL mRNA levels in HepG2 cells and this was associated with phosphorylation of AKT and nuclear export of FOXA2. In summary, the data of the present study combined with previous findings on the role of FOXA2 in HDL metabolism and gluconeogenesis, suggest that FOXA2 is a key regulator of lipid and glucose homeostasis in the adult liver. Understanding the mechanisms by which FOXA2 exerts its functions in hepatocytes may open the way to novel therapeutic strategies for patients with metabolic diseases such as dyslipidemia, diabetes and the metabolic syndrome.

Analysis of stereoselective drug interactions with serum proteins by high-performance affinity chromatography: A historical perspective

The interactions of drugs with serum proteins are often stereoselective and can affect the distribution, activity, toxicity and rate of excretion of these drugs in the body. A number of approaches based on affinity chromatography, and particularly high-performance affinity chromatography (HPAC), have been used as tools to study these interactions. This review describes the general principles of affinity chromatography and HPAC as related to their use in drug binding studies. The types of serum agents that have been examined with these methods are also discussed, including human serum albumin, α1-acid glycoprotein, and lipoproteins. This is followed by a description of the various formats based on affinity chromatography and HPAC that have been used to investigate drug interactions with serum proteins and the historical development for each of these formats. Specific techniques that are discussed include zonal elution, frontal analysis, and kinetic methods such as those that make use of band-broadening measurements, peak decay analysis, or ultrafast affinity extraction.

Possible molecular basis for macromolecular antigen attachment to host cells: their immune complex with plasma antibodies have unoccupied binding sites enabling binding to smaller ligands

Macromolecules such as lipoprotein(a) and antigens of Streptococcus mutans that cause dental infections accumulate in perivascular cells in inflammatory vascular disorders though they do not bind host cells directly. Suspecting a role for cognate circulating antibodies in the molecular mechanisms of macromolecular antigen deposition in tissues we investigated the ligand binding properties of de novo immune complexes (IC) formed between these two entities. Negatively charged Streptococcus mutans antigens (NSMAg) from cultured bacteria and apo(a) subunit separated from human plasma lipoprotein (a) were used as antigens to interact with anti β-glucan (ABG) and anti-α-galactoside (anti-Gal) antibodies respectively of normal plasma. Binding of antigen to antibody was measured in terms of the enhancement of fluorescence of FITC-labeled antibody that accompanies antigen binding. Unoccupied binding sites on macromolecular antigen-antibody ICs were demonstrated by capturing them on smaller ligands immobilized on microplates. Biotin-labeled macromolecular antigens were detected in ICs using HRP-labeled avidin. While total Streptococcus mutans antigens and a synthetic glycoprotein in which an ABG-specific disaccharide (cellobiose) had been covalently attached were equal in avidity towards ABG, NSMAg was superior. When binding of antibody to plate-coated ligands was monitored using HRP-labeled anti-immunoglobulin preincubation of ABG or anti-Gal with respective macromolecular antigens significantly enhanced binding response whereas antibodies preincubated with small sugars were fully inhibited from binding, indicating that macromolecular antigen binding had activated the Fc part of antibodies and that the resulting ICs in turn bound to other ligands using binding sites in the antibody spared by the large antigens. Presence of biotinylated macromolecular antigen in IC bound to immobilized ligands was confirmed using avidin-HRP probe. Unoccupied binding sites in the antibodies involved may enable homing of macromolecule ICs on vascular cells possessing smaller ligands. This along with an activated Fc could enhance their inflammatory potential.

Electromagnetic Simulation for the Diagnosis of Lipoprotein Density in Human Blood, a Non-Invasive Approach

With the rise in prevalence of Type II diabetes throughout the world, an increasing need for a portable monitoring system for both blood glucose and lipoprotein concentrations is in demand. Recent work has led to non-invasive wearable devices for monitoring changes in blood glucose concentrations using electromagnetic (EM) waves. However, this still fall short as a means of monitoring cholesterol levels in diabetic patients. The EM study on human tissues emphasized here may also relate to the safety guidelines applied to cellular communications, power lines, and other EM applications. The specific absorption rate (SAR) for the power of the non-ionizing frequency must not exceed a threshold as it impacts DNA and can lead to cancerous tissues. In this study, we used COMSOL software for the investigation of the viability of using EM within the frequency range of 64 MHz-1 GHz as a means of monitoring the transmission properties of human blood and lipoprotein. In this approach, wave equations were solved within blood and lipoprotein boundaries. Research parameters, including frequency range, Power input (SAR), and lipoprotein densities, were investigated. The transmission properties, produced by the electrical and thermal characteristics of these physiological parameters, have led to proper diagnosis of lipoprotein density. Within the frequency range of 64 MHz to 1 GHz, and for a power range of 0.1 to 0.6 SAR, lipoprotein density from 1.00 g/mL to 1.20 g/mL was considered. A 2D model, with an antenna source that supplied the electromagnetic waves to human tissues, was created for the simulations. These were used for the study of the transmission properties of the EM energy into the blood and lipoprotein tissues. While the range of magnetic flux values between simulations varies only slightly or not at all, the distribution of these values is impacted by given parameters. As such, a device capable of comparing magnetic flux values and penetration depths could easily distinguish between samples of different lipoprotein densities. The results obtained in this study can be accommodated non-invasively by human tissues, and can be produced in a practical model using wearable devices. A practical model is proposed for future consideration.

Transcriptome analyses unveils the unique requirement for human lipoproteins for optimal ex-vivo expansion of cultured red blood cells for transfusion.

Cultured red blood cells (cRBCs) generated from discarded stem cell sources are being considered as alternative transfusion products. We developed a humanized media (HEMAdef) composed of clinical grade components and dexamethasone (Dex) that sustain amplifications of cRBCs from stem cells discarded from regular blood donations in numbers sufficient for transfusion [1]. These cells, however, have an altered morphology of their plasma membrane which suggests that may reduce their survival in vivo. Since ~50% of the cRBC mass is constituted by lipid, we hypothesized that the plasma membrane abnormalities were caused by insufficient lipid supply. cRBCs produce their plasma membranes starting from lipids supplemented by the media and through a dedicated biosynthetic pathway, well known to be affected by Dex. Comparison of the expression profiling of cRBCs generated with/without Dex [2] identified remarkable similarities in gene expression. The majority of the differences were however detected in genes involved in lipid metabolism. In particular genes involved in lipid synthesis (GPAM and PRKACB) and efflux/degradation (HMGCL and ABCA1) were respectively down and up-regulated in cRBCs obtained with Dex, suggesting that in cultures with Dex cRBCs are exquisitely dependent on exogenous lipids for their membrane biosynthesis. This hypothesis led us to optimize the lipid formulation of HEMAdef by replacing the synthetic liposomes with lipoproteins purified from human plasma which represents the natural carriers for delivering lipids to the cells. These experiments compared the levels of amplification/maturation of cRBCs in HEMAdef supplemented with either synthetic liposomes or the total lipoproteins contained in human plasma (TLP) or its high density (HDL), low density (LDL) and very low density (VLDL) lipoprotein fraction. Addition of LDL and VLDL both increased by 3-2-fold the numbers of cRBCs generated in HEMAdef. TPLF has modest effects on the number of cRBCs generated in HEMAdef but drastically reduced the frequency of cRBCs with membrane abnormalities. More importantly, addition of TLP increased both the number (by 2-3-fold) and the membrane quality of cRBCs generated in HEMAdef. These results confirm the importance of an appropriate lipid supply for correct generation of RBC and identify culture conditions which assure maximal expansion of morphologically normal cRBCs for transfusion.

Investigation of cardiotoxic effect of A-1 apo + D actinomycin complex on isolated rat heart

Aim. Chemotherapy is one of the main methods of treating malformations. However, this technique causes a great deal of side effects, among which are complications related to cardiac toxicity. Development of new anticancer drugs is directly related to a decrease in the general toxic effect on the organism and the cardiovascular system in particular. The aim of the study was to investigate the capability of apolipoprotein A-1 together with an anticancer drug, actinomycin D, to induce functional disturbances in the performance of an isolated rat heart and to compare the effect with that of pure actinomycin D. Methods. For experiments use was made of Wistar male rats weighing 220-250 g. A modified Krebbs-Henseleit buffer saturated with carbogene (95 % O2 and 5 % CO2, solution temperature 37.5 °C) was used as perfusate. Experiments were conducted on isolated rat hearts. The isolated rat hearts were perfused retrogradely by a standard procedure, with isovolumic pressure registered in the left ventricle. Apolipoprotein A-I was isolated of human high-density lipoprotein by delipidating followed by separation of the proteins mix by means of column chromatography. In the experiment, apolipoprotein A-I- was mixed with actinomycin D, with concentration of the anticancer drug in the complex being 0.1 µg/ml.Results. Actinomycin D with concentration 1 µg/ml aggravates the functional indicators of the heart and enhances energy load on the myocardium. Decreasing the concentration of cytostatic down to 0.1 µg/ml of perfusate brings the indicators to the baseline values. Apolipoprotein A-I combined with actinomycin D offsets the changes introduced by the cytostatic without a carrier. The complex improves hemodynamics, which can be interpreted by the presence of a protein component.Conclusion. An isolated rat heart model shows that apolipoprotein A-I in combination with 0.1 µg/ml actinomycin D does not cause cardio toxicity. The complex improves myocardial efficiency through the use of the protein component as a carrier of the anticancer drug.Received 15 November 2016. Accepted 30 November 2016.Funding: The research was supported by the federal budget project implemented by Research Institute of Biochemistry, Reg. R-085.Conflict of interest: The authors declare no conflict of interest.Author contributionsConceptualization and study design: Kolpakov AR. Сбор и обработка материала: Knyazev R.A., Trifonova N.V.Statistical data processing: Knyazev R.A.Article writing: Knyazev R.A.Review & editing: Polyakov L.M.

Phenotypic Characterization of Genetically Lowered Human Lipoprotein(a) Levels

BackgroundGenomic analyses have suggested that the LPA gene and its associated plasma biomarker, lipoprotein(a) (Lp[a]), represent a causal risk factor for coronary heart disease (CHD). As such, lowering Lp(a) levels has emerged as a therapeutic strategy. Beyond target identification, human genetics may contribute to the development of new therapies by defining the full spectrum of beneficial and adverse consequences and by developing a dose–response curve of target perturbation. ObjectivesThe goal of this study was to establish the full phenotypic impact of LPA gene variation and to estimate a dose–response curve between genetically altered plasma Lp(a) and risk for CHD. MethodsWe leveraged genetic variants at the LPA gene from 3 data sources: individual-level data from 112,338 participants in the U.K. Biobank; summary association results from large-scale genome-wide association studies; and LPA gene sequencing results from case subjects with CHD and control subjects free of CHD. ResultsOne SD genetically lowered Lp(a) level was associated with a 29% lower risk of CHD (odds ratio [OR]: 0.71; 95% confidence interval [CI]: 0.69 to 0.73), a 31% lower risk of peripheral vascular disease (OR: 0.69; 95% CI: 0.59 to 0.80), a 13% lower risk of stroke (OR: 0.87; 95% CI: 0.79 to 0.96), a 17% lower risk of heart failure (OR: 0.83; 95% CI: 0.73 to 0.94), and a 37% lower risk of aortic stenosis (OR: 0.63; 95% CI: 0.47 to 0.83). We observed no association with 31 other disorders, including type 2 diabetes and cancer. Variants that led to gain of LPA gene function increased the risk for CHD, whereas those that led to loss of gene function reduced the CHD risk. ConclusionsBeyond CHD, genetically lowered Lp(a) levels are associated with a lower risk of peripheral vascular disease, stroke, heart failure, and aortic stenosis. As such, pharmacological lowering of plasma Lp(a) may influence a range of atherosclerosis-related diseases.

Significance of Lipid-Free and Lipid-Associated ApoA-I in Cellular Cho-lesterol Efflux.

The structure and stability of apolipoprotein (apo)A-I, the major apolipoprotein of human plasma high-density lipoproteins (HDL), determine the efficiency of the protein in the process of HDL generation and affect HDL properties in binding and exchanging its constituents, thus playing an essential role in reverse cholesterol transport. The equilibrium stability of an apoA-I molecule at the lipid interface (12.7 kcal/mol) predicted by a thermodynamic cycle for apolipoprotein folding-unfolding in water and at interface, largely exceeds apoA-I helix stability in HDL against chemical denaturation (3-5 kcal/mol). An ensemble of structures of lipid-bound apoA-I with different stabilities is assumed to exist. The conformational transitions between apoA-I conformers in water and lipid phases correspond to Lumry-Eyring model OL ⇔ CL ⇒ MW, where OL and CL are open and closed structures of HDLbound apoA-I, and MW is the molten globule in water. The model includes the reversible foldingunfolding transitions of N- and C-domains at HDL interface and apolipoprotein irreversible dissociation. We gathered published data on cholesterol efflux for apoA-I proteins with missense mutations in C-domain and calculated the stability of these mutants as a change of free energy relative to a wild type protein. Significant negative correlation was found between this stability and the efficiency of cAMP-stimulated cholesterol efflux. Thus, besides the known role of C-domain hydrophobicity, structure-destabilizing changes may significantly contribute to ABCA1-mediated cholesterol efflux by free apolipoprotein.

Neo High-Density Lipoprotein Produced by the Streptococcal Serum Opacity Factor Activity against Human High-Density Lipoproteins Is Hepatically Removed via Dual Mechanisms

Injection of streptococcal serum opacity factor (SOF) into mice reduces the plasma cholesterol level by ∼40%. In vitro, SOF converts high-density lipoproteins (HDLs) into multiple products, including a small HDL, neo HDL. In vitro, neo HDL accounts for ∼60% of the protein mass of the SOF reaction products; in vivo, the accumulated mass of neo HDL is <1% of that observed in vitro. To identify the underlying cause of this difference, we determined the fate of neo HDL in plasma in vitro and in vivo. Following incubation with HDL, neo HDL-PC rapidly transfers to HDL, giving a small remnant, which fuses with HDL. An increased level of SR-B1 expression in Huh7 hepatoma cells and a reduced level of LDLR expression in CHO cells had little effect on neo HDL-[3H]CE uptake. Thus, the dominant receptors for neo HDL uptake are not LDLR or SR-B1. The in vivo metabolic fates of neo HDL-[3H]CE and HDL-[3H]CE were different. Thirty minutes after the injection of neo HDL-[3H]CE and HDL-[3H]CE into mice, plasma [3H]CE counts were 40 and 53%, respectively, of injected counts, with 10 times more [3H]CE appearing in the livers of neo HDL-[3H]CE-injected than in those of HDL-[3H]CE-injected mice. These data support a model of neo HDL-[3H]CE clearance by two parallel pathways. At early post-neo HDL-[3H]CE injection times, some neo HDL is directly removed by the liver; the remainder transfers its PC to HDL, leaving a remnant that fuses with HDL, which is also hepatically removed more slowly. Given that SR-B1 and SOF both remove CE from HDL, this novel mechanism may also underlie the metabolism of remnants released by hepatocytes following selective SR-B1-mediated uptake of HDL-CE.

Mechanisms of selective delivery of xanthophylls to retinal pigment epithelial cells by human lipoproteins

The xanthophylls, lutein and zeaxanthin, are dietary carotenoids that selectively accumulate in the macula of the eye providing protection against age-related macular degeneration. To reach the macula, carotenoids cross the retinal pigment epithelium (RPE). Xanthophylls and β-carotene mostly associate with HDL and LDL, respectively. HDL binds to cells via a scavenger receptor class B1 (SR-B1)-dependent mechanism, while LDL binds via the LDL receptor. Using an in-vitro, human RPE cell model (ARPE-19), we studied the mechanisms of carotenoid uptake into the RPE by evaluating kinetics of cell uptake when delivered in serum or isolated LDL or HDL. For lutein and β-carotene, LDL delivery resulted in the highest rates and extents of uptake. In contrast, HDL was more effective in delivering zeaxanthin and meso-zeaxanthin leading to the highest rates and extents of uptake of all four carotenoids. Inhibitors of SR-B1 suppressed zeaxanthin delivery via HDL. Results show a selective HDL-mediated uptake of zeaxanthin and meso-zeaxanthin via SR-B1 and a LDL-mediated uptake of lutein. This demonstrates a plausible mechanism for the selective accumulation of zeaxanthin greater than lutein and xanthophylls over β-carotene in the retina. We found no evidence of xanthophyll metabolism to apocarotenoids or lutein conversion to meso-zeaxanthin.

Orlistat response to missense mutations in lipoprotein lipase.

The human lipoprotein lipase (LPL) is a therapeutic target for obesity, and inhibition of LPL with the approved small molecule agent orlistat has been widely used in clinic to treat obesity-related health problems such as diabetes and cardiovascular diseases. However, a variety of missense mutations in LPL protein have been observed, which may cause resistance or sensitization for orlistat, largely limiting the clinical applications of orlistat in obesity therapy. Here, we integrated molecular dynamics simulations and enzyme inhibition to investigate orlistat response to 16 disorder-associated missense mutations in LPL catalytic domain. It was found that most mutations have a modest effect on orlistat binding, and only few can exert strong impact to the binding. Three unfavorable (Trp86Arg, Ile194Thr, and Glu242Lys) and two favorable (His136Arg and Gly188Glu) mutations were identified, which can alter the binding affinity and inhibitory activity of orlistat considerably. Structural and energetic analysis revealed that these potent mutations induce orlistat resistance and sensitization by directly influencing the intermolecular interaction between LPL and orlistat or by indirectly addressing allosteric effect on LPL structure.

Development of Monoclonal Antibody Against Chlorinated 192tyrosine Containing ApoAI Peptide to Screen Quality of Human High Density Lipoprotein (HDL).

ApoAI, a major protein component of HDL is considered to be a key factor which contributes for HDL's cardio- protective functions. ApoAI is sensitive to myeloperoxidase mediated oxidative modifications under chronic inflammatory conditions such as atherosclerosis. The amino acid tyrosine at position 192 of ApoAI is considered to be one of the vulnerable sites for oxidation which impairs HDL functions and its quality. The presence of oxidized ApoAI in plasma may serve as a useful indicator of CVD risks in humans, but its detection in the clinical settings requires monoclonal antibodies (mAbs) with high specificity. In this study, we have developed mAbs against chloro- tyrosine at position 192 of ApoAI. We designed a small synthetic peptide of 7 amino acids length containing modified tyrosine residue (189-LAE-3-Cl-Y-HAK-195) based on antigenicity prediction. We coupled this peptide to Keyhole Limpet Hemocyanin (KLH) for generating mAbs. Hybridoma clones, KLH-ApoAI-F2 and KLH-ApoAI-H9, were found to be highly specific to chloro-192Tyr containing peptide of ApoAI and not to either nitro-192Tyr containing or to chloro-166Tyr and nitro-166Tyr containing peptides of ApoAI. The utility of these mAbs for screening the quality of HDL as an indicator of CVD risks in humans is discussed.

Acylation of lysine residues in human plasma high density lipoprotein increases stability and plasma clearance in vivo

Although human plasma high density lipoproteins (HDL) concentrations negatively correlate with atherosclerotic cardiovascular disease, underlying mechanisms are unknown. Thus, there is continued interest in HDL structure and functionality. Numerous plasma factors disrupt HDL structure while inducing the release of lipid free apolipoprotein (apo) AI. Given that HDL is an unstable particle residing in a kinetic trap, we tested whether HDL could be stabilized by acylation with acetyl and hexanoyl anhydrides, giving AcHDL and HexHDL respectively. Lysine analysis with fluorescamine showed that AcHDL and HexHDL respectively contained 11 acetyl and 19 hexanoyl groups. Tests with biological and physicochemical perturbants showed that HexHDL was more stable than HDL to perturbant-induced lipid free apo AI formation. Like the reaction of streptococcal serum opacity factor against HDL, the interaction of HDL with its receptor, scavenger receptor class B member 1 (SR-B1), removes CE from HDL. Thus, we tested and validated the hypothesis that selective uptake of HexHDL-[3H]CE by Chinese Hamster Ovary cells expressing SR-B1 is less than that of HDL-[3H]CE; thus, selective SR-B1 uptake of HDL-CE depends on HDL instability. However, in mice, plasma clearance, hepatic uptake and sterol secretion into bile were faster from HexHDL-[3H]CE than from HDL-[3H]CE. Collectively, our data show that acylation increases HDL stability and that the reaction of plasma factors with HDL and SR-B1-mediated uptake are reduced by increased HDL stability. In vivo data suggest that HexHDL promotes charge-dependent reverse cholesterol transport, by a mechanism that increases hepatic sterol uptake via non SR-B1 receptors, thereby increasing bile acid output.

The role of ANGPTL3 in human lipoprotein metabolism

The role of ANGPTL3 in human lipoprotein metabolism

High intake of sodium chloride exacerbates degradation of human lipoproteins and impairment of reproductive capacity via testicular toxicity in zebrafish

High intake of sodium chloride exacerbates degradation of human lipoproteins and impairment of reproductive capacity via testicular toxicity in zebrafish

Short-term cold exposure modulates human VLDL and HDL metabolism

Objectives: Upon cold activation, brown adipose tissue (BAT) combusts intracellular triglycerides (TG) into heat. Mouse studies showed that BAT consequently internalizes VLDL-TG-derived fatty acids to replenish intracellular TG stores (Hoeke, Circ Res 2016). Since the effect of cold-induced BAT activation on human lipoprotein metabolism remained elusive, we now assessed the effect of short-term mild cold exposure on the lipoprotein profile in men.