Lecithin:cholesterol Acyltransferase Activity Research Articles

Myrtenal alleviates hyperglycaemia, hyperlipidaemia and improves pancreatic insulin level in STZ-induced diabetic rats

Context: Myrtenal is monoterpene a constituent of essential oils found mainly in herbs such as mint, pepper, cumin, etc. It exerts admirable pharmacological activities against many diseases including diabetes. Hyperlipidaemia is a secondary complication of diabetes and also a major risk factor for cardiovascular diseases.Objective: The present study investigated the possible antihyperlipidaemic efficacy of myrtenal on plasma glucose, pancreatic insulin, plasma and tissue lipid levels in streptozotocin (STZ) induced diabetic rats.Materials and methods: Diabetes was induced in male Wistar rats by a single intraperitoneal injection of STZ (40 mg/kg b.w.). Myrtenal (80 mg/kg) was administered orally to diabetic rats for a period of 28 d. Plasma glucose, pancreatic insulin, TC, TGs, FFAs, PLs, LDL-C, HDL-C, VLDL, atherogenic index, (HMG-CoA) reductase, LPL, LCAT and liver histology were analyzed.Results: Diabetic rats showed significantly (p < 0.05) increased plasma glucose (273.18 mg/dL), total cholesterol (142 mg/dL), triglycerides (126 mg/dL), free fatty acids (118 mg/dL), phospholipids (153 mg/dL), low-density lipoprotein (88.07 mg/dL), very low-density lipoprotein (25.2 mg/dL), atherogenic index, whereas a decrease in the levels of pancreatic insulin (97.48 ng/mg) and high-density lipoprotein (29.12 mg/dL). In addition, the activity of 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase (0.94 HMG-CoA ratio/(mevalonate) increased significantly in contrast to the activities of lipoprotein lipase (4.87 μmoles of glycerol liberated/h/L) and lecithin cholesterol acyltransferase (54.61 μmoles of cholesterol esterified/h/L) in diabetic rats. Treatment with myrtenal significantly (p < 0.05) improved the levels of plasma glucose, pancreatic insulin and lipid profiles. Moreover, the histopathological analysis of liver was also in agreement with the biochemical findings.Discussion and conclusions: The present study indicates that myrtenal possess antihyperglycaemic and antihyperlipidemic properties, and could potentially be a useful phytochemical in treating diabetes.

Abstract 226: Mechanism of LCAT Activation by Compound A

Background: Lecithin:cholesterol acyltransferase (LCAT) catalyzes cholesteryl ester (CE) production from free cholesterol (FC) and phosphatidylcholine (lecithin), promoting HDL formation. Objective: To investigate activation of LCAT by Compound A (Amgen), a previously described small-molecule activator of LCAT, with the ultimate goal of developing novel LCAT activators for therapeutic use. Methods: LCAT activity in plasma from Familial LCAT Deficiency (FLD) patients with different mutations was quantitated by TLC before and after addition of Compound A. HEK293 cells were transiently transfected with plasmids containing wild-type (WT) or mutant LCAT cDNA. Media was then incubated with either vehicle or Compound A and LCAT activity was quantitated using a novel plate assay utilizing Methylumbelliferyl Palmitate as a substrate. Results: Compound A increased LCAT activity for a subset of FLD mutations to a level above which renal disease may occur. Mutations of Cys31 in vitro strongly affected basal LCAT activity as well as activation by Compound A. Charged residues at position 31 profoundly decreased activity whereas bulky hydrophobic groups increased LCAT activity up to 3-fold (p &lt; 0.005, all). Mass spectrometry of WT LCAT incubated with Compound A revealed a +103.017 m/z adduct to the tryptic peptide containing Cys31, indicative of a cyanopyrazine adduct to LCAT Cys31. Molecular modeling identified potential binding sites of Compound A to LCAT. Conclusions: Our findings yield important mechanistic insight into LCAT activation that can be used to design novel LCAT activators for therapeutic use.

Abstract 223: Optimization of Apolipoprotein A-1 Mimetic Peptides for Lipid Binding and Lecithin-cholesterol Acyltransferase Activation

Introduction: Apolipoprotein A-I (apoA-I) mimetic peptides present an exciting avenue in the clinical development of synthetic HDL (sHDL), offering a more feasible and economical approach in sHDL production. One such example is ETC-642, or 22A—a 22 amino acid ApoA-I mimetic peptide tested in single and multiple dose clinical trials. The goal of this study is to understand how specific amino acid substitutions affect physico-chemical properties of 22-mer peptides and correlate them with the ability to form stable nano-sized sHDL particles and promote LCAT activity. Methods/Results: For this study, we used the 22A peptide (PVLDLFNELLNELLEALKQKLK or ETC-642) and designed 10 variants comprised of a single amino acid substitution, denoted 22A-1-10. We used computational programs FMAP (Folding of Membrane-Associated Peptides), PPM2.0 and FRAMEWORK to select peptides that are predicted to form α-helices, have good membrane penetration (&gt;7.8 Å) with low transfer energy to membranes (ΔG transfer &lt;-16.1 kcal/mol), and form stable α-helices (ΔG helix &lt;-23.5 kcal/mol) with strong intermolecular LCAT interactions. The calculated ΔG helix varied from -18.4 to -24.4 kcal/mol, and G transfer varied from -11.1 to -17.2 kcal/mol for the synthesized peptides. The percent peptide α-helicity in lipid bound form was measured by circular dichroism spectroscopy and was found to vary dramatically between 19 and 82%. The lipid solubilization kinetics were determined using UV absorbance to measure turbidity. The particle size and stability of resulting HDL particles were measured by dynamic light scattering and gel permeation chromatography. The peptides with low predicted energy of lipid binding exhibited high helicity and formed stable HDL particles with sizes less than 20 nm. The HDLs formed from apoA-I peptides with high helicity were also able to activate LCAT and facilitate sterol esterification with a K cat of 5 to 30 min -1 . Conclusions: Our data shows that that minimalistic changes in primary structure of ApoA-1 peptide can lead significant changes in physico-chemical properties. Peptides with degree of α-helicity exhibited superior lipid binding and formed stable HDL particles of small size able to interact with LCAT.

Structural and Biochemical Studies on the Activation Mechanism of Lecithin:Cholesterol Acyltransferase

Coronary heart disease (CHD) is the leading cause of death in the United States and is strongly associated with low levels of high density lipoprotein‐cholesterol (HDL‐C), thus there is significant clinical interest in methods that raise HDL‐C in order to treat and prevent CHD. Lecithin:cholesterol acyltransferase (LCAT) esterifies cholesterol on high density lipoprotein (HDL), which promotes HDL maturation and reverse cholesterol transport, the process by which cholesterol is transported from arterial plaques to the liver. Mutations in LCAT are known to cause two genetic diseases. Fish eye disease (FED) is characterized by partial loss of LCAT activity, whereas familial LCAT deficiency (FLD) is characterized by complete loss of LCAT. Individuals with either FED or FLD have low HDL‐C levels and corneal opacities, while FLD patients ultimately develop renal failure. Consequently, administration of recombinant LCAT and LCAT‐activating compounds are being investigated as treatments to increase HDL in both CHD and FLD, and have already shown promise in animal models. However, improvement of these therapeutics is limited due to a lack of structural information on LCAT and its interactions with HDL. LCAT is 50% identical to lysosomal phospholipase A2 (LPLA2). We previously solved a 1.84 Å structure of LPLA2, as well as a low‐resolution structure of LCAT (8.7 Å), both of which are in an active “lid open” conformation. In addition, structures of LPLA2 bound to inhibitors that mimic substrate binding led to models for how LPLA2 and LCAT bind to phospholipids and subsequently transfer an acyl group to the appropriate acceptor. Here we present a new “closed” crystal structure of LCAT at 3.3 Å wherein a retractable lid appears to block substrate access to the active site. Interestingly, the lid interacts with residues on the catalytic core that are mutated in LCAT genetic disease and are hypothesized to be important for activation by apolipoprotein A‐I (ApoA‐I), the major protein component of HDL particles. We therefore hypothesized that ApoA‐I binding will compete with the lid and thereby stimulate LCAT activity. We have probed the role of the lid by examining disease‐causing and lid mutations for esterase activity on a soluble substrate, binding to synthetic HDLs, and transacylase activity on HDLs. Overall, our results confirm the importance of the retractable lid and its observed intramolecular contacts in proper HDL binding and cholesterol esterification. Overall, these studies are expected to guide the design of more potent small molecule LCAT activators as well as more catalytically efficient recombinant LCAT that could be used for enzyme replacement therapy.Support or Funding InformationThis work was supported by US National Institutes of Health (NIH) grant F037815, an American Heart Association (AHA) Postdoctoral Fellowship 15POST24870001 (K.A.M.), and a University of Michigan Chemical Biology Postdoctoral Fellowship (A.G.).

A Systematic Investigation of Structure/Function Requirements for the Apolipoprotein A-I/Lecithin Cholesterol Acyltransferase Interaction Loop of High-density Lipoprotein

The interaction of lecithin-cholesterol acyltransferase (LCAT) with apolipoprotein A-I (apoA-I) plays a critical role in high-density lipoprotein (HDL) maturation. We previously identified a highly solvent-exposed apoA-I loop domain (Leu(159)-Leu(170)) in nascent HDL, the so-called "solar flare" (SF) region, and proposed that it serves as an LCAT docking site (Wu, Z., Wagner, M. A., Zheng, L., Parks, J. S., Shy, J. M., 3rd, Smith, J. D., Gogonea, V., and Hazen, S. L. (2007) Nat. Struct. Mol. Biol. 14, 861-868). The stability and role of the SF domain of apoA-I in supporting HDL binding and activation of LCAT are debated. Here we show by site-directed mutagenesis that multiple residues within the SF region (Pro(165), Tyr(166), Ser(167), and Asp(168)) of apoA-I are critical for both LCAT binding to HDL and LCAT catalytic efficiency. The critical role for possible hydrogen bond interaction at apoA-I Tyr(166) was further supported using reconstituted HDL generated from apoA-I mutants (Tyr(166) → Glu or Asn), which showed preservation in both LCAT binding affinity and catalytic efficiency. Moreover, the in vivo functional significance of NO2-Tyr(166)-apoA-I, a specific post-translational modification on apoA-I that is abundant within human atherosclerotic plaque, was further investigated by using the recombinant protein generated from E. coli containing a mutated orthogonal tRNA synthetase/tRNACUA pair enabling site-specific insertion of the unnatural amino acid into apoA-I. NO2-Tyr(166)-apoA-I, after subcutaneous injection into hLCAT(Tg/Tg), apoA-I(-/-) mice, showed impaired LCAT activation in vivo, with significant reduction in HDL cholesteryl ester formation. The present results thus identify multiple structural features within the solvent-exposed SF region of apoA-I of nascent HDL essential for optimal LCAT binding and catalytic efficiency.

Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.

THU-382 - Lecithin-Cholesterol Acyltransferase Activity and Apolipoprotein A1 Levels Predict Progression to Organ Failure in ACLF Patients and 3 Month Mortality Following Acute Cirrhosis Decompensation

THU-382 - Lecithin-Cholesterol Acyltransferase Activity and Apolipoprotein A1 Levels Predict Progression to Organ Failure in ACLF Patients and 3 Month Mortality Following Acute Cirrhosis Decompensation

High-Density Lipoprotein, Lecithin: Cholesterol Acyltransferase, and Atherosclerosis.

Epidemiological data clearly show the existence of a strong inverse correlation between plasma high-density lipoprotein cholesterol (HDL-C) concentrations and the incidence of coronary heart disease. This relation is explained by a number of atheroprotective properties of HDL, first of all the ability to promote macrophage cholesterol transport. HDL are highly heterogeneous and are continuously remodeled in plasma thanks to the action of a number of proteins and enzymes. Among them, lecithin:cholesterol acyltransferase (LCAT) plays a crucial role, being the only enzyme able to esterify cholesterol within lipoproteins. LCAT is synthetized by the liver and it has been thought to play a major role in reverse cholesterol transport and in atheroprotection. However, data from animal studies, as well as human studies, have shown contradictory results. Increased LCAT concentrations are associated with increased HDL-C levels but not necessarily with atheroprotection. On the other side, decreased LCAT concentration and activity are associated with decreased HDL-C levels but not with increased atherosclerosis. These contradictory results confirm that HDL-C levels per se do not represent the functionality of the HDL system.

Chronic vitamin A-enriched diet feeding regulates hypercholesterolaemia through transcriptional regulation of reverse cholesterol transport pathway genes in obese rat model of WNIN/GR-Ob strain.

Background & objectives:Hepatic scavenger receptor class B1 (SR-B1), a high-density lipoprotein (HDL) receptor, is involved in the selective uptake of HDL-associated esterified cholesterol (EC), thereby regulates cholesterol homoeostasis and improves reverse cholesterol transport. Previously, we reported in euglycaemic obese rats (WNIN/Ob strain) that feeding of vitamin A-enriched diet normalized hypercholesterolaemia, possibly through hepatic SR-B1-mediated pathway. This study was aimed to test whether it would be possible to normalize hypercholesterolaemia in glucose-intolerant obese rat model (WNIN/GR/Ob) through similar mechanism by feeding identical vitamin A-enriched diet.Methods:In this study, 30 wk old male lean and obese rats of WNIN/GR-Ob strain were divided into two groups and received either stock diet or vitamin A-enriched diet (2.6 mg or 129 mg vitamin A/kg diet) for 14 wk. Blood and other tissues were collected for various biochemical analyses.Results:Chronic vitamin A-enriched diet feeding decreased hypercholesterolaemia and normalized abnormally elevated plasma HDL-cholesterol (HDL-C) levels in obese rats as compared to stock diet-fed obese groups. Further, decreased free cholesterol (FC) and increased esterified cholesterol (EC) contents of plasma cholesterol were observed, which were reflected in higher EC to FC ratio of vitamin A-enriched diet-fed obese rats. However, neither lecithin-cholesterol acyltransferase (LCAT) activity of plasma nor its expression (both gene and protein) in the liver were altered. On the contrary, hepatic cholesterol levels significantly increased in vitamin A-enriched diet fed obese rats. Hepatic SR-B1 expression (both mRNA and protein) remained unaltered among groups. Vitamin A-enriched diet fed obese rats showed a significant increase in hepatic low-density lipoprotein receptor mRNA levels, while the expression of genes involved in HDL synthesis, namely, ATP-binding cassette protein 1 (ABCA1) and apolipoprotein A-I, were downregulated. No such response was seen in vitamin A-supplemented lean rats as compared with their stock diet-fed lean counterparts.Interpretation & conclusions:Chronic vitamin A-enriched diet feeding decreased hypercholesterolaemia and normalized HDL-C levels, possibly by regulating pathways involved in HDL synthesis and degradation, independent of hepatic SR-B1 in this glucose-intolerant obese rat model.

Role of LCAT in Atherosclerosis.

Lecithin:cholesterol acyltransferase (LCAT) is the only enzyme capable of esterifying cholesterol in plasma, thus determining the maturation of high-density lipoproteins. Because it maintains an unesterified cholesterol gradient between peripheral cells and extracellular acceptors, for a long time, LCAT has been considered as a key enzyme in reverse cholesterol transport. However, despite the fact that it has been more than 50 years since the identification of LCAT, the role of this enzyme in the pathogenesis of atherosclerosis is still debated. A number of studies have been conducted in different animal models, with contradictory results. Studies in humans, in particular in the general population, in subjects at high cardiovascular risk, and in carriers of genetic LCAT deficiency in an excellent model to evaluate the correlation between the reduction of LCAT activity and atherosclerosis also gave conflicting results. This review provides a comprehensive overview of the controversial findings obtained in animals and humans, strengthening the necessity of further investigation to establish how LCAT could be regulated in a promising therapeutic strategy to reduce cardiovascular risk.

P-087: Effect of changing the type of fat on markers of cardiometabolic risk in obese rat

Background The purpose of this study is to verify if replacing margarine by sardine oil attenuates lipid peroxidation and improves reverse cholesterol transport in obese rats. Methods Obesity was induced by feeding a high-fat-diet during 3 months. At 400 ± 10 g the obese rats (n = 24) were divided into 2 groups and consumed during 1 month (d30) 20% margarine or sardine oil. At d30, 6 rats from each group were sacrificed and the remaining rats were then subjected to a change in diet for 1 month (d60): margarine was replaced by sardine oil and inversely. Results The substitution of sardine oil by margarine enhanced cholesterol -very low density lipoprotein (C-VLDL) (+21%) and C-low density lipoprotein (LDL-HDL1) (+17%) and decreased C-high density lipoprotein (HDL2 and HDL3) subfractions by 38% and 15%, respectively. Indeed, lipid peroxidation was enhanced in LDL-HDL1 (+26%) and HDL2 and HDL3 (+41%). When margarine was replaced by sardine oil, C-VLDL, C-LDL-HDL1, C-HDL2 concentrations were deceased by –58, –81 and –35%, respectively and lecithin: cholesterol acyltransferase (LCAT) activity was significantly higher (+74%). Indeed, HDL2 and HDL3 lipid peroxidation was decreased respectively by 28% and 14%. Conclusions In obese rats, the replacement of sardine oil by margarine seems to increase atherogenic markers and lipoperoxidation and consequently seemed to enhance cardiovascular diseases. However, the substitution of margarine by sardine oil can reduce lipid peroxidation and improves reverse cholesterol transport by increasing the activity of LCAT and thus ensuring a fortification in HDL2 cholesteryl esters. Sardine oil compared to margarine may have a protective effect against cardiovascular risk by improving the anti-atherogenic metabolic pathway of cholesterol.

Serum- and HDL3-serum amyloid A and HDL3-LCAT activity are influenced by increased CVD-burden

BackgroundHigh density lipoproteins (HDL) protect against cardiovascular disease (CVD). However, increased serum amyloid-A (SAA) related inflammation may negate this property. This study investigated if SAA was related to CVD-burden. MethodsSubjects referred to the rapid chest pain clinic (n = 240) had atherosclerotic burden assessed by cardiac computerised tomography angiography. Subjects were classified as: no-CVD (n = 106), non-obstructive-CVD, stenosis<50% (n = 58) or moderate/significant-CVD, stenosis ≥50% (n = 76). HDL was subfractionated into HDL2 and HDL3 by rapid-ultracentrifugation. SAA-concentration was measured by ELISA and lecithin cholesterol acyltransferase (LCAT) activity measured by a fluorimetric assay. ResultsWe illustrated that serum-SAA and HDL3-SAA-concentration were higher and HDL3-LCAT-activity lower in the moderate/significant-CVD-group, compared to the no-CVD and non-obstructive-CVD-groups (percent differences: serum-SAA, +33% & +30%: HDL3-SAA, +65% and +39%: HDL3-LCAT, −6% & −3%; p < 0.05 for all comparisons). We also identified a positive correlation between serum-SAA and HDL3-SAA (r = 0.698; p < 0.001) and a negative correlation between HDL3-SAA and HDL3-LCAT-activity (r = −0.295; p = 0.003), while CVD-burden positively correlated with serum-SAA (r = 0.150; p < 0.05) and HDL3-SAA (r = 0.252; p < 0.001) and negatively correlated with HDL3-LCAT-activity (r = −0.182; p = 0.006). Additionally, multivariate regression analysis adjusted for age, gender, CRP and serum-SAA illustrated that HDL3-SAA was significantly associated with modifying CVD-risk of moderate/significant CVD-risk (p < 0.05). ConclusionThis study has demonstrated increased SAA-related inflammation in subjects with moderate/significant CVD-burden, which appeared to impact on the antiatherogenic potential of HDL. We suggest that SAA may be a useful biomarker to illustrate increased CVD-burden, although this requires further investigation.

Effect of sardine proteins on hyperglycaemia, hyperlipidaemia and lecithin:cholesterol acyltransferase activity, in high-fat diet-induced type 2 diabetic rats.

Type 2 diabetes (T2D) is a major risk factor of CVD. The effects of purified sardine proteins (SP) were examined on glycaemia, insulin sensitivity and reverse cholesterol transport in T2D rats. Rats fed a high-fat diet (HFD) for 5 weeks, and injected with a low dose of streptozotocin, were used. The diabetic rats were divided into four groups, and they were fed casein (CAS) or SP combined with 30 or 5% lipids, for 4 weeks. HFD-induced hyperglycaemia, insulin resistance and hyperlipidaemia in rats fed HFD, regardless of the consumed protein. In contrast, these parameters lowered in rats fed SP combined with 5 or 30% lipids, and serum insulin values reduced in SP v. CAS. HFD significantly increased total cholesterol and TAG concentrations in the liver and serum, whereas these parameters decreased with SP, regardless of lipid intake. Faecal cholesterol excretion was higher with SP v. CAS, combined with 30 or 5% lipids. Lecithin:cholesterol acyltransferase (LCAT) activity and HDL3-phospholipids (PL) were higher in CAS-HF than in CAS, whereas HDL2-cholesteryl esters (CE) were lower. Otherwise, LCAT activity and HDL2-CE were higher in the SP group than in the CAS group, whereas HDL3-PL and HDL3-unesterified cholesterol were lower. Moreover, LCAT activity lowered in the SP-HF group than in the CAS-HF group, when HDL2-CE was higher. In conclusion, these results indicate the potential effects of SP to improve glycaemia, insulin sensitivity and reverse cholesterol transport, in T2D rats.

Lecithin-cholesterol acyltransferase in brain: Does oxidative stress influence the 24-hydroxycholesterol esterification?

24-Hydroxycholesterol (24OH-C) is esterified by the enzyme lecithin-cholesterol acyltransferase (LCAT) in the cerebrospinal fluid (CSF). We report here that the level of 24OH-C esters was lower in CSF of patients with amyotrophic lateral sclerosis than in healthy subjects (54% vs 68% of total 24OH-C, p=0.0005; n=8). Similarly, the level of 24OH-C esters in plasma was lower in patients than in controls (62% vs 77% of total 24OH-C; p=0.0076). The enzyme amount in CSF, as measured by densitometry of the protein band revealed by immunoblotting, was about 4-fold higher in patients than in controls (p=0.0085). As differences in the concentration of the LCAT stimulator Apolipoprotein E were not found, we hypothesized that the reduced 24OH-C esterification in CSF of patients might depend on oxidative stress. We actually found that oxidative stress reduced LCAT activity in vitro, and 24OH-C effectively stimulated the enzyme secretion from astrocytoma cells in culture. Enhanced LCAT secretion from astrocytes might represent an adaptive response to the increase of non-esterified 24OH-C percentage, aimed to avoid the accumulation of this neurotoxic compound. The low degree of 24OH-C esterification in CSF or plasma might reflect reduced activity of LCAT during neurodegeneration.

Alterations in lipid transfers to HDL associated with the presence of coronary artery disease in patients with type 2 diabetes mellitus.

BackgroundWe previously showed that unesterified-cholesterol transfer to high-density lipoprotein (HDL), a crucial step in cholesterol esterification and role in reverse cholesterol transport, was diminished in non-diabetic patients with coronary artery disease (CAD). The aim was to investigate whether, in patients with type 2 diabetes mellitus (T2DM), the occurrence of CAD was also associated with alterations in lipid transfers and other parameters of plasma lipid metabolism.MethodsSeventy-nine T2DM with CAD and 76 T2DM without CAD, confirmed by cineangiography, paired for sex, age (40–80 years), BMI and without statin use, were studied. In vitro transfer of four lipids to HDL was performed by incubating plasma of each patient with a donor emulsion containing radioactive lipids during 1 h at 37 °C. Lipids transferred to HDL were measured after chemical precipitation of non-HDL fractions and the emulsion. Results are expressed as % of total radioactivity of each lipid in HDL.ResultsIn T2DM + CAD, LDL-cholesterol and apo B were higher than in T2DM. T2DM + CAD also showed diminished transfer to HDL of unesterified cholesterol (T2DM + CAD = 7.6 ± 1.2; T2DM = 8.2 ± 1.5 %, p < 0.01) and of cholesteryl-esters (4.0 ± 0.6 vs 4.3 ± 0.7, p < 0.01). Unesterified cholesterol in the non-HDL serum fraction was higher in T2DM + CAD (0.93 ± 0.20 vs 0.85 ± 0.15, p = 0.02) and CETP concentration was diminished (2.1 ± 1.0 vs 2.5 ± 1.1, p = 0.02). Lecithin-cholesterol acyltransferase activity, HDL size and lipid composition were equal.ConclusionReduction in T2DM + CAD of cholesterol transfer to HDL may impair cholesterol esterification and reverse cholesterol transport and altogether with simultaneous increased plasma unesterified cholesterol may facilitate CAD development in T2DM.

A novel protein glycan biomarker and LCAT activity in metabolic syndrome.

The cholesterol-esterifying enzyme, lecithin:cholesterol acyltransferase (LCAT), is instrumental in high-density lipoprotein (HDL) remodelling. LCAT may also modify oxidative and inflammatory processes, as supported by an inverse relationship with HDL antioxidative functionality and a positive relationship with high-sensitivity C-reactive protein (hsCRP). GlycA is a recently developed proton nuclear magnetic resonance (NMR) spectroscopy-measured biomarker of inflammation whose signal originates from a subset of N-acetylglucosamine residues on the most abundant glycosylated acute-phase proteins. Plasma GlycA correlates positively with hsCRP and may predict cardiovascular disease even independent of hsCRP. Here, we tested the extent to which plasma GlycA is elevated in metabolic syndrome (MetS), and determined its relationship with LCAT activity. Plasma GlycA, hsCRP, serum amyloid A (SAA), tumour necrosis factor-α (TNF-α) and LCAT activity were measured in 58 subjects with MetS (including 46 subjects with type 2 diabetes mellitus (T2DM)) and in 45 nondiabetic subjects without MetS. Plasma GlycA was higher in MetS coinciding with higher hsCRP and LCAT activity (P < 0.01 for each). In all subjects combined, GlycA was correlated positively with hsCRP, SAA and LCAT activity (P < 0.001 for each), but not with TNF-α. Age- and sex-adjusted multivariable linear regression analysis revealed that GlycA was positively associated with LCAT activity (P = 0.029), independent of the presence of MetS, T2DM, hsCRP and SAA. GlycA was unrelated to diabetes status. A pro-inflammatory glycoprotein biomarker, GlycA, is higher in MetS. Higher plasma levels of this glycoprotein biomarker relate to increased LCAT activity in the setting of MetS.

Association of Lecithin Cholesterol Acyltransferase rs5923 Polymorphism in Iranian Individuals with Extremely Low High-Density Lipoprotein Cholesterol: Tehran Lipid and Glucose Study.

Background:The serum concentration of high-density lipoprotein cholesterol (HDL-C) is one of the important heritable risk factors for cardiovascular disease and is a target for therapeutic intervention. In this study, we aimed to evaluate the effects of lecithin cholesterol acyltransferase (LCAT) gene polymorphism rs5923 on LCAT enzyme activity and serum HDL-C concentration. Methods: The study population was selected from consecutive individuals with HDL-C ≤ 5th percentile (n = 73) and extremely high HDL-C ≥ 95th percentile (n = 57) who had participated in the Tehran Lipid and Glucose Study. The rs5923 polymorphism was genotyped using direct sequencing. LCAT activity was measured by fluorometric assay kit, and lipid concentrations were measured using the enzymatic colorimetric method. Results: The genotype frequencies were significantly different between the high HDL-C group (CC 94.7%, CT 5.3%) and the low HDL-C group (CC 83.6%, CT 16.4%) (P = 0.048). The T-allele frequencies in subjects with low and high HDL-C were 0.082 and 0.026, respectively (P = 0.16). The association of the single-nucleotide polymorphism rs5923 with low HDL-C was not statistically significant after adjustment for age, sex, and BMI (odd ratio = 2.65, 95% confidence interval = 0.32-21.5, P = 0.36, regression logistic analysis). Also, the effects of LCAT enzyme activity did not depend on the HDL-C level (P = 0.24). Conclusion:rs5923 polymorphism is not associated with low HDL-C levels in Iranian population.

Response of the cholesterol metabolism to a negative energy balance in dairy cows depends on the lactational stage.

The response of cholesterol metabolism to a negative energy balance (NEB) induced by feed restriction for 3 weeks starting at 100 days in milk (DIM) compared to the physiologically occurring NEB in week 1 postpartum (p.p.) was investigated in 50 dairy cows (25 control (CON) and 25 feed-restricted (RES)). Blood samples, liver biopsies and milk samples were taken in week 1 p.p., and in weeks 0 and 3 of feed restriction. Plasma concentrations of total cholesterol (C), phospholipids (PL), triglycerides (TAG), very low density lipoprotein-cholesterol (VLDL-C) and low density lipoprotein-cholesterol (LDL-C) increased in RES cows from week 0 to 3 during feed restriction and were higher in week 3 compared to CON cows. In contrast, during the physiologically occurring NEB in week 1 p.p., C, PL, TAG and lipoprotein concentrations were at a minimum. Plasma phospholipid transfer protein (PLTP) and lecithin:cholesterol acyltransferase (LCAT) activities did not differ between week 0 and 3 for both groups, whereas during NEB in week 1 p.p. PLTP activity was increased and LCAT activity was decreased. Milk C concentration was not affected by feed restriction in both groups, whereas milk C mass was decreased in week 3 for RES cows. In comparison, C concentration and mass in milk were elevated in week 1 p.p. Hepatic mRNA abundance of sterol regulatory element-binding factor-2 (SREBF-2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), and ATP-binding cassette transporter (ABCA1) were similar in CON and RES cows during feed restriction, but were upregulated during NEB in week 1 p.p. compared to the non-lactating stage without a NEB. In conclusion, cholesterol metabolism in dairy cows is affected by nutrient and energy deficiency depending on the stage of lactation.

Differential antioxidative and hypocholesterolemic responses to two fish protein hydrolysates (Sardina pilchardus and Boops boops) in cholesterol-fed rats

Purpose– The purpose of this study was to evaluate the antioxidant and hypocholesterolemic activities of sardine and bogue protein hydrolysates in cholesterol-fed rats.Design/methodology/approach– In total, 18 male Wistar rats (220 ± 10 g) fed 20 per cent casein, 1 per cent cholesterol and 0.5 per cent cholic acid were divided into three groups and received a daily gavage of 250 mg of sardine (SPH) or bogue (BPH) protein hydrolysates for 30 days. The third group, named control group (CG), received in the same conditions water. Lipoproteins were fractionated by size-exclusion fast protein liquid chromatography, and serum lipids, apolipoproteins and lipoproteins were assayed.Findings– In SPH and BPH groups, serum total cholesterol concentrations were −66 per cent lower than in CG. This corresponded to the decreased very low-density lipoprotein-C in the former groups. Moreover, BPH treatment reduced low-density lipoprotein-C compared with CG and SPH groups. Compared with CG, serum phospholipids were reduced by SPH and BPH. Furthermore, BPH increased significantly APOA4 and sphingomyelin but lowered phosphatidylcholine. In the latter group, serum lecithin cholesterol acyltransferase activity was +23 per cent higher, but with SPH, this activity was −35 per cent reduced compared with CG. Apolipoprotein A-I contents were similar in the three groups. Compared with CG, hydroperoxide and lipid peroxidation contents in serum and lipoprotein fractions were reduced by SPH and BPH. Compared with CG, serum superoxide dismutase and glutathione peroxidase activities were increased in the treated groups, particularly in the BPH group.Originality/value– These results suggest that sardine protein hydrolysates and particularly those of bogue could be a very useful natural compound to prevent hypercholesterolemia by both improving the lipid profile and modulating oxidative stress in cholesterol-fed rats.

9. AAV-Mediated Gene Transfer as a Therapeutic Approach for Familial LCAT Deficiency

Lecithin Cholesterol Acyltransferase (LCAT) is a plasma enzyme secreted by the liver and responsible for esterification of free cholesterol to cholesteryl ester in the blood compartment. Familial LCAT deficiency (FLD) is an autosomal recessive disorder caused by loss-of-function mutations of the LCAT gene and is characterized by extremely low levels of high density lipoprotein cholesterol (HDL-C), anemia, corneal opacities, and chronic progressive renal disease leading to end-stage kidney disease as early as the third decade of life. Currently, there is no specific treatment for FLD. The fact that patients with even very low levels of plasma LCAT activity do not develop kidney disease make FLD an attractive candidate for liver-directed gene therapy with adeno-associated viral (AAV) vectors. LCAT is activated by apolipoprotein A-I (apoA-I) and this interaction is highly species specific. Therefore, LCAT KO mice were crossed with human apoA-I transgenic (hApoAITg) mice in order to facilitate experiments involving expression of human LCAT. LCAT KO/hApoAI transgenic mice received a single IP injection of AAV8 encoding human LCAT in a dose range of 5e10 GC/kg to 5e11 GC/kg. HDL-C increased in a dose-dependent fashion, from 32 mg/dL in the control group to 103 mg/dL at the dose of 1.5e11 GC/kg and 473 mg/dL at the dose of 5e11 GC/kg. Plasma% cholesterol ester, a sensitive indicator of LCAT activity, was normalized at the low dose of 1.5e11 GC/kg. Thus, we demonstrate that low doses of AAV8-hLCAT provide stable expression of the LCAT enzyme and correct dyslipidemia in a ‘humanized’ mouse model of LCAT deficiency. Studies designed to demonstrate prevention of kidney disease in this mouse model are ongoing. These proof-of-concept studies provide a foundation for further development of AAV-based gene therapy for familial LCAT deficiency.