Mining the LIPG allelic spectrum reveals the contribution of rare and common regulatory variants to HDL cholesterol.

Genome-wide association studies (GWAS) have successfully identified loci associated with quantitative traits, such as blood lipids. Deep resequencing studies are being utilized to catalogue the allelic spectrum at GWAS loci. The goal of these studies is to identify causative variants and missing heritability, including heritability due to low frequency and rare alleles with large phenotypic impact. Whereas rare variant efforts have primarily focused on nonsynonymous coding variants, we hypothesized that noncoding variants in these loci are also functionally important. Using the HDL-C gene LIPG as an example, we explored the effect of regulatory variants identified through resequencing of subjects at HDL-C extremes on gene expression, protein levels, and phenotype. Resequencing a portion of the LIPG promoter and 5' UTR in human subjects with extreme HDL-C, we identified several rare variants in individuals from both extremes. Luciferase reporter assays were used to measure the effect of these rare variants on LIPG expression. Variants conferring opposing effects on gene expression were enriched in opposite extremes of the phenotypic distribution. Minor alleles of a common regulatory haplotype and noncoding GWAS SNPs were associated with reduced plasma levels of the LIPG gene product endothelial lipase (EL), consistent with its role in HDL-C catabolism. Additionally, we found that a common nonfunctional coding variant associated with HDL-C (rs2000813) is in linkage disequilibrium with a 5' UTR variant (rs34474737) that decreases LIPG promoter activity. We attribute the gene regulatory role of rs34474737 to the observed association of the coding variant with plasma EL levels and HDL-C. Taken together, the findings show that both rare and common noncoding regulatory variants are important contributors to the allelic spectrum in complex trait loci.

A naturally occurring variant of endothelial lipase associated with elevated HDL exhibits impaired synthesis.

Human endothelial lipase (EL) is a member of a family of lipases and phospholipases that are involved in the metabolism of plasma lipoproteins. EL displays a preference to hydrolyze lipids in HDL. We report here that a naturally occurring low frequency coding variant in the EL gene (LIPG), glycine-26 to serine (G26S), is significantly more common in African-American individuals with elevated HDL cholesterol (HDL-C) levels. To test the hypothesis that this variant results in reduced EL function, we extensively characterized and compared the catalytic and noncatalytic functions of the G26S variant and wild-type (WT) EL. While the catalytic-specific activity of G26S EL is similar to WT EL, its secretion is markedly reduced. Consistent with this observation, we found that carriers of the G26S variant had significantly reduced plasma levels of EL protein. Thus, this N-terminal variant results in reduced secretion of EL protein, plausibly leading to increased HDL-C levels.

Endothelial lipase is increased in vivo by inflammation in humans.

BACKGROUND: Endothelial lipase (EL) is a plasma lipase that we previously reported to be significantly correlated with all features of the metabolic syndrome in humans, including directly with measures of adiposity and inversely with high-density lipoprotein cholesterol levels. We hypothesized that inflammation associated with obesity results in upregulation of EL. We determined the relationship between inflammatory markers and EL levels in a cohort of healthy persons recruited on the basis of family history of coronary disease. Furthermore, we directly tested the hypothesis that plasma EL concentrations would increase with induction of an inflammatory state by low-dose endotoxin in humans. METHODS AND RESULTS: High-sensitivity C-reactive protein, interleukin 6, soluble tumor necrosis factor receptor II, soluble intercellular adhesion molecule 1, leptin, and adiponectin were measured in plasma of 858 subjects. Significant direct correlations (P<0.001 for all) were found between EL concentrations and high-sensitivity C-reactive protein (r=0.28), interleukin-6 (r=0.22), soluble tumor necrosis factor receptor II (r=0.22), soluble intercellular adhesion molecule 1 (r=0.24), and leptin (r=0.20). An inverse correlation was present with adiponectin (r=-0.15, P<0.001). Adiponectin inhibited the tumor necrosis factor-alpha-stimulated EL secretion from cultured human coronary endothelial cells in a dose-dependent manner. Experimental low-dose endotoxemia in 20 subjects resulted in a 2.5-fold increase in EL concentrations 12 to 16 hours after injection, which correlated temporally with decreases in both total and high-density lipoprotein phospholipid. CONCLUSIONS: In humans, plasma inflammatory markers are directly correlated with plasma EL concentrations, and experimental endotoxemia significantly increases plasma EL concentrations, proving that EL is upregulated by inflammation in humans. This mechanism may partially explain the low high-density lipoprotein cholesterol levels seen in obesity and metabolic syndrome.

Vitamin D and the bariatric surgical patient: a review.

OBJECTIVE: Adiposity has been inversely associated with vitamin D concentration across a range of body mass index values and cultural groups. As obesity has increased markedly worldwide, a greater number of patients with severe obesity have been treated with gastric restrictive and/or malabsorptive surgical procedures. The purpose of this review was to describe current knowledge about vitamin D and severe obesity, and the impact of obesity surgery on vitamin D status. RESEARCH METHODS AND PROCEDURES: A systematic review was conducted with search terms obesity, vitamin D, osteoporosis, bone disease, gastric bypass, and obesity surgery in various combinations. Publications were limited to those since 2000 to control for similarity in vitamin D assays and obesity prevalence levels. RESULTS: Mean 25-hydroxy vitamin D was <80 nmol/l in more than 1,900 patients preoperatively, and was not restored to the optimal concentration of >80 nmol/l postoperatively. Both secondary hyperparathyroidism and bone loss were common, particularly when the obesity surgery included a malabsorptive component. Standard postsurgical supplementation with vitamin D and calcium have not been adequate to suppress secondary hyperparathyroidism or to restore 25-hydroxy vitamin D status. DISCUSSION: The mechanisms behind vitamin D deficiency in severe obesity and evidence-based corrective actions have not been well-defined. Of particular concern are adolescents who qualify for and elect surgical treatment of their obesity, where subsequent metabolic bone disease may be long-standing.

Obesity and inflammation: lessons from bariatric surgery.

BACKGROUND: Obesity is associated with a series of comorbid conditions that are characterized by an inflammatory state. The purpose of this review is to update knowledge about obesity, adipose tissue, and inflammation. METHODS: Review of the published literature using search terms of adipose, inflammation, obesity, and insulin resistance in combinations. RESULTS: Adipose tissue elaborates proinflammatory cytokines such as interleukin-6 and tumor necrosis factor-alpha, with greater secretion from the stromal vascular fraction than from adipocytes and with greater secretion from visceral than subcutaneous adipose tissue sites. This proinflammatory state is associated with insulin resistance and ameliorated by weight loss, with concurrent increase in production of the anti-inflammatory adipokine adiponectin. CONCLUSION: Although these associations between obesity and inflammation are clearly important, many questions remain unresolved. It is unclear if benefits of weight loss pertain only to those with a proinflammatory profile, who receive a particular type of obesity surgical procedure, or whether these benefits are sustained over a lifetime. The outcomes associated with anti-inflammatory nutrient supplementation, with or without weight loss, in the obese would also increase our understanding.

High-dose statin therapy for secondary prevention of stroke: stroke prevention by aggressive reduction in cholesterol levels study review.

It has been shown that HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors (statins) lower the incidence of a first stroke in patients with coronary heart disease, diabetes, or risk factors for cardiovascular disease. However, it is unknown whether statin therapy could reduce the incidence of a second stroke in patients without evidence of heart disease. This article reviews the results of the Stroke Prevention by Aggressive Reduction in Cholesterol Levels trial, a prospective, randomized, multicentered, double-blind, placebo-controlled, international trial designed to examine the effect of high-dose atorvastatin on secondary stroke prevention. Trial participants (4,731) had experienced a stroke or transient ischemic attack within 1 to 6 months before randomization into the study. Over the 5-year follow-up period, incidence of second stroke or transient ischemic attack was significantly reduced in the atorvastatin treatment group compared with the placebo group. In addition, high-dose atorvastatin therapy significantly decreased major coronary artery and other negative cardiovascular events. The reduction in incidence of secondary stroke was specific to ischemic stroke as opposed to hemorrhagic stroke. Results of the trial are clinically significant and support extension of the latest secondary stroke prevention guidelines to include statin therapy for those patients without coronary heart disease.

Visceral adiposity and endothelial lipase.

CONTEXT: Overexpression of endothelial lipase (EL) has been shown to reduce plasma high-density lipoprotein cholesterol levels in animal models. However, the extent to which EL contributes to modulate the deteriorated high-density lipoprotein profile observed in obesity in humans is less clear. OBJECTIVES: The objectives of this study were to investigate the association between levels of obesity and visceral adiposity in particular and plasma EL concentrations. METHODS: Postheparin plasma EL concentrations were measured by ELISA and visceral adiposity by computed tomography in a sample of 80 sedentary men in good health. EL mRNA levels in abdominal sc and omental adipose tissues obtained during abdominal hysterectomies were measured in another sample of 14 women. RESULTS: Plasma EL levels were positively correlated with body mass index (R = 0.46, P < 0.0001), visceral adipose tissue accumulation (R = 0.44, P < 0.0001), and a proatherogenic lipid profile including increased plasma cholesterol and triglycerides. However, EL mRNA levels were similar in sc and omental AT and were 10,000-fold lower than lipoprotein lipase mRNA levels in those tissues. CONCLUSIONS: Increased visceral adiposity is significantly correlated with elevated plasma EL levels, but this association is unlikely to be causal and may reflect other common metabolic alterations.

Endothelial lipase concentrations are increased in metabolic syndrome and associated with coronary atherosclerosis.

BACKGROUND: Endothelial lipase (EL), a new member of the lipase family, has been shown to modulate high-density lipoprotein (HDL-C) metabolism and atherosclerosis in mouse models. We hypothesized that EL concentrations would be associated with decreased HDL-C and increased atherosclerosis in humans. METHODS AND FINDINGS: Healthy individuals with a family history of premature coronary heart disease (n = 858) were recruited as part of the Study of the Inherited Risk of Atherosclerosis. Blood was drawn in the fasting state before and, in a subgroup (n = 510), after administration of a single dose of intravenous heparin. Plasma lipids were measured enzymatically, lipoprotein subclasses were assessed by nuclear magnetic resonance, and coronary artery calcification (CAC) was quantified by electron beam computed tomography. Plasma EL mass was measured using a newly developed enzyme-linked immunosorbent assay. Median EL mass in pre-heparin plasma was 442 (interquartile range = 324-617) ng/ml. Median post-heparin mass was approximately 3-fold higher, 1,313 (888-1,927) ng/ml. The correlation between pre-heparin EL mass and post-heparin EL mass was 0.46 (p < 0.001). EL mass concentrations in both pre- and post-heparin plasma significantly correlated with all NCEP ATPIII-defined metabolic syndrome factors: waist circumference (r = 0.28 and 0.22, respectively, p < 0.001 for each), blood pressure (r = 0.18 and 0.24, p < 0.001 for each), triglycerides (r = 0.22, p < 0.001; and 0.13, p = 0.004), HDL cholesterol (r = -0.11, p = 0.002; and -0.18, p < 0.001), and fasting glucose (r = 0.11 and 0.16, p = 0.001 for both). EL mass in both routine (odds ratio [OR] = 1.67, p = 0.01) and post-heparin (OR = 2.42, p = 0.003) plasma was associated with CAC as determined by ordinal regression after adjustment for age, gender, waist circumference, vasoactive medications, hormone replacement therapy (women), and established cardiovascular risk factors. CONCLUSIONS: We report, to our knowledge for the first time, that human plasma EL concentrations, in both post-heparin and routine pre-heparin plasma, are significantly associated with metabolic syndrome features and with subclinical atherosclerosis. EL may be a pro-atherogenic factor in humans, especially in overweight individuals and those with metabolic syndrome.

Effect of fenofibrate on plasma lipoprotein composition and kinetics in patients with complete hepatic lipase deficiency.

OBJECTIVE: The goal of this study was to characterize the effect of microcoated fenofibrate (160 mg/day for 6 months) on plasma lipoprotein composition and kinetics in 2 patients with complete hepatic lipase (HL) deficiency. METHODS AND RESULTS: Fenofibrate treatment normalized the plasma lipoprotein profile of patients with complete HL deficiency, as evidenced by significant reductions in the plasma concentration of cholesterol (-49%) and triglycerides (-82%) and a significant increase in low-density lipoprotein (LDL) size (251.5+/-1.8 versus 263.5+/-0.7 A). The in vivo kinetics of very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and LDL apolipoprotein (apo)B-100 and plasma apoA-I and apoA-II were studied using a primed-constant infusion of L-[5,5,5-D3]-leucine for 12 hours in the fasted state. Fenofibrate treatment in complete HL-deficient patients substantially decreased plasma concentrations of VLDL, IDL, and LDL apoB-100 attributable to important increases in VLDL (+325%), IDL (+129%), and LDL (+218%) apoB-100 fractional catabolic rates (FCR). IDL apoB-100 FCR nevertheless remained 60% lower after treatment compared with values obtained in controls (n=5). The kinetics of plasma apoA-I and apoA-II as well as the capacity of total plasma and of high-density lipoprotein particles to efflux cellular cholesterol from normal human skin fibroblasts was not altered by fenofibrate. CONCLUSIONS: Fenofibrate therapy exerts a pronounced antiatherogenic effect on triglyceride-rich lipoproteins even in the complete absence of HL.

Identification of the active form of endothelial lipase, a homodimer in a head-to-tail conformation.

Endothelial lipase (EL) is a member of a subfamily of lipases that act on triglycerides and phospholipids in plasma lipoproteins, which also includes lipoprotein lipase and hepatic lipase. EL has a tropism for high density lipoprotein, and its level of phospholipase activity is similar to its level of triglyceride lipase activity. Inhibition or loss-of-function of EL in mice results in an increase in high density lipoprotein cholesterol, making it a potential therapeutic target. Although hepatic lipase and lipoprotein lipase have been shown to function as homodimers, the active form of EL is not known. In these studies, the size and conformation of the active form of EL were determined. Immunoprecipitation experiments suggested oligomerization. Ultracentrifugation experiments showed that the active form of EL had a molecular weight higher than the molecular weight of a simple monomer but less than a dimer. A construct encoding a covalent head-to-tail homodimer of EL (EL-EL) was expressed and had similar lipolytic activity to EL. The functional molecular weights determined by radiation inactivation were similar for EL and the covalent homodimer EL-EL. We previously showed that EL could be cleaved by proprotein convertases, such as PC5, resulting in loss of activity. In cells overexpressing PC5, the covalent homodimeric EL-EL appeared to be more stable, with reduced cleavage and conserved lipolytic activity. A comparative model obtained using other lipase structures suggests a structure for the head-to-tail EL homodimer that is consistent with the experimental findings. These data confirm the hypothesis that EL is active as a homodimer in head-to-tail conformation.

Endothelial lipase is associated with inflammation in humans.

The aim of this study was to investigate the extent to which inflammation is linked with plasma endothelial lipase (EL) concentrations among healthy sedentary men. Plasma C-reactive protein (CRP) concentrations were measured with a highly sensitive commercial immunoassay, plasma interleukin-6 (IL-6) concentrations were measured using a commercial ELISA, and plasma secretory phospholipase A(2) type IIA (sPLA(2)-IIA) concentrations were measured using a commercial assay in a sample of 74 moderately obese men (mean body mass index, 29.8 +/- 5.2 kg/m(2)). Plasma EL concentrations were positively correlated with various indices of obesity, fasting plasma insulin, and plasma CRP, IL-6, and sPLA(2)-IIA concentrations. Multiple regression analyses revealed that plasma CRP concentrations explained 14.5% (P = 0.0008) of the variance in EL concentrations. When entered into the model, LPL activity accounted for 16.1% (P < 0.0001) and plasma CRP concentrations accounted for 20.9% (P < 0.0001) of the variance in EL concentrations. The combined impact of visceral adipose tissue (VAT) and of an inflammation score on EL concentrations was investigated. Among subjects with high or low VAT, those having a high inflammation score based on plasma CRP, IL-6, and sPLA(2)-IIA concentrations had increased plasma EL concentrations (P = 0.0005). In conclusion, our data reveal a strong association between proinflammatory cytokines and plasma EL concentrations among healthy people with low or high VAT levels.

The role of endothelial lipase in high-density lipoprotein metabolism.

PURPOSE OF REVIEW: Elevating high-density lipoprotein levels is increasingly being identified as an essential strategy for the prevention of atherosclerosis. Plasma levels of high-density lipoprotein cholesterol and its major protein, apoAI, are largely influenced by the rate of turnover. Lipases play an important role in modulating the metabolism of high-density lipoprotein. In particular, endothelial lipase has been shown to be an important determinant of high-density lipoprotein metabolism and levels in murine models. This article reviews new developments in our understanding of the biology of endothelial lipase and its relation to high-density lipoprotein metabolism. RECENT FINDINGS: Inhibition of the endothelial lipase gene, either by antibody injection or by targeted gene deletion, results in an approximately 50% increase in high-density lipoprotein cholesterol levels in mice. As many as 31 single-nucleotide polymorphisms have been identified in the endothelial lipase gene. The 584 C/T mutation, which results in a threonine-to-isoleucine amino acid change, has been associated with higher high-density lipoprotein cholesterol levels in three separate studies. SUMMARY: Increasing evidence suggests that endothelial lipase plays a significant role in high-density lipoprotein metabolism. Endothelial lipase could be an important new target for novel therapies to raise high-density lipoprotein levels.

Allosteric modification of factor XIa functional activity upon binding to polyanions.

The effects of several polyanions on the hydrolysis of the chromogenic substrate L-pyroglutamyl-L-prolyl-L-arginyl-p-nitroaniline (S-2366) and on the activation of factor IX by factor XIa have been investigated. Two forms of dextran sulfate (M(r) approximately 500000 and M(r) approximately 10000, DX10) and two forms of heparin (64 disaccharide units, M(r) approximately 14000, and hypersulfated heparin, S-Hep, M(r) approximately 12000) inhibited both factor XIa amidolytic activity and factor IX activation in a concentration-dependent manner. The inhibitory effect was not due to binding of either substrate by the polyanions since only a decrease in V(max) without any effect on K(m) was observed in kinetic assays. Steric inhibition is unlikely since the concentrations of polyanions required for inhibition of small peptide hydrolysis were lower than those required for macromolecular substrate cleavage. In contrast, an allosteric inhibitory mechanism was supported by an enhancement of the dansyl fluorescence of 5-(dimethylamino)-1-(naphthalenesulfonyl)glutamylglycylarginyl- (DEGR-) factor XIa observed when the fluorophore was in complex with either DX10 or S-Hep. Moreover, in the presence of a polyanion the fluorophore was far more resistant to quenching by acrylamide. These results provide compelling evidence that factor XIa binding to the polyanions, dextran sulfate and heparin, results in inhibition of the enzyme by an allosteric mechanism.

RETRACTED: Factor XI binding to the platelet glycoprotein Ib-IX-V complex promotes factor XI activation by thrombin.

Factor XI binds to high affinity sites on the surface of stimulated platelets where it is efficiently activated by thrombin. Here, we provide evidence that the factor XI binding site on platelets is in the glycoprotein (GP) Ibalpha subunit of the GP Ib-IX-V complex as follows. 1) Bernard-Soulier platelets, lacking the complex, are deficient in factor XI binding; 2) two GP Ibalpha ligands, SZ-2 (a monoclonal antibody) and bovine von Willebrand factor, inhibit factor XI binding to platelets; 3) by surface plasmon resonance, factor XI bound specifically to glycocalicin (the extracellular domain of GP Ibalpha) in Zn(2+)-dependent fashion (K(d)( app) approximately 52 nm). We then investigated whether glycocalicin could promote factor XI activation by thrombin, another GP Ibalpha ligand. In the presence of high molecular weight kininogen (45 nm), Zn(2+) and Ca(2+) ions, thrombin activated factor XI in the presence of glycocalicin at rates comparable with those seen in the presence of dextran sulfate (1 microg/ml). With higher high molecular weight kininogen concentrations (360 nm), the rate of thrombin-catalyzed factor XI activation in the presence of glycocalicin was comparable with that on activated platelets. Thus, factor XI binds to the GP Ib-IX-V complex, promoting its activation by thrombin.

Role of N-linked glycosylation in the secretion and activity of endothelial lipase.

Human endothelial lipase (EL), a member of the triglyceride lipase gene family, has five potential N-linked glycosylation sites, two of which are conserved in both lipoprotein lipase and hepatic lipase. Reduction in molecular mass of EL after treatment with glycosidases and after treatment of EL-expressing cells with the glycosylation inhibitor tunicamycin demonstrated that EL is a glycosylated protein. Each putative glycosylation site was examined by site-directed mutagenesis of the asparagine (Asn). Mutation of Asn-60 markedly reduced secretion and slightly increased specific activity. Mutation of Asn-116 did not influence secretion but increased specific activity. In both cases, this resulted from decreased apparent K(m) and increased apparent V(max). Mutation of Asn-373 did not influence secretion but significantly reduced specific activity, as a result of a decrease in apparent V(max). Mutation of Asn-471 resulted in no reduction in secretion or specific activity. Mutation of Asn-449 resulted in no change in secretion, activity, or molecular mass, indicating that the site is not utilized. The ability of mutants secreted at normal levels to mediate bridging between LDL and cell surfaces was examined. The Asn-373 mutant demonstrated a 3-fold decrease in bridging compared with wild-type EL, whereas Asn-116 and Asn-471 were similar to wild-type EL.