Endothelial lipase and HDL metabolism

PURPOSE OF REVIEW: In the past year, several laboratories taking independent approaches have provided compelling evidence that endothelial lipase, a relatively recent addition to the triglyceride lipase gene family, is a major determinant of HDL metabolism. This review summarizes recent findings from experiments in mice with altered levels of endothelial lipase, from an examination of endothelial lipase catalytic and non-catalytic functions in vitro, and from human genetic studies. RECENT FINDINGS: An analysis of lipids and lipoproteins in endothelial lipase knockout and transgenic mice and in mice with adenovirus-driven hepatic overexpression of endothelial lipase shows, without exception, that total cholesterol, phospholipid and HDL-cholesterol all vary inversely with the endothelial lipase gene dosage, and primarily depend on endothelial lipase catalytic activity. Endothelial lipase participates in HDL metabolism by promoting the turnover of HDL components and increasing the catabolism of apolipoprotein A-I. The measurement of lipase activity on lipoprotein substrates in vitro demonstrates that endothelial lipase is distinct from other triglyceride lipases in showing the highest activity on HDL. Endothelial lipase gene polymorphisms in humans appear to be associated with HDL-cholesterol or HDL3-cholesterol concentrations. SUMMARY: A low HDL-cholesterol level in humans is a risk factor for coronary heart disease. Although not yet demonstrated, it is possible that the action of endothelial lipase on HDL may promote atherogenesis, in which case endothelial lipase may represent an attractive target for pharmaceutical intervention.     

Hepatic proprotein convertases modulate HDL metabolism

The risk of atherosclerosis is inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). However, HDL metabolism is incompletely understood, and there are few effective approaches to modulate HDL-C levels. Here we show that inhibition in the liver of the classical proprotein convertases (PCs), but not the atypical PCs S1P and PCSK9, decreases plasma HDL-C levels. This metabolic effect of hepatic PCs is critically dependent on expression of endothelial lipase (EL), an enzyme that directly hydrolyzes HDL phospholipids and promotes its catabolism. Hepatic PCs reduce EL function through direct inactivating cleavage of EL as well as through activating cleavage of angiopoietin-like protein 3 (ANGPTL3), an endogenous inhibitor of EL. Thus, inhibition of hepatic PCs results in increased EL activity, leading to reduced HDL-C as well as impaired reverse cholesterol transport. The hepatic PC-ANGPTL3-EL-HDL pathway is therefore a novel mechanism controlling HDL metabolism and cholesterol homeostasis.     

Metabolic and genetic determinants of HDL metabolism and hepatic lipase activity in normolipidemic females.

The metabolic and genetic determinants of HDL cholesterol (HDL-C) levels and HDL turnover were studied in 36 normolipidemic female subjects on a whole-food low-fat metabolic diet. Lipid, lipoprotein, and apolipoprotein levels, lipoprotein size, and apolipoprotein turnover parameters were determined, as were genetic variation at one site in the hepatic lipase promoter and six sites in the apolipoprotein AI/CIII/AIV gene cluster. Menopause had no significant effect on HDL-C or turnover. Stepwise multiple regression analysis revealed that HDL-C was most strongly correlated with HDL size, apolipoprotein A-II (apoA-II), and apolipoprotein A-I (apoA-I) levels, which together could account for 90% of the variation in HDL-C. HDL size was inversely correlated with triglycerides, body mass index, and hepatic lipase activity, which together accounted for 82% of the variation in HDL size. The hepatic lipase promoter genotype had a strong effect on hepatic lipase activity and could account for 38% of the variation in hepatic lipase activity. The apoA-I transport rate (AI-TR) was the major determinant of apoA-I levels, but AI-TR was not associated with six common genetic polymorphism in the apoAI/CIII/AIV gene cluster.A simplified model of HDL metabolism is proposed, in which A-I and apoA-II levels combined with triglycerides, and hepatic lipase activity could account for 80% of the variation in HDL-C.     

Hepatic lipase gene -514C>T variant is associated with exercise training-induced changes in VLDL and HDL by lipoprotein lipase

Our objective was to test the hypothesis that a common polymorphism in the hepatic lipase (HL) gene (LIPC -514C>T, rs1800588) influences aerobic exercise training-induced changes in TG, very-low-density lipoprotein (VLDL), and high-density lipoprotein (HDL) through genotype-specific increases in lipoprotein lipase (LPL) activity and that sex may affect these responses. Seventy-six sedentary overweight to obese men and women aged 50-75 yr at risk for coronary heart disease (CHD) underwent a 24-wk prospective study of the LIPC -514 genotype-specific effects of exercise training on lipoproteins measured enzymatically and by nuclear magnetic resonance, postheparin LPL and HL activities, body composition by dual energy x-ray absorptiometry and computer tomography scan, and aerobic capacity. CT genotype subjects had higher baseline total cholesterol, HDL-C, HDL(2)-C, large HDL, HDL particle size, and large LDL than CC homozygotes. Exercise training elicited genotype-specific decreases in VLDL-TG (-22 vs. +7%; P < 0.05; CC vs. CT, respectively), total VLDL and medium VLDL, and increases in HDL-C (7 vs. 4%; P < 0.03) and HDL(3)-C with significant genotype×sex interactions for the changes in HDL-C and HDL(3)-C (P values = 0.01-0.02). There were also genotype-specific changes in LPL (+23 vs. -6%; P < 0.05) and HL (+7 vs. -24%; P < 0.01) activities, with LPL increasing only in CC subjects (P < 0.006) and HL decreasing only in CT subjects (P < 0.007). Reductions in TG, VLDL-TG, large VLDL, and medium VLDL and increases in HDL(3)-C and small HDL particles correlated significantly with changes in LPL, but not HL, activity only in CC subjects. This suggests that the LIPC -514C>T variant significantly affects training-induced anti-atherogenic changes in VLDL-TG, VLDL particles, and HDL through an association with increased LPL activity in CC subjects, which could guide therapeutic strategies to reduce CHD risk.     

Novel roles of hepatic lipase and phospholipid transfer protein in VLDL as well as HDL metabolism

Objective

Elevated plasma phospholipid transfer protein (PLTP) expression may increase atherosclerosis in mice by reducing plasma HDL and increasing hepatic VLDL secretion. Hepatic lipase (HL) is a lipolytic enzyme involved in several aspects of the same pathways of lipoprotein metabolism. We investigated whether the effects of elevated PLTP activity are compromised by HL deficiency.

Methods and results

HL deficient mice were crossbred with PLTP transgenic (PLTPtg) mice and studied in the fasted state. Plasma triglycerides were decreased in HL deficiency, explained by reduced hepatic triglyceride secretion. In PLTPtg mice, a redistribution of HL activity between plasma and tissue was evident and plasma triglycerides were also decreased. HL deficiency mitigated or even abolished the stimulatory effect of elevated PLTP activity on hepatic triglyceride secretion. HL deficiency had a modest incremental effect on plasma HDL, which remained present in PLTP transgenic/HL^−/− mice, thereby partially compensating the decrease in HDL caused by elevation of PLTP activity. HDL decay experiments showed that the fractional turnover rate of HDL cholesteryl esters was delayed in HL deficient mice, increased in PLTPtg mice and intermediate in PLTPtg mice in an HL^−/− background.

Conclusions

HL affects hepatic VLDL. Elevated PLTP activity lowers plasma HDL-cholesterol by stimulating the plasma turnover and hepatic uptake of HDL cholesteryl esters. HL is not required for the increase in hepatic triglyceride secretion or for the lowering of HDL-cholesterol induced by PLTP overexpression.     

Hepatic lipase purification and characterization

Hepatic lipase has been purified to homogeneity from rat liver homogenates. The purified enzyme exhibits a single band on SDS-polyacrylamide gel electrophoresis. The molecular size of the native hepatic lipase is 200000, while on SDS-polyacrylamide gel electrophoresis the apparent minimum molecular weight of the enzyme is 53000, suggesting that the active enzyme is composed of four subunits. The relationship between triacylglycerol, monoacylglycerol and phospholipid hydrolyzing activities of the purified rat liver enzyme was studied. All three activities had a pH optimum of 8.5. The maximal reaction rates obtained with triolein, monoolein and dipalmitoylphosphatidylcholine were 55000, 66000 and 2600 μmol fatty acid/mg per h with apparent Michaelis constant (K_m) values of 0.4, 0.25 and 1.0 mM, respectively. Hydrolysis of triolein and monoolein probably takes place at the same site on the enzyme molecule, since competitive inhibition between these two substrates was observed, and a similar loss of hydrolytic activity occurred in the presence of diisopropylfluorophosphate. Addition of apolipoproteins C-II and C-I had no effect on the hydrolytic activity of the enzyme with the three substrates tested. However, the triacylglycerol hydrolyzing activity was inhibited by the addition of apolipoprotien C-III. Monospecific antiserum to the pure hepatic lipase has been raised in a rabbit.     

Endothelial lipase is less effective at influencing HDL metabolism in vivo in mice expressing apoA-II

Endothelial lipase (EL) plays an important physiological role in modulating HDL metabolism. Data suggest that plasma contains an inhibitor of EL, and previous studies have suggested that apolipoprotein A-II (apoA-II) inhibits the activity of several enzymes involved in HDL metabolism. Therefore, we hypothesized that apoA-II may reduce the ability of EL to influence HDL metabolism. To test this hypothesis, we determined the effect of EL expression on plasma phospholipase activity and HDL metabolism in human apoA-I and human apoA-I/A-II transgenic mice. Expression of EL in vivo resulted in lower plasma phospholipase activity and significantly less reduction of HDL-cholesterol, phospholipid, and apoA-I levels in apoA-I/A-II double transgenic mice compared with apoA-I single transgenic mice. We conclude that the presence of apoA-II on HDL particles inhibits the ability of EL to influence the metabolism of HDL in vivo.     

Hepatic lipase. Purification and characterization

Hepatic lipase has been purified to homogeneity from rat liver homogenates. The purified enzyme exhibits a single band on SDS-polyacrylamide gel electrophoresis. The molecular size of the native hepatic lipase is 200 000, while on SDS-polyacrylamide gel electrophoresis the apparent minimum molecular weight of the enzyme is 53 000, suggesting that the active enzyme is composed of four subunits. The relationship between triacylglycerol, monoacylglycerol and phospholipid hydrolyzing activities of the purified rat liver enzyme was studied. All three activities had a pH optimum of 8.5. The maximal reaction rates obtained with triolein, monoolein and dipalmitoylphosphatidylcholine were 55 000, 66 000 and 2600 mumol fatty acid/mg per h with apparent Michaelis constant (Km) values of 0.4, 0.25 and 1.0 mM, respectively. Hydrolysis of triolein and monoolein probably takes place at the same site on the enzyme molecule, since competitive inhibition between these two substrates was observed, and a similar loss of hydrolytic activity occurred in the presence of diisopropylfluorophosphate. Addition of apolipoproteins C-II and C-I had no effect on the hydrolytic activity of the enzyme with the three substrates tested. However, the triacylglycerol hydrolyzing activity was inhibited by the addition of apolipoprotein C-III. Monospecific antiserum to the pure hepatic lipase has been raised in a rabbit.     

Evidence that hepatic lipase deficiency in humans is not associated with proatherogenic changes in HDL composition and metabolism

The aim of the present study was to characterize the composition and metabolism of HDL in subjects with complete hepatic lipase (HL) deficiency. Analyses were carried out in three complete and three partial HL-deficient subjects as well as in eight normotriglyceridemic (NTG) and two hypertriglyceridemic controls. Complete HL deficiency was associated with hypertriglyceridemia and with a 3.5-fold increase in HDL-triglyceride (TG) levels. The in vivo kinetics of apolipoprotein A-I (apoA-I) and apoA-II (d < 1.25 g/l) were studied in the fasted state using a primed-constant infusion of l-(5,5,5-D3)leucine for 12 h. Complete HL deficiency was associated with a reduced fractional catabolic rate of apoA-I in the HL-deficient female proband (-47%) and in her two brothers (-21%) compared with gender- and TG-matched controls. Total plasma and HDL from complete HL-deficient patients were able to mediate normal cholesterol efflux from human skin fibroblasts labeled with [3H]cholesterol. Complete HL deficiency was also associated with normal levels of prebeta-migrating apoA-I-containing HDL separated by two-dimensional gel electrophoresis and with an accumulation of large HDL particles compared with NTG controls. These results suggest that HL activity is important for adequate HDL metabolism, although its presence may not be necessary for normal HDL-mediated reverse cholesterol transport.     

Inherited disorders of HDL metabolism and atherosclerosis

PURPOSE OF REVIEW: Genetic disorders of HDL metabolism are rare and, as a result, the assessment of atherosclerosis risk in individuals suffering from these disorders has been difficult. Ultrasound imaging of carotid arteries has provided a tool to assess the risk in hereditary hypo and hyperalphalipoproteinemia. This review gives a comprehensive summary. RECENT FINDINGS: Epidemiological studies have unequivocally shown that HDL cholesterol levels are inversely related to coronary artery disease risk, but the literature concerning genetic disorders of HDL metabolism provides less convincing information. Fortuitously, we were able to directly compare carotid intima media thickness data of substantial numbers of individuals with mutations in either apolipoprotein A-I (apoA-I), ATP binding cassette AI (ABCA1), lecithin: cholesterol acyltransferase (LCAT) or cholesteryl ester transfer protein. These data show that carriers of an apoA-I mutation exhibit the most pronounced accelerated atherosclerosis compared with those carrying mutations in ABCA1 and LCAT. Heterozygosity for a non-sense mutation in cholesteryl ester transfer protein did, by contrast, not distinguish carriers from controls in terms of intima media thickness progression. We will discuss these results in the context of the current literature. SUMMARY: Intima media thickness studies have provided evidence that hypoalphalipoproteinemia due to mutations in apoA-I, ABCA1, and LCAT is associated with increased progression of atherosclerosis. In contrast, hyperalphalipoproteinemia as a result of loss of cholesteryl ester transfer protein function is associated with unaltered atherosclerosis progression compared with family controls. This insight is of interest, since it can assist in the prioritizing of antiatherogenic therapy by increasing HDL cholesterol levels.     

Apolipoprotein A-II regulates HDL stability and affects hepatic lipase association and activity

The effect of apolipoprotein A-II (apoA-II) on the structure and stability of HDL has been investigated in reconstituted HDL particles. Purified human apoA-II was incorporated into sonicated, spherical LpA-I particles containing apoA-I, phospholipids, and various amounts of triacylglycerol (TG), diacylglycerol (DG), and/or free cholesterol. Although the addition of PC to apoA-I reduces the thermodynamic stability (free energy of denaturation) of its alpha-helices, PC has the opposite effect on apoA-II and significantly increases its helical stability. Similarly, substitution of apoA-I with various amounts of apoA-II significantly increases the thermodynamic stability of the particle alpha-helical structure. ApoA-II also increases the size and net negative charge of the lipoprotein particles. ApoA-II directly affects apoA-I conformation and increases the immunoreactivity of epitopes in the N and C termini of apoA-I but decreases the exposure of central domains in the molecule (residues 98-186). ApoA-II appears to increase HL association with HDL and inhibits lipid hydrolysis. ApoA-II mildly inhibits PC hydrolysis in TG-enriched particles but significantly inhibits DG hydrolysis in DG-rich LpA-I. In addition, apoA-II enhances the ability of reconstituted LpA-I particles to inhibit VLDL-TG hydrolysis by HL. Therefore, apoA-II affects both the structure and the dynamic behavior of HDL particles and selectively modifies lipid metabolism.     

HDL: structure, function and metabolism.

The major advances in our knowledge of the structure, function and metabolism of the plasma lipoproteins have occurred as a result of the rapid increase in our knowledge of the structure and function of the apolipoproteins, lipoproteins, and the heterogeneity of the individual classes of lipoproteins. Over the last decade, there has been a tremendous increase in our knowledge of the structure and molecular properties of ApoA-I and ApoA-II which has permitted an analysis of the functions of these apolipoproteins in lipid and lipoprotein metabolism and the initiation of kinetic studies of HDL metabolism. The elucidation of the structures of the ApoA-I and ApoA-II genes has permitted the determination of genetic defects resulting in decreased levels of HDL and premature cardiovascular disease, as well as the identification of new diseases (e.g. hereditary systemic amyloidosis). The future focus of research on HDL will be the analysis of the individual lipoprotein particles within HDL which have different physiological functions and important roles in reverse cholesterol transport. An improved understanding of the role of HDL in the transport of cellular cholesterol to the liver and the exchange of cholesterol between plasma lipoproteins will provide critical information on cholesterol metabolism in normal subjects and permit the elucidation of the molecular defects of new genetic diseases which may be associated with the development of premature cardiovascular disease.     

A high-throughput screen for endothelial lipase using HDL as substrate

Endothelial lipase (EL) is a 482-amino-acid protein from the triglyceride lipase gene family that uses a Ser-His-Asp triad for catalysis. Its expression in endothelial cells and preference for phospholipids rather than triglycerides are unique. Animal models in which it is overexpressed or knocked out indicate EL levels are inversely correlated with high-density lipoprotein cholesterol (HDL-C). HDL-C is commonly referred to as the good form of cholesterol because it is involved in the reverse cholesterol transport pathway, in which excess cholesterol is effluxed from peripheral tissues for excretion or reabsorption. Thus, EL inhibition in humans is expected to lead to increases in HDL levels and possibly a decrease in cardiovascular disease. To discover inhibitors of EL, a coupled assay for EL has been developed, using its native substrate, HDL. Hydrolysis of HDL by EL yields free fatty acids, which are coupled through acyl-CoA synthetase, acyl-CoA oxidase, and horseradish peroxidase to produce the fluorescent species resorufin. This assay was developed into a 5-microL, 1536-well assay format, and a high-throughput screen was executed against the GSK collection. In addition to describing the screening results, novel post-HTS mechanism-of-action studies were developed for EL and applied to 1 of the screening hits as an example.     

Effect of rosiglitazone on HDL metabolism in subjects with metabolic syndrome and low HDL

Treatment with the peroxisome proliferator-activated receptor γ agonist rosiglitazone has been reported to increase HDL-cholesterol (HDL-C) levels, although the mechanism responsible for this is unknown. We sought to determine the effect of rosiglitazone on HDL apolipoprotein A-I (apoA-I) and apoA-II metabolism in subjects with metabolic syndrome and low HDL-C. Subjects were treated with placebo followed by rosiglitazone (8 mg) once daily. At the end of each 8 week treatment, subjects (n = 15) underwent a kinetic study to measure apoA-I and apoA-II production rate (PR) and fractional catabolic rate. Rosiglitazone significantly reduced fasting insulin and high-sensitivity C-reactive protein (hsCRP) and increased apoA-II levels. Mean apoA-I and HDL-C levels were unchanged following rosiglitazone treatment, although there was considerable individual variability in the HDL-C response. Rosiglitazone had no effect on apoA-I metabolism, whereas the apoA-II PR was increased by 23%. The change in HDL-C in response to rosiglitazone was significantly correlated with the change in apoA-II concentration but not to changes in apoA-I, measures of glucose homeostasis, or hsCRP. Treatment with rosiglitazone significantly increased apoA-II production in subjects with metabolic syndrome and low HDL-C but had no effect on apoA-I metabolism. The change in HDL-C in response to rosiglitazone treatment was unrelated to effects on apoA-I, instead being related to the change in the metabolism of apoA-II.     

Hepatic and extrahepatic uptake of intravenously injected lipoprotein lipase

Rats were injected intravenously with 125I-labeled bovine lipoprotein lipase. The lipase disappeared within minutes from the blood due to uptake both in the liver (about 50% of the injected dose) and in extrahepatic tissues. Lipase enzyme activity disappeared in parallel to the 125I radioactivity. Thus, there was no inactivation of lipase in the circulating blood. Similar results were obtained when lipoprotein lipase purified from guinea pigs was injected into guinea pigs. Using supradiphragmatic rats we could show that the extrahepatic uptake was saturable and that the amounts of lipase that could be bound far exceeded the amounts of endogenous lipase expected to be present on the endothelium. When the lipase was denatured before injection, its removal in supradiaphragmatic rats became slower, and in intact rats the fraction of the uptake that occurred in extrahepatic tissues was much decreased. It is concluded that recognition by the extrahepatic receptors depends on the native conformation of the lipase. The extrahepatic uptake was strongly impeded by injection of heparin prior to injection of the lipase, and the uptake could to a large extent be reversed by injection of heparin after the lipase. Even after 1 h lipase that had been taken up by extrahepatic tissues reappeared immediately in the blood on injection of heparin. This was true both for enzyme activity and for enzyme radioactivity. Thus, internalization-inactivation-degradation occur only slowly in extrahepatic tissues. It is possible that the extrahepatic binding occurs to the enzyme's physiological receptors. The hepatic uptake was not dependent on the native conformation of the lipase, was less sensitive to heparin, could not be reversed by heparin and was not saturable. The enzyme was not rapidly inactivated after uptake; its activity could be detected in liver homogenates even after 1 h. Degradation to acid-soluble products in the liver was relatively slow; the t1/2 for native lipase was about 1 h. In comparison, in parallel experiments asialofetuin was degraded with a t1/2 of about 15 min.     

Hepatic endothelial lipase activity in neonatal rat liver

Hepatic endothelial lipase (HEL) activity is as high in the neonatal (1-day old) rat liver as in adults. Most of the HEL activity is located at the capillaries since 75% of the total activity is released by heparin or collagenase perfusion. The residual activity (non-releasable) is located in hepatocytes and not in hemopoietic cells, which are the major cell type in neonatal liver. Per mg of protein, the HEL activity is 50% higher in neonatal than in adult hepatocytes. We suggest that neonatal hepatocytes have an increased capacity to synthesize and secrete HEL activity, so maintaining a high activity in the whole organ. it might contribute to the hepatic uptake of cholesterol from circulating lipoproteins, in a period in which endogenous cholesterol synthesis is known to be inhibited in the liver.