Gene_locus Report for: human-LIPC

Human hepatic lipase (hHL) is a cell surface associated enzyme that hydrolyzes triacylglycerols and phospholipids within circulating lipoproteins. We hypothesized that an amino acid sequence mimicking the major heparin binding domain (HBD) of hHL will displace hHL from cell surfaces. To test this hypothesis, we generated a recombinant protein of thioredoxin linked with a cleavable, tagged sequence containing amino acids 442 to 476 of the mature hHL sequence, which contains the major HBD of hHL. The recombinant protein associated with heparin-sepharose, and its peak elution from heparin-sepharose occurred in the presence of 0.5 M NaCl. We cleaved and purified the tagged sequence containing the HBD from the recombinant protein and tested the ability of the peptide to displace full-length hHL from HEK-293 cells. The peptide indeed displaced hHL from cell surfaces, while no significant displacement was observed in the presence of a peptide with a scrambled sequence. Finally, we obtained structural information for the peptide containing the HBD. (1) H- and (15) N-NMR spectra of the peptide indicate the peptide is largely unstructured, although not completely random coil. The addition of heparin to the peptide induced some changes in chemical shift, suggesting changes in peptide structure and/or specific interactions with heparin. Molecular simulations confirm the largely unstructured nature of the isolated peptide, but they also indicate weak tendencies for both alpha- and beta-structure formation in different parts of the chain. Overall, these data provide a proof-of-principle for the use of mimetic peptides for the displacement of cell surface associated lipases.

BACKGROUND: The relationship between plasma lipoprotein lipase (LPL), hepatic triglyceride lipase (HTGL), glycosylphosphatidylinositol anchored HDL binding protein1 (GPIHBP1) concentration and the metabolism of remnant lipoproteins (RLP) and small dense LDL (sdLDL) in patients with coronary artery disease (CAD) is not fully elucidated. METHODS: One hundred patients who underwent coronary angiography were enrolled. The plasma LPL, HTGL and GPIHBP1 concentrations were determined by ELISA. The time dependent changes in those lipases, lipids and lipoproteins were studied at a time-point just before, and 15min, 4h and 24h after heparin administration. RESULTS: The LPL concentration exhibited a significant positive correlation with HDL-C, and inversely correlated with TG and RLP-C. The HTGL concentration was positively correlated with RLP-C and sdLDL-C. The HTGL ratio of the pre-heparin/post-heparin plasma concentration and sdLDL-C/LDL-C ratio were significantly greater in CAD patients than in non-CAD patients. GPIHBP1 was positively correlated with LPL and inversely correlated with RLP-C and sdLDL-C. CONCLUSION: The HTGL concentration was positively correlated with RLP-C and sdLDL-C, while LPL and GPIHBP1 were inversely correlated with RLP-C and sdLDL-C. These results suggest that elevated HTGL is associated with increased CAD risk, while elevated LPL is associated with a reduction of CAD risk.

BACKGROUND: Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol levels. Only a few genes causally related to plasma LDL cholesterol levels have been identified so far, and only 1 gene, ANGPTL3, has been causally related to combined hypocholesterolemia. Here, our aim was to elucidate the genetic origin of an unexplained combined hypocholesterolemia inherited in 4 generations of a French family. METHODS: Using next-generation sequencing, we identified a novel dominant rare variant in the LIPC gene, encoding for hepatic lipase, which cosegregates with the phenotype. We characterized the impact of this LIPC-E97G variant on circulating lipid and lipoprotein levels in family members using nuclear magnetic resonance-based lipoprotein profiling and lipidomics. To uncover the mechanisms underlying the combined hypocholesterolemia, we used protein homology modeling, measured triglyceride lipase and phospholipase activities in cell culture, and studied the phenotype of APOE*3.Leiden.CETP mice after LIPC-E97G overexpression. RESULTS: Family members carrying the LIPC-E97G variant had very low circulating levels of LDL cholesterol and high-density lipoprotein cholesterol, LDL particle numbers, and phospholipids. The lysophospholipids/phospholipids ratio was increased in plasma of LIPC-E97G carriers, suggestive of an increased lipolytic activity on phospholipids. In vitro and in vivo studies confirmed that the LIPC-E97G variant specifically increases the phospholipase activity of hepatic lipase through modification of an evolutionarily conserved motif that determines substrate access to the hepatic lipase catalytic site. Mice overexpressing human LIPC-E97G recapitulated the combined hypocholesterolemic phenotype of the family and demonstrated that the increased phospholipase activity promotes catabolism of triglyceride-rich lipoproteins by different extrahepatic tissues but not the liver. CONCLUSIONS: We identified and characterized a novel rare variant in the LIPC gene in a family who presents with dominant familial combined hypocholesterolemia. This gain-of-function variant makes LIPC the second identified gene, after ANGPTL3, causally involved in familial combined hypocholesterolemia. Our mechanistic data highlight the critical role of hepatic lipase phospholipase activity in LDL cholesterol homeostasis and suggest a new LDL clearance mechanism.

Human hepatic lipase (hHL) is a cell surface associated enzyme that hydrolyzes triacylglycerols and phospholipids within circulating lipoproteins. We hypothesized that an amino acid sequence mimicking the major heparin binding domain (HBD) of hHL will displace hHL from cell surfaces. To test this hypothesis, we generated a recombinant protein of thioredoxin linked with a cleavable, tagged sequence containing amino acids 442 to 476 of the mature hHL sequence, which contains the major HBD of hHL. The recombinant protein associated with heparin-sepharose, and its peak elution from heparin-sepharose occurred in the presence of 0.5 M NaCl. We cleaved and purified the tagged sequence containing the HBD from the recombinant protein and tested the ability of the peptide to displace full-length hHL from HEK-293 cells. The peptide indeed displaced hHL from cell surfaces, while no significant displacement was observed in the presence of a peptide with a scrambled sequence. Finally, we obtained structural information for the peptide containing the HBD. (1) H- and (15) N-NMR spectra of the peptide indicate the peptide is largely unstructured, although not completely random coil. The addition of heparin to the peptide induced some changes in chemical shift, suggesting changes in peptide structure and/or specific interactions with heparin. Molecular simulations confirm the largely unstructured nature of the isolated peptide, but they also indicate weak tendencies for both alpha- and beta-structure formation in different parts of the chain. Overall, these data provide a proof-of-principle for the use of mimetic peptides for the displacement of cell surface associated lipases.

INTRODUCTION AND AIM: Hepatocellular carcinoma (HCC) is a lethal malignancy, but the molecular mechanisms of hepatocarcinogenesis remain undefined. The present study aims to investigate the relationship between polymorphisms of the hepatic lipase (HL) gene promoters and risk of HCC. MATERIAL AND METHODS: Totally, 279 HCC patients and 200 healthy individuals were enrolled. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) was used to analyze the genotypes of HL gene. Logistic regression analysis was conducted to identify risk factors of HCC. RESULTS: There was significant difference in the distribution of smoking history, drinking history, and family history of subjects between the case and control groups (all p < 0.05). Difference in the -250G/A (p = 0.011; OR = 1.61; 95%CI: 1.11-2.34) and -514C/T (p = 0.007; OR = 1.65; 95%CI: 1.14-2.38) genotypes and allele frequencies between two groups was significant. A higher risk of HCC was identified in those with polymorphisms in the - 250G/A (p = 0.007; OR = 1.45; 95%CI: 1.11-1.89) and -514C/T (p = 0.003; OR = 1.51; 95%CI: 1.15-2.00). Polymorphisms at - 250G/A (GA + AA) (p = 0.025; OR = 1.55; 95%CI: 1.06-2.28), -514C/T (CT + TT) (p = 0.021; OR = 1.57; 95%CI: 1.07-2.29), smoking history (p = 0.017; OR = 1.70; 95%CI: 1.10-2.63) and drinking history (p = 0.003; OR = 2.04; 95%CI: 1.27-3.27) were significantly related to the risk of HCC (all p < 0.05). CONCLUSION: The results obtained from this study indicated that polymorphisms of -250G/A and -514C/T in HL gene promoters were associated with the risk of HCC.

KEY POINTS: Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) are the key enzymes involved in intramuscular triglyceride (IMTG) lipolysis. In isolated rat skeletal muscle, HSL translocates to IMTG-containing lipid droplets (LDs) following electrical stimulation, but whether HSL translocation occurs in human skeletal muscle during moderate-intensity exercise is currently unknown. Perilipin-2 (PLIN2) and perilipin-5 (PLIN5) proteins have been implicated in regulating IMTG lipolysis by interacting with HSL and ATGL in cell culture and rat skeletal muscle studies. This study investigated the hypothesis that HSL (but not ATGL) redistributes to LDs during moderate-intensity exercise in human skeletal muscle, and whether the localisation of these lipases with LDs was affected by the presence of PLIN proteins on the LDs. HSL preferentially redistributed to PLIN5-associated LDs whereas ATGL distribution was not altered with exercise; this is the first study to illustrate the pivotal step of HSL redistribution to PLIN5-associated LDs following moderate-intensity exercise in human skeletal muscle. ABSTRACT: Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) control skeletal muscle lipolysis. ATGL is present on the surface of lipid droplets (LDs) containing intramuscular triglyceride (IMTG) in both the basal state and during exercise. HSL translocates to LD in ex vivo electrically stimulated rat skeletal muscle. Perilipin-2- and perilipin-5-associated lipid droplets (PLIN2+ and PLIN5+ LDs) are preferentially depleted during exercise in humans, indicating that these PLINs may control muscle lipolysis. We aimed to test the hypothesis that in human skeletal muscle in vivo HSL (but not ATGL) is redistributed to PLIN2+ and PLIN5+ LDs during moderate-intensity exercise. Muscle biopsies from 8 lean trained males (age 21 +/- 1 years, BMI 22.6 +/- 1.2 kg m(-2) and VO2 peak 48.2 +/- 5.0 ml min(-1) kg(-1) ) were obtained before and immediately following 60 min of cycling exercise at approximately 59% VO2 peak . Cryosections were stained using antibodies targeting ATGL, HSL, PLIN2 and PLIN5. LDs were stained using BODIPY 493/503. Images were obtained using confocal immunofluorescence microscopy and object-based colocalisation analyses were performed. Following exercise, HSL colocalisation to LDs increased (P < 0.05), and was significantly greater to PLIN5+ LDs (+53%) than to PLIN5- LDs (+34%) (P < 0.05), while the increases in HSL colocalisation to PLIN2+ LDs (+16%) and PLIN2- LDs (+28%) were not significantly different. Following exercise, the fraction of LDs colocalised with ATGL (0.53 +/- 0.04) did not significantly change (P < 0.05) and was not affected by PLIN association to the LDs. This study presents the first evidence of exercise-induced HSL redistribution to LDs in human skeletal muscle and identifies PLIN5 as a facilitator of this mechanism.

BACKGROUND: The incidental finding of severe hypertriglyceridemia (HyperTG) in a child may suggest the diagnosis of familial chylomicronemia syndrome (FCS), a recessive disorder of the intravascular hydrolysis of triglyceride (TG)-rich lipoproteins. FCS may be due to pathogenic variants in lipoprotein lipase (LPL), as well as in other proteins, such as apolipoprotein C-II and apolipoprotein A-V (activators of LPL), GPIHBP1 (the molecular platform required for LPL activity on endothelial surface) and LMF1 (a factor required for intracellular formation of active LPL). OBJECTIVE: Molecular characterization of 5 subjects in whom HyperTG was an incidental finding during infancy/childhood. METHODS: We performed the parallel sequencing of 20 plasma TG-related genes. RESULTS: Three children with severe HyperTG were found to be compound heterozygous for rare pathogenic LPL variants (2 nonsense, 3 missense, and 1 splicing variant). Another child was found to be homozygous for a nonsense variant of APOA5, which was also found in homozygous state in his father with longstanding HyperTG. The fifth patient with a less severe HyperTG was found to be heterozygous for a frameshift variant in LIPC resulting in a truncated Hepatic Lipase. In addition, 1 of the patients with LPL deficiency and the patient with APOA-V deficiency were also heterozygous carriers of a pathogenic variant in LIPC and LPL gene, respectively, whereas the patient with LIPC variant was also a carrier of a rare APOB missense variant. CONCLUSIONS: Targeted parallel sequencing of TG-related genes is recommended to define the molecular defect in children presenting with an incidental finding of HyperTG.

BACKGROUND: The relationship between plasma lipoprotein lipase (LPL), hepatic triglyceride lipase (HTGL), glycosylphosphatidylinositol anchored HDL binding protein1 (GPIHBP1) concentration and the metabolism of remnant lipoproteins (RLP) and small dense LDL (sdLDL) in patients with coronary artery disease (CAD) is not fully elucidated. METHODS: One hundred patients who underwent coronary angiography were enrolled. The plasma LPL, HTGL and GPIHBP1 concentrations were determined by ELISA. The time dependent changes in those lipases, lipids and lipoproteins were studied at a time-point just before, and 15min, 4h and 24h after heparin administration. RESULTS: The LPL concentration exhibited a significant positive correlation with HDL-C, and inversely correlated with TG and RLP-C. The HTGL concentration was positively correlated with RLP-C and sdLDL-C. The HTGL ratio of the pre-heparin/post-heparin plasma concentration and sdLDL-C/LDL-C ratio were significantly greater in CAD patients than in non-CAD patients. GPIHBP1 was positively correlated with LPL and inversely correlated with RLP-C and sdLDL-C. CONCLUSION: The HTGL concentration was positively correlated with RLP-C and sdLDL-C, while LPL and GPIHBP1 were inversely correlated with RLP-C and sdLDL-C. These results suggest that elevated HTGL is associated with increased CAD risk, while elevated LPL is associated with a reduction of CAD risk.

Familial combined hyperlipidemia (FCH) is a complex and common familial dyslipidemia characterized by elevated total cholesterol and/or triglyceride levels with over five-fold risk of coronary heart disease. The genetic architecture and contribution of rare Mendelian and common variants to FCH susceptibility is unknown. In 53 Finnish FCH families, we genotyped and imputed nine million variants in 715 family members with DNA available. We studied the enrichment of variants previously implicated with monogenic dyslipidemias and/or lipid levels in the general population by comparing allele frequencies between the FCH families and population samples. We also constructed weighted polygenic scores using 212 lipid-associated SNPs and estimated the relative contributions of Mendelian variants and polygenic scores to the risk of FCH in the families. We identified, across the whole allele frequency spectrum, an enrichment of variants known to elevate, and a deficiency of variants known to lower LDL-C and/or TG levels among both probands and affected FCH individuals. The score based on TG associated SNPs was particularly high among affected individuals compared to non-affected family members. Out of 234 affected FCH individuals across the families, seven (3%) carried Mendelian variants and 83 (35%) showed high accumulation of either known LDL-C or TG elevating variants by having either polygenic score over the 90th percentile in the population. The positive predictive value of high score was much higher for affected FCH individuals than for similar sporadic cases in the population. FCH is highly polygenic, supporting the hypothesis that variants across the whole allele frequency spectrum contribute to this complex familial trait. Polygenic SNP panels improve identification of individuals affected with FCH, but their clinical utility remains to be defined.

UNLABELLED: Protein phosphorylation is one of the most common post-translational modifications. It plays key roles in regulating diverse biological processes of liver tissues. To better understand the role of protein phosphorylation in liver functions, it is essential to perform in-depth phosphoproteome analysis of human liver. Here, an enzyme assisted reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach with both RPLC separations operated with optimized acidic mobile phase was developed. High orthogonal separation was achieved by trypsin digestion of the Glu-C generated peptides in the fractions collected from the first RPLC separation. The phosphoproteome coverage was further improved by using two types of instruments, i.e. TripleTOF 5600 and LTQ Orbitrap Velos. A total of 22,446 phosphorylation sites, corresponding to 6526 nonredundant phosphoproteins were finally identified from normal human liver tissues. Of these sites, 15,229 sites were confidently localized with Ascore>/=13. This dataset was the largest phosphoproteome dataset of human liver. It can be a public resource for the liver research community and holds promise for further biology studies. BIOLOGICAL SIGNIFICANCE: The enzyme assisted approach enabled the two RPLC separations operated both with optimized acidic mobile phases. The identifications from TripleTOF 5600 and Orbitrap Velos are highly complementary. The largest phosphoproteome dataset of human liver was generated.

OBJECTIVE: To assess the phospholipase activity of endothelial (EL) and hepatic lipase (HL) in postheparin plasma of subjects with metabolic syndrome (MS)/obesity and their relationship with atherogenic and antiatherogenic lipoproteins. Additionally, to evaluate lipoprotein lipase (LPL) and HL activity as triglyceride (TG)-hydrolyses to complete the analyses of SN1 lipolytic enzymes in the same patient. APPROACH AND
RESULTS: Plasma EL, HL, and LPL activities were evaluated in 59 patients with MS and 36 controls. A trend toward higher EL activity was observed in MS. EL activity was increased in obese compared with normal weight group (P=0.009) and was negatively associated with high-density lipoprotein-cholesterol (P=0.014 and P=0.005) and apolipoprotein A-I (P=0.045 and P=0.001) in control and MS group, respectively. HL activity, as TG-hydrolase, was increased in MS (P=0.025) as well as in obese group (P=0.017); directly correlated with low-density lipoprotein-cholesterol (P=0.005) and apolipoprotein B (P=0.003) and negatively with high-density lipoprotein-cholesterol (P=0.021) in control group. LPL was decreased in MS (P<0.001) as well as in overweight and obese compared with normal weight group (P=0.015 and P=0.004, respectively); inversely correlated %TG-very low-density lipoproteins (P=0.04) and TG/apolipoprotein B index (P=0.013) in control group. These associations were not found in MS.
CONCLUSIONS: We describe for the first time EL and HL activity as phospholipases in MS/obesity, being both responsible for high-density lipoprotein catabolism. Our results elucidate part of the remaining controversies about SN1 lipases activity in MS and different grades of obesity. The impact of insulin resistance on the activity of the 3 enzymes determines the lipoprotein alterations observed in these states.

Hepatic lipase (HL) and endothelial lipase (EL) share overlapping and complementary roles in lipoprotein metabolism. The deletion of HL and EL alleles in mice raises plasma total cholesterol and phospholipid concentrations. However, the influence of HL and EL in vivo on individual molecular species from each class of lipid is not known. We hypothesized that the loss of HL, EL, or both in vivo may affect select molecular species from each class of lipids. To test this hypothesis, we performed lipidomic analyses on plasma and livers from fasted female wild-type, HL-knockout, EL-knockout, and HL/EL-double knockout mice. Overall, the loss of HL, EL, or both resulted in minimal changes to hepatic lipids; however, select species of CE were surprisingly reduced in the livers of mice only lacking EL. The loss of HL, EL, or both reduced the plasma concentrations for select molecular species of triacylglycerol, diacylglycerol, and free fatty acid. On the other hand, the loss of HL, EL, or both raised the plasma concentrations for select molecular species of phosphatidylcholine, cholesteryl ester, diacylglycerol, sphingomyelin, ceramide, plasmanylcholine, and plasmenylcholine. The increased plasma concentration of select ether phospholipids was evident in the absence of EL, thus suggesting that EL might exhibit a phospholipase A2 activity. Using recombinant EL, we showed that it could hydrolyse the artificial phospholipase A2 substrate 4-nitro-3-(octanoyloxy)benzoic acid. In summary, our study shows for the first time the influence of HL and EL on individual molecular species of several classes of lipids in vivo using lipidomic methods.

BACKGROUND: Variants in the CETP and the LIPC genes, encoding cholesteryl ester transfer protein and hepatic lipase, respectively, are associated with high levels of HDL-cholesterol or hyperalphalipoproteinemia (HALP). Recently, we have identified three novel variants in the CETP promoter and two novel variants in LIPC in Thai subjects with HALP. In this study, we investigated the functions of these 5 variants in vitro.
METHODS: For CETP promoter variants, we used site-directed mutagenesis, transient expression in HepG2 cells and luciferase reporter assay. For LIPC variants, cDNA was cloned and mutagenesis for missense variants was performed before expression in HepG2 cells.
RESULTS: The transcriptional activities of -49G>T,-70C>T, and -372C>T CETP promoter variants were markedly reduced (5%, 8% and 30%, respectively, compared to that of the wild-type, P<0.001). For LIPC variants, hepatic lipase activities in the lysates of cells transfected with c.421A>G (p.G141S) and c.517G>A (p.V173M) variants were 41% and 46%, respectively, compared to that of the wild-type (P<0.05).
CONCLUSIONS: The recently-identified variants in the CETP promoter and in the LIPC gene may contribute to HALP. Our result may have a diagnostic application in the genetic evaluation of subjects with high HDL-cholesterol levels.

BACKGROUND The T allele of the hepatic lipase HL C-514T polymorphism was previously found to be associated with lower plasma HL activity Here we examined the association between this polymorphism and plasma HDL-cholesterol concentrations in patients with coronary arteries stenosis METHODS We studied 342 subjects undergoing coronary angiography in two groups of non CAD n=146 and CAD n=196 514C-->T polymorphism was determined using polymerase chain reaction and restriction fragment length polymorphism PCR-RFLP RESULTS After adjustment for age smoking and body mass index HDL-cholesterol concentrations were significantly higher in men with the C/T&T/T genotype than those with the C/C genotype(mean 38.6 and 34.7 respectively P=0.01 The frequency of T allele in non CAD was 0.136 and 0.226 in female and male respectively and 0.170 and 0.223 for female and male in CAD subjects There was no difference in T allele frequency in CAD and none CAD groups in male and female P=0.466 and 0.722 respectively CONCLUSION 514C-->T of LIPC gene have a positive effect on HDL-C concentration especially in male gender However no difference was determined in frequency of T allele between CAD and normal arteries subjects.

Genetic factors associated with hyperalphalipoproteinemia (HALP; or high levels of high-density lipoprotein cholesterol) are incompletely understood. The aim of this study was to resequence 3 candidate genes, CETP, LIPC, and LIPG, which encode cholesteryl ester transfer protein, hepatic lipase, and endothelial lipase, respectively, in Thai subjects with HALP and compare them to normolipidemic controls. Sequence variants of CETP, LIPC, and LIPG were identified by sequencing exons and exon-intron junctions in 64 subjects with high-density lipoprotein cholesterol levels >/=2.59 mmol/L (100 mg/dl) and compared to those of 113 normolipidemic subjects. Two heterozygous frameshift mutations in CETP (p.Leu262ProfsX31 and p.Val411ArgfsX6) and two heterozygous missense mutations in LIPC (p.Gly141Ser and p.Val173Met) were found. One deletion mutation and 3 point mutations in the CETP promoter were also identified. Collectively, these rare mutations were found only in the HALP group but not in the control group (8% vs 0%, p = 0.0056). One common variant of CETP (p.Asp459Gly) was found at a higher frequency in the HALP group (23% vs 4%, p = 0.000074). Altogether, rare variants of CETP or LIPC and/or the common CETP p.Asp459Gly variant were found in 30% of the HALP group and 4% of the controls (p = 0.0000014). No rare variant of LIPG was identified. In conclusion, common and rare genetic variants in CETP and LIPC, but not LIPG, were more commonly found in the Thai HALP group, which could potentially contribute to high high-density lipoprotein cholesterol phenotypes in this population.

Reverse cholesterol transport (RCT) constitutes a key part of the atheroprotective properties of high-density lipoproteins (HDL). Hepatic lipase (HL) and endothelial lipase (EL) are negative regulators of plasma HDL cholesterol levels. Although overexpression of EL decreases overall macrophage-to-feces RCT, knockout of both HL and EL leaves RCT essentially unaffected. With respect to important individual steps of RCT, current data on the role of EL and HL in cholesterol efflux are not conclusive. Both enzymes increase hepatic selective cholesterol uptake; however, this does not translate into altered biliary cholesterol secretion, which is regarded the final step of RCT. Also, the impact of HL and EL on atherosclerosis is not clear cut; rather it depends on respective experimental conditions and chosen models. More mechanistic insights into the diverse biological properties of these enzymes are therefore required to firmly establish EL and HL as targets for the treatment of atherosclerotic cardiovascular disease.

Hepatic lipase (HL) deficiency is a rare genetic disorder that has been associated with premature atherosclerosis despite high plasma high-density lipoprotein (HDL) cholesterol concentrations in the affected individuals. The authors describe the clinical and biochemical features of HL deficiency in a young male of Middle-Eastern-Arabic origin. This is the first report of cholesterol ester transfer protein (CETP) activity and mass in HL deficiency in a patient from this ethnic group. While the CETP mass was high, its activity was low, a discrepancy likely due to the abnormal composition of patient's HDL particles.

RATIONALE: Hepatic lipase (HL) and endothelial lipase (EL) are extracellular lipases that both hydrolyze triglycerides and phospholipids and display potentially overlapping or complementary roles in lipoprotein metabolism. OBJECTIVE: We sought to dissect the overlapping roles of HL and EL by generating mice deficient in both HL and EL (HL/EL-dko) for comparison with single HL-knockout (ko) and EL-ko mice, as well as wild-type mice. METHODS AND RESULTS: Reproduction and viability of the HL/EL-dko mice were impaired compared with the single-knockout mice. The plasma levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, non-HDL cholesterol, and phospholipids in the HL/EL-dko mice were markedly higher than those in the single-knockout mice. Most notably, the HL/EL-dko mice exhibited an unexpected substantial increase in small low-density lipoproteins. Kinetic studies with [(3)H]cholesteryl ether-labeled very-low-density lipoproteins demonstrated that the HL/EL-dko mice accumulated counts in the smallest low-density lipoprotein-sized fractions, as assessed by size exclusion chromatography, suggesting that it arises from lipolysis of very-low-density lipoproteins. HDL from all 3 lipase knockout models had an increased cholesterol efflux capacity but reduced clearance of HDL cholesteryl esters versus control mice. Despite their higher HDL cholesterol levels, neither HL-ko, EL-ko, nor HL/EL-dko mice demonstrated an increased rate of macrophage reverse cholesterol transport in vivo. CONCLUSIONS: These studies reveal an additive effect of HL and EL on HDL metabolism but not macrophage reverse cholesterol transport in mice and an unexpected redundant role of HL and EL in apolipoprotein B lipoprotein metabolism.

Hepatic lipase (LIPC) and cholesteryl ester transfer protein (CETP) are important components of high-density lipoprotein (HDL) metabolism and reverse cholesterol transport. Therefore, their genes are promising candidate genes for cardiovascular disease. The aim of the present study was to investigate whether combined LIPC -514C/T and CETP I405V polymorphisms correlate with angiographically documented coronary artery disease (CAD). Genotyping was performed in 317 patients who underwent clinically indicated coronary angiography. The patients were classified with significantly diseased arteries if one or more coronary arteries had a stenosis >50% and with minimally diseased arteries if there was no significant stenosis (<40%) in any artery. There were no significant associations of individual polymorphisms with the risk of significant CAD. In a multivariate logistic regression analysis including cardiovascular risk factors, simultaneous presence of both LIPC -514T and CETP 405V alleles was an independent predictor of significantly diseased arteries (odds ratio = 2.04; p = 0.022). This association was not significant in women with combined genotype who had the highest HDL-cholesterol. In conclusion, the combined T allele of LIPC -514C/T and V allele of CETP I405V are associated with the risk of CAD. Further, the higher HDL-cholesterol and female gender may reduce the effect of combined genotype on CAD risk.

Previous studies have shown that CETP and LIPC mutations contribute to hyperalphalipoproteinemia (HALP) in some populations. We investigated whether activities in cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) contribute to HALP in the Thai population and performed genetic analyses of the CETP and LIPC genes. We recruited 38 individuals with high-density lipoprotein cholesterol (HDL-C) levels of at least 2.59 mmol/L (100 mg/dL) (HALP group) and an equal number of individuals with normal serum HDL-C levels (control group). The CETP and HL activities were determined in both groups. Genetic analyses covering all the coding regions and exon-intron junctions of the CETP and LIPC genes were performed in subjects who had low CETP activity and HL activity, respectively. The mean CETP and HL activities were significantly lower in the HALP group than in the control group (34 +/- 4 vs 44 +/- 3 pmol/[microL h], P = .04 and 150 +/- 17 vs 227 +/- 16 nmol free fatty acid/[mL min] P = .002, respectively). Of the 38 individuals with HALP, 19 and 16 were found to have low CETP activity and HL activity, respectively. Of the 19 subjects with low CETP activity, 6 subjects were found to be heterozygous for a known functionally relevant c.1325A>G (D442G) mutation. The other subject was found to be heterozygous for a novel deletion mutation, c.734_737delTCCC mutation. Of the 16 subjects with low HL activity, 8 and 2 subjects were found to be heterozygous for known variants, c.283 G>A (V73M) and c.1068A>C (L334F), respectively. These variants have previously been shown not to be associated with HALP. Another subject was found to be heterozygous for a novel missense mutation, c.421G>A (G119S). Its amino acid change, absence in controls, evolutionary conservation, occurrence in functionally important domain, and predicted damaging function suggested that the G119S mutation is functionally relevant. Two novel mutations in the CETP and LIPC genes found in this study are likely to be the causes of low enzyme activities and elevated HDL-C levels.

OBJECTIVE: To investigate the association of hepatic lipase -250G/A gene promoter polymorphism with type 2 diabetes mellitus combining with coronary heart disease. METHODS: Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), we detected the genotypes of the hepatic lipase gene promoter -250G/A, the effect of this polymorphism on plasma lipids, lipoproteins and apolipoproteins in 364 patients with type 2 diabetes mellitus and coronary heart disease(T2DM+CHD), 357 patients with type 2 diabetes mellitus alone(T2DM) and 356 healthy controls. RESULTS: The frequencies of alleles and genotypes in the T2DM group were not significantly different from that of controls. However, the AA and GA genotypes in the T2DM+CHD group were lower than those in controls (0.431vs 0.618, P=0.031). The frequencies of both allele and genotype were not related to gender, family history, smoking and BMI. When adjusted by factors such as gender, age, BMI, history of smoking, family history of coronary atherosclerosis and systemic hypertension, Spearmanos correlation and linear regression analyses showed that the A allele is related positively to the levels of HDL-C and apoA1 in T2DM and T2DM+CHD patients. However, logistic regression analysis showed that the A allele is one risk factor for the presence of coronary heart disease. CONCLUSION: The hepatic lipase gene promoter -250G/A polymorphisms is associated with type 2 diabetes mellitus with coronary heart disease, its polymorphisms may affect the levels of HDL-C and apoA1.

BACKGROUND AND AIM: The -250G/A promoter polymorphism of the hepatic lipase gene has been associated with changes in the activity of the enzyme. We investigated whether this polymorphism modifies the postprandial response of triacylglycerol-rich lipoproteins (TRL) in young normolipemic males. METHODS AND RESULTS: Fifty-one healthy apolipoprotein (apo) E3/E3 male volunteers (30 G/G and 21 carriers of the A allele) underwent a vitamin A fat-loading test and blood samples were drawn every hour until the 6th, and every 2h and 30 min until the 11th. Total plasma cholesterol and triacylglycerols (TG), as well as cholesterol, TG and retinyl palmitate (RP) in TRL, isolated by ultracentrifugation, were determined. Carriers of the A allele showed a higher response (P=0.008), a higher area under the curve (AUC; P=0.022) and a lower RP peak time (P=0.029) in small TRL during the postprandial response, as well as a lower peak time in total plasma TG levels (P=0.034) and large TRL-TG (P=0.033) than subjects who were homozygous for the G allele. CONCLUSION: Our data indicate that the presence of the A allele in the -250G/A promoter polymorphism of the hepatic lipase gene is associated with a higher postprandial lipemic response.

AIM: This study aimed to investigate the association between a polymorphism in the hepatic lipase (LIPC) gene promoter and the presence of peripheral arterial disease (PAD) in persons with type 2 diabetes. PATIENT AND METHODS: We evaluated 120 type 2 diabetics and identified those with PAD according to the ankle-arm index. The G-250A polymorphisms in the promoter of the LIPC gene were studied by PCR restriction. A logistic regression analysis was performed to determine the association between the rare allele and PAD. RESULTS: The prevalence of PAD was 19%. The frequency of the -250A allele was 0.211 in the group without PAD and 0.395 in the group with PAD (P<.05). Carriers of the -250A allele differed only in the ankle-arm index (0.92+/-0.12 for carriers vs. 1.00+/-0.12 for noncarriers, P<.05), with the difference remaining significant after adjustment for covariates (age; sex; waist-to-hip ratio; body mass index; duration of diabetes; smoking; hypertension; glycated hemoglobin; triglycerides; HDL cholesterol; LDL cholesterol; small, dense LDL cholesterol). Only smoking [odds ratio (OR)=6.93, 95% confidence interval (CI)=2.12-22.69, P=.001] and the -250A allele (OR=2.89, 95% CI=1.07-7.84, P=.036) were significantly associated with vascular disease in the logistic regression analysis. CONCLUSIONS: Patients with type 2 diabetes who are carriers of the rare -250A allele in the promoter of the hepatic lipase gene are susceptible to PAD.

Hei Yi Zhuang is an isolated subgroup of the Zhuang minority in China. This study was designed to compare the difference in the hepatic lipase gene (LIPC) -514C/T polymorphism and its association with lipid profiles between the Guangxi Hei Yi Zhuang and Han populations. Genotyping of the LIPC -514C/T was performed in 873 subjects of Hei Yi Zhuang and 867 participants of Han Chinese. The frequency of -514T allele was 43.47% in Hei Yi Zhuang, and 36.10% in Han (P < 0.001). The frequencies of CC, CT and TT genotypes were 30.01, 53.04 and 16.95% in Hei Yi Zhuang, and 40.95, 45.91 and 13.14% in Han (P < 0.001); respectively. Serum high-density lipoprotein cholesterol (HDL-C) and apolipoprotein B levels in both ethnic groups were higher in LIPC -514T carriers than in C carriers. In addition, serum triglyceride levels in Han were higher in TT genotype individuals than in CC genotype subjects (P < 0.05). Serum HDL-C levels were positively correlated with age, alcohol consumption and LIPC -514C/T genotypes, and negatively associated with hypertension and cigarette smoking in Hei Yi Zhuang (P < 0.05-0.01), whereas HDL-C levels were positively correlated with age, alcohol consumption and LIPC -514C/T genotypes, and negatively associated with body mass index and cigarette smoking in Han (P < 0.05-0.001). The differences in serum HDL-C levels between the two ethnic groups might partially attribute to the differences in the LIPC -514C/T polymorphism.

OBJECTIVE: To study the prevalence of the hepatic lipase gene (LIPC) promoter polymorphism (at position -514) in patients with nonalcoholic fatty liver disease (NAFLD), and its relationship with the susceptibility to NAFLD.
METHODS: Genotype of LIPC promoter was detected with PCR-RFLP in 106 patients with NAFLD. Body mass index, waist-to-hip ratio (WHR), blood pressure, CHOL, HDL, LDL, TG, FPG and FINS of the patients were measured. Index of insulin resistance was determined using the homeostasis model assessment (HOMA) method. One hundred six healthy subjects matched for age and sex served as controls.
RESULTS: The frequency of CC genotype and C allele in the NAFLD group were significantly higher than those in the control group (31.1% vs 26.4%, 62.7% vs 54.2%, P<0.05). Compared with TT genotype, both CC genotype and CT genotypes had higher relative risk of NAFLD (OR: 3.73, 95% CI: 1.31, 10.63; OR: 3.60, 95% CI: 1.35, 9.60). At the same time, the non-carriers of T allele in -514 had higher WHR than the T carriers (0.877+/-0.06 vs 0.848+/-0.06, t=2.072, P<0.05)). Logistic regression analysis showed that T substitution in LIPC-514 position (OR: 1.28, 95% CI 0.10-0.74) had a lower susceptibility to NAFLD. CONCLUSION: The LIPC-514C/T polymorphism is associated with WHR, and the T substitution of LIPC-514 may lower the susceptibility to NAFLD.

We previously selected a group of hypertension candidate genes by a key word search using the OMIM database of NCBI and validated 525 coding single nucleotide polymorphisms (SNPs) in 179 hypertension candidate genes by DNA sequencing in a Japanese population. In the present study, we examined the association between 61 non-synonymous SNPs and blood pressure variations and hypertension. We used DNA samples taken from 1,880 subjects in the Suita study, a population-based study using randomly selected subjects. Analyses of covariance adjusting for age, body mass index, hyperlipidemia, diabetes, smoking, drinking, and antihypertensive medication revealed that 17 polymorphisms in 16 genes (APOB, CAST, CLCNKB, CTNS, GHR, GYS1, HF1, IKBKAP, KCNJ11, LIPC, LPL, P2RY2, PON2, SLC4A1, TRH, VWF) were significantly associated with blood pressure variations. Multivariate logistic regression analysis with adjustment for the same factors revealed that 11 polymorphisms in 11 genes (CAST, CTLA4, F5, GC, GHR, LIPC, PLA2G7, SLC4A1, SLCI8A1, TRH, VWF) showed significant associations with hypertension. Five polymorphisms in five genes, CAST(calpastatin), LIPC (hepatic lipase), SLC4A1 (band 3 anion transporter), TRH (thyrotropin-releasing hormone), and VWF (von Willebrand factor), were significantly associated with both blood pressure variation and hypertension. Thus, our study suggests that these five genes were susceptibility genes for essential hypertension in this Japanese population.

Here we present a finished sequence of human chromosome 15, together with a high-quality gene catalogue. As chromosome 15 is one of seven human chromosomes with a high rate of segmental duplication, we have carried out a detailed analysis of the duplication structure of the chromosome. Segmental duplications in chromosome 15 are largely clustered in two regions, on proximal and distal 15q; the proximal region is notable because recombination among the segmental duplications can result in deletions causing Prader-Willi and Angelman syndromes. Sequence analysis shows that the proximal and distal regions of 15q share extensive ancient similarity. Using a simple approach, we have been able to reconstruct many of the events by which the current duplication structure arose. We find that most of the intrachromosomal duplications seem to share a common ancestry. Finally, we demonstrate that some remaining gaps in the genome sequence are probably due to structural polymorphisms between haplotypes; this may explain a significant fraction of the gaps remaining in the human genome.

A number of studies have evaluated the effect of the -514C/T polymorphism of the hepatic lipase (HL) gene on concentration and composition of the plasma lipoproteins. However, the effect of this polymorphism on the response to hypolipaemic treatment has not been addressed. We evaluated the effect of this polymorphism on baseline lipids and lipoproteins and their response to treatment comprising 20mg/day pravastatin for 3 months. The study was multi-centred, prospective and interventional in 236 hypercholesterolaemic subjects (mean age 57.3 years; 45% males) from 21 Primary Health-care Centres. The lipid and genotype measurements were conducted centrally. The genotype distribution was 60.2% homozygous for the C allele and 36.0% heterozygous. No significant differences in baseline lipid concentrations between the genotypes were observed, except that carriers of the T allele had higher concentrations of triglycerides (p=0.021). Post-pravastatin, concentrations of HDL-C increased by 6.9% (95% CI: 2.9-10.8%) in those carrying the T allele with almost no change (0.8%) in the CC genotypes (95% CI: -2.3-4.0%) (p=0.019). The significance remained (p=0.014) following adjustment for other confounding factors (age, basal HDL-cholesterol, LDL-cholesterol and triglycerides). We conclude that the -514C/T polymorphism modulates significantly the HDL-C response to pravastatin, irrespective of the baseline lipoprotein concentrations.

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.

Hepatic lipase (HL) is a glycoprotein that plays a major role in remodeling high-density lipoprotein (HDL). The effect of the -250G/A promoter polymorphism on coronary artery disease (CAD) and lipid levels was studied in 231 male CAD patients and in a population-based sample of men and women (n = 514). A sample of 140 men was chosen among those included in the population-based sample as controls for the CAD sample. In the total group of CAD patients, the frequency of the -250A allele was somewhat lower (25% in CAD patients and 32% in controls; p = 0.06), but when the control samples were compared only with the CAD(+) sample (more than 60% of luminal stenosis in at least one coronary artery or major branch segment) the -250A allele was significantly less frequent (23% in the patients vs 32% in controls; p = 0.02). A multiple logistic regression analysis showed that this association was independent of classical CAD risk factors [odds ratio (OR) = 1.79, p = 0.025]. Using multiple linear regression analyses, it has been shown that this polymorphism was a significant factor affecting HDL-C levels in men from the population-based sample (p = 0.001), an interaction between -250G/A variant and wine consumption was also detected (p = 0.001). Thus, our results show that the -250G/A polymorphism in the HL gene is associated with significant variations in HDL-C levels and CAD risk in males.

This study explored the genetic basis of the combination of extreme blood levels of HDL-C and LDL-C, a well-studied endophenotype for CVD, which has several attractive features as a target for genetic analysis: (1) the trait is moderately heritable; (2) non-genetic risk factors account for a significant but still limited portion of the phenotypic variance; (3) it is known to be moderated by a number of gene products. We exhaustively surveyed 11 candidate genes for allelic variation in a random population-based sample characterized for known CVD risk factors and blood lipid profiles. With the goal of generating specific etiological hypotheses, we compared two groups of subjects with extreme lipid phenotypes, from the same source population, using a case-control design. Cases (n=186) were subjects, within the total sample of 1708 people, who scored in the upper tertile of LDL-C and the lowest tertile of HDL-C, while controls (n=185) scored in the lowest tertile of LDL-C and the upper tertile of HDL-C. We used logistic regression and a four-tiered, systematic model building strategy with internal cross-validation and bootstrapping to investigate the relationships between the trait and 275 genetic variants in the presence of 10 non-genetic risk factors. Our results implicate a subset of nine genetic variants, spanning seven candidate genes, together with five environmental risk factors, in the etiology of extreme lipoprotein phenotypes. We propose a model involving these 14 genetic and non-genetic risk factors for evaluation in future independent studies.

Individuals with hepatic lipase (HL) deficiency are often characterized by elevated levels of triglycerides (TGs) and cholesterol. The aim of the present study was to characterize the molecular defect leading to severe HL deficiency in a Quebec-based kindred. In the proband and two of her brothers, the very low to undetectable HL activity resulted from compound heterozygosity for two rare HL gene mutations, a previously unknown missense mutation in exon 5 designated A174T and the previously reported T383M mutation in exon 8 of the HL gene. The mutation at codon 174 resulted in the substitution of alanine for threonine, a polar amino acid, in a highly conserved nonpolar region of the protein involved in the catalytic activity of the enzyme. The severe HL deficiency among the three related compound heterozygotes was associated with a marked TG enrichment of LDL and HDL particles. The two men with severe HL deficiency also presented with abdominal obesity, which appeared to amplify the impact of HL deficiency on plasma TG-rich lipoprotein levels. Our results demonstrated that HL deficiency in this Quebec kindred is associated with an abnormal lipoprotein-lipid profile, which may vary considerably in the presence of secondary factors such as abdominal obesity.

Familial combined hyperlipidemia (FCHL) is a heritable lipid disorder characterized by multiple lipoprotein phenotypes within a single family. Previously, we have shown an increased incidence of mutations in the LPL gene which was associated with elevated levels of very low density lipoprotein (VLDL) and decreased levels of high density lipoprotein among the families studied. Now, we report the results of our study on the hepatic lipase gene. We found the HL V73M variant to be present in four FCHL families. By means of a pedigree-based maximum log-likelihood method we analyzed the effect of this variant on the lipid levels in these families. Carriers of the HL V73M variant revealed significantly higher levels of total cholesterol (P < 0.01) and apoB (P <0.01). These findings show that the HL V73M mutant explains another part of the variability in the phenotype observed among FCHL family members, compared with mutations in the LPL gene. Family analysis shows that in these FCHL families, carriers of mutations in the LPL or HL genes have an increased risk for FCHL compared with their non-carrier relatives.

A major goal in human genetics is to understand the role of common genetic variants in susceptibility to common diseases. This will require characterizing the nature of gene variation in human populations, assembling an extensive catalogue of single-nucleotide polymorphisms (SNPs) in candidate genes and performing association studies for particular diseases. At present, our knowledge of human gene variation remains rudimentary. Here we describe a systematic survey of SNPs in the coding regions of human genes. We identified SNPs in 106 genes relevant to cardiovascular disease, endocrinology and neuropsychiatry by screening an average of 114 independent alleles using 2 independent screening methods. To ensure high accuracy, all reported SNPs were confirmed by DNA sequencing. We identified 560 SNPs, including 392 coding-region SNPs (cSNPs) divided roughly equally between those causing synonymous and non-synonymous changes. We observed different rates of polymorphism among classes of sites within genes (non-coding, degenerate and non-degenerate) as well as between genes. The cSNPs most likely to influence disease, those that alter the amino acid sequence of the encoded protein, are found at a lower rate and with lower allele frequencies than silent substitutions. This likely reflects selection acting against deleterious alleles during human evolution. The lower allele frequency of missense cSNPs has implications for the compilation of a comprehensive catalogue, as well as for the subsequent application to disease association.

Hepatic lipase (HL) is an endothelial enzyme involved in the metabolism of intermediate density lipoproteins (IDL) and high density lipoproteins (HDL) in plasma. In a Finnish pedigree consisting of 18 members belonging to three generations two missense mutations RI86H and L334F in exons 5 and 7 of the HL gene co-segregated with low post-heparin HL activity. Haplotype analysis of the HL gene in family members revealed a high degree of genetic variation and demonstrated that the two missense mutations reside on the same chromosome. In vitro site-directed mutagenesis and expression of the cDNA constructs in COS-1 cells revealed that the R186H mutation leads to a protein that is not secreted while the L334F mutation results in the production of a HL protein that is secreted but has only about 30% of wild type HL activity. Carriers of the mutated HL gene exhibited clearly reduced HL activity and mass in post-heparin plasma. Probably due to their heterozygous carrier status they had only moderate elevation of total triglycerides, IDL, and LDL-triglycerides. The LDL-particles were enriched in triglycerides and depleted of cholesterol. Also their HDL2- and HDL3-particles were enriched in triglycerides.

Overlapping bacterial phage and cosmid genomic clones were isolated spanning an area of approximately 60 kilobases that contains the human hepatic lipase (HL) gene. It is composed of 9 exons spanning approximately 35 kilobases of DNA. The entire coding regions, the 5'-flanking sequences, and the exon-intron junctions were sequenced. The intron positions correspond to those of human lipoprotein lipase and canine pancreatic lipase, supporting the concept that these genes constitute a dispersed gene family of lipases and have evolved by duplication of a common ancestral gene. A region of the HL gene, which displays a significant homology with various other lipolytic enzymes and contains the putative catalytic site serine residue of HL, was encoded by exon 4. A major transcription start site of the human HL gene was located by primer extension analysis, 43 nucleotides upstream of the translation initiation codon. Two possible promoter elements were located 25 and 63 nucleotides upstream of the transcription initiation site: a "TATA" box-like sequence, TAATA, and a sequence found in the promoter region of many liver-specific genes, AGGTTAATTATTAAT. In addition, sequences homologous to glucocorticoid and cAMP-responsive elements were identified in the 5'-nontranscribed region.

The structure of the human hepatic triglyceride lipase gene was determined from multiple cosmid clones. All the exons, exon-intron junctions, and 845 bp of the 5' and 254 bp of the 3' flanking DNA were sequenced. Comparison of the exon sequences to three previously published cDNA sequences revealed differences in the sequence of the codons for residues 133, 193, 202, and 234 that may represent sequence polymorphisms. By primer extension, hepatic lipase mRNA initiates at an adenine 77 bases upstream of the translation initiation site. The hepatic lipase gene spans over 60 kb containing 9 exons and 8 introns, the latter being all located within the region encoding the mature protein. The exons are all of average size (118-234 bp). Exon 1 encodes the signal peptide, exon 4, a region that binds to the lipoprotein substrate, and exon 5, an evolutionarily highly conserved region of potential catalytic function, and exons 6 and 9 encode sequences rich in basic amino acids thought to be important in anchoring the enzyme to the endothelial surface by interacting with acidic domains of the surface glycosaminoglycans. The human lipoprotein lipase gene has been recently reported to have an identical exon-intron organization containing the analogous structural domains [Deeb & Peng (1989) Biochemistry 28, 4131-4135]. Our observations strongly support the common evolutionary origin of these two lipolytic enzymes.

Human hepatic lipase is an important enzyme in high density lipoprotein (HDL) metabolism, being implicated in the conversion of HDL2 to HDL3. Three human hepatic lipase cDNA clones were identified in two lambda gt11 libraries from human liver. The cDNA-derived amino acid sequence predicts a protein of 476 amino acid residues, preceded by a 23-residue signal peptide. Four potential N-glycosylation sites are identified, two of which are conserved in rat hepatic lipase. On alignment with human, mouse, and bovine lipoprotein lipase, the same two sites were also conserved in lipoprotein lipase in all three species. Stringent conservation of the cysteine residues was also evident. Comparative analysis of amino acid sequences shows that hepatic lipase evolves at a rapid rate, 2.07 x 10(-9) substitutions/site/year, about four times that in lipoprotein lipase and half that in pancreatic lipase. Further, hepatic lipase and pancreatic lipase appear to be evolutionarily closer to each other than either of them is to lipoprotein lipase. Southern blot analysis revealed high frequency restriction fragment length polymorphisms of the hepatic lipase gene for the enzymes HindIII and MspI. these polymorphisms will be useful for haplotype and linkage analysis of the hepatic lipase gene. Using cloned human hepatic lipase cDNA as a hybridization probe, we performed Southern blot analysis of a panel of 13 human-rodent somatic cell hybrids. Concordance analysis of the various hybrid clones indicates that the hepatic lipase gene is located on the long arm of human chromosome 15. Analysis of hybrids containing different translocations of chromosome 15 localized the gene to the region 15q15----q22.

Hepatic triglyceride lipase (H-TGL) was isolated from human postheparin plasma by column chromatography on heparin-Sepharose and phenyl-Sepharose and immunoaffinity chromatography with monoclonal antibodies. The purified enzyme had an apparent molecular weight of 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an amino-terminal sequence of Leu-Gly-Gln-Ser-Leu-Lys-Pro-Glu. Partial amino acid sequences of seven cyanogen bromide peptides were obtained. A human hepatoma cDNA library was screened with synthetic oligonucleotides derived from the partial protein sequence. The cloned H-TGL cDNA of 1569 nucleotides predicts a mature protein of 477 amino acids plus a leader sequence of 22 amino acids. Blot hybridization analysis of poly(A)+ mRNA with a putative H-TGL cDNA clone gave a single hybridizing band of 1.7 kilobases. The protein contains four consensus N-glycosylation sequences based on the cDNA sequence. Comparison of the enzyme sequence with that of other lipases reveals highly conserved sequences in regions of putative lipid and heparin binding. The carboxyl terminus of H-TGL contains a highly basic sequence which is not reported to be present in rat H-TGL or other members of the lipase gene family.

By immunoscreening of a human cDNA expression library and hybridization of colonies, four partially overlapping cDNA clones of human hepatic triglyceride lipase (HTGL) mRNA were isolated. The clones included the complete coding sequence, the 3'- and at least part of the 5'-untranslated region. The length of the composite HTGL cDNA segment (1.7 kb) was consistent with the size of the mRNA identified in an established human hepatoma cell line. DNA-sequence analysis of cDNAs of partially unspliced mRNAs, and of cloned genomic DNA indicated that the HTGL coding sequence comprises at least six exons. As predicted from the cDNA, the unprocessed HTGL protein has a molecular weight of 56, three potential glycosylation sites, and a signal peptide of 23 amino acids. Sequence comparison with cDNA of other lipases, including rat hepatic lipase, revealed 30%-75% protein-sequence homology. The data establish that HTGL is a secretory protein produced in the hepatocyte, and that its synthesis can be continued in permanent cell lines of hepatoma origin. Our studies also showed that HTGL is another member of a lipase gene family which has interfacial binding sites and possibly other functional domains in common.