Human Lipoprotein Lipase Gene Research Articles

Regulation of the human lipoprotein lipase gene by the forkhead box transcription factor FOXA2 in hepatic cells

Regulation of the human lipoprotein lipase gene by the forkhead box transcription factor FOXA2 in hepatic cells

Physical and functional interactions between nuclear receptor LXRα and the forkhead box transcription factor FOXA2 regulate the response of the human lipoprotein lipase gene to oxysterols in hepatic cells

Lipoprotein lipase (LPL) catalyzes the hydrolysis of triglycerides from triglyceride-rich lipoproteins such as VLDL and chylomicrons in the circulation. Mutations in LPL or its activator apolipoprotein C-II cause hypertriglyceridemia in humans and animal models. The levels of LPL in the liver are low but they can be strongly induced by a high cholesterol diet or by synthetic ligands of Liver X Receptors (LXRs). However, the mechanism by which LXRs activate the human LPL gene is unknown. In the present study we show that LXR agonists increased the mRNA and protein levels as well as the promoter activity of human LPL in HepG2 cells. A promoter deletion analysis defined the proximal −109/−28 region, which contains a functional FOXA2 element, as essential for transactivation by ligand-activated LXRα/RXRα heterodimers. Silencing of endogenous FOXA2 in HepG2 cells by siRNAs or by treatment with insulin compromised the induction of the LPL gene by LXR agonists whereas mutations in the FOXA2 site abolished the synergistic transactivation of the LPL promoter by LXRα/RXRα and FOXA2. Physical and functional interactions between LXRα and FOXA2 were established in vitro and ex vivo. In summary, the present study revealed a novel mechanism of human LPL gene induction by oxysterols in the liver with is based on physical and functional interactions between transcription factors LXRα and FOXA2. This mechanism, which may not be restricted to the LPL gene, is critically important for a better understanding of the regulation of cholesterol and triglyceride metabolism in the liver under healthy or pathological states.

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

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

AAVR: A Multi-Serotype Receptor for AAV

Adeno-associated virus (AAV) continues to be a promising viral vector for delivery of therapeutic genes in human gene therapy. In 2012, the European Commission approved Glybera (alipogene tiparvovec),1 an AAV vector carrying the human lipoprotein lipase gene for commercial use in treatment of lipoprotein lipase deficiency. In addition, within the past five years there have been numerous successes in preclinical, early, and late-stage clinical trials of AAV for treatment of a variety of diseases, including muscular dystrophy, hemophilia, Leber’s congenital amaurosis retinopathy, age-related macular degeneration, cardiac disorders, cancer, and neurodegenerative disorders, such as Parkinson’s disease and Canavan disease. However, despite this progress, the infectious pathway of AAV vectors is an evolving story. In a study recently reported in Nature, Pillay and colleagues2 employed a powerful genome-wide haploid genetic screen to gain a better understanding of the AAV infectious entry pathway. Among other factors consistent with the known pathways of entry and infection, the authors identified a new cellular factor that is required for infection of multiple AAV serotypes, which they term AAV receptor (AAVR).

Over-Expression of Human Lipoprotein Lipase in Mouse Mammary Glands Leads to Reduction of Milk Triglyceride and Delayed Growth of Suckling Pups

BackgroundThe mammary gland is a conserved site of lipoprotein lipase expression across species and lipoprotein lipase attachment to the luminal surface of mammary gland vascular endothelial cells has been implicated in the direction of circulating triglycerides into milk synthesis during lactation.Principal FindingsHere we report generation of transgenic mice harboring a human lipoprotein lipase gene driven by a mammary gland-specific promoter. Lipoprotein lipase levels in transgenic milk was raised to 0.16 mg/ml, corresponding to an activity of 8772.95 mU/ml. High lipoprotein lipase activity led to a significant reduction of triglyceride concentration in milk, but other components were largely unchanged. Normal pups fed with transgenic milk showed inferior growth performances compared to those fed with normal milk.ConclusionOur study suggests a possibility to reduce the triglyceride content of cow milk using transgenic technology.

PPARα L162V polymorphism alters the potential of n‐3 fatty acids to increase lipoprotein lipase activity

Omega-3 fatty acids (FAs) may accelerate plasma triglyceride (TG) clearance by altering lipoprotein lipase (LPL) activity. Yet, the ability of n-3 FAs to increase LPL activity is dependent on transcription factors such as peroxisome proliferator-activated receptor alpha (PPARalpha). The objective was to examine the effects of n-3 FAs on LPL activity considering the occurrence of PPARalpha L162V polymorphism. First, 14 pairs of men either L162 homozygotes or carriers of the V162 allele were supplemented with n-3 FAs. Second, transient transfections in HepG2 cells, for the L162- and V162-PPARalpha variants with the peroxisome proliferator-response element from the human LPL gene, were transactivated with n-3 FAs. In vivo results demonstrate that the LPL activity increased non-significantly by 14.4% in L162 homozygotes compared with 6.6% in carriers of the PPARalpha-V162 allele, after n-3 FA supplementation. Additionally, the L162 homozygotes tended towards an inverse correlation between LPL activities and plasma TG levels. Conversely, carriers of the V162 allele showed no such relationship. In vitro data demonstrates that transcription rates of LPL tended to be higher for the L162-PPARalpha than V162-PPARalpha after n-3 FAs activation. Overall, these results indicate that n-3 FA supplementation increases the transcription rate of LPL to a greater extent in L162-PPARalpha than V162-PPARalpha.

Information-theoretic identification of predictive SNPs and supervised visualization of genome-wide association studies

The size, dimensionality and the limited range of the data values makes visualization of single nucleotide polymorphism (SNP) datasets challenging. The purpose of this study is to evaluate the usefulness of 3D VizStruct, a novel multi-dimensional data visualization technique for SNP datasets capable of identifying informative SNPs in genome-wide association studies. VizStruct is an interactive visualization technique that reduces multi-dimensional data to three dimensions using a combination of the discrete Fourier transform and the Kullback–Leibler divergence. The performance of 3D VizStruct was challenged with several diverse, biologically relevant published datasets including the human lipoprotein lipase (LPL) gene locus, the human Y-chromosome in several populations and a multi-locus genotype dataset of coral samples from four populations. In every case, the SNPs and or polymorphic markers identified by the 3D VizStruct mapping were predictive of the underlying biology.

VizStruct for visualization of genome-wide SNP analyses

The size, dimensionality and the limited range of the data values make visualization of single nucleotide polymorphism (SNP) datasets challenging. The purpose of this study is to evaluate the usefulness of 3D VizStruct, a novel multi-dimensional data visualization technique for analyzing patterns in SNP datasets. VizStruct is an interactive visualization technique that reduces multi-dimensional data to two dimensions using the complex-valued harmonics of the discrete Fourier transform (DFT). In the 3D VizStruct extension, the multi-dimensional SNP data vectors are reduced to three dimensions using a combination of the DFT and the Kullback-Leibler divergence. The performance of 3D VizStruct was challenged with several biologically relevant published datasets that included human Chromosome 21, the human lipoprotein lipase (LPL) gene locus and the multi-locus genotypes of coral populations. In every case, the 3D VizStruct mapping provided an intuitive visual description of the key characteristics of the underlying multi-dimensional genotype.

Genotyping of the Human Lipoprotein Lipase Gene by Ferrocenylnaphthalene Diimide-based Electrochemical Hybridization Assay

A ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay (FND-EHA) was applied to the detection of two mutations in human lipoprotein lipase (LPL) gene, G188E (one base transition) and Arita (one base deletion). A probe oligodeoxyribonucleotide of 13 bases representing the wild type (WT) sequence of LPL was immobilized on a gold electrode, followed by hybridization with a sample PCR product of 350 base pairs under conditions in which both WT and mutated (MT) sequences could form a duplex with the probe. The hybridized electrodes were soaked in an electrolyte containing FND under conditions in which only the mismatched duplex could undergo dissociation. FND was concentrated in proportion to the amount of the duplex remaining on the electrode to give rise to a current signal. Blind tests were run to judge the genotype (WT/WT, WT/MT, or MT/MT) of 10 samples each for the G188E and Arita mutations and then, 8 and 10 of them were judged correctly, respectively.

Macrophage-derived lipoprotein lipase increases aortic atherosclerosis in cholesterol-fed Tg rabbits

Lipoprotein lipase (LPL) produced by macrophages is upregulated in the atherosclerotic lesions; however, it is not fully understood whether increased macrophage-derived LPL is pro-atherogenic. To examine the hypothesis that macrophage-derived LPL in the arterial wall enhances atherosclerotic lesion formation, we generated transgenic (Tg) rabbits that express the human LPL transgene under the control of the human scavenger receptor enhancer/promoter, which drives macrophage-specific expression of the human LPL gene. We fed Tg and non-Tg littermate rabbits a diet containing 0.3% cholesterol for 16 weeks and compared their lipoproteins and aortic atherosclerosis. We found that there was no difference in plasma lipid or lipoprotein profiles between Tg and non-Tg rabbits; however, atherosclerotic lesions were significantly increased in Tg compared to non-Tg rabbits. There was a 1.4-fold increase in total aortic en face lesions and a 2-fold increase in intimal lesions evaluated by image analysis system. Furthermore, immunohistochemical staining revealed that the increased atherosclerotic lesions present in Tg rabbits were characterized by marked accumulation of macrophage-derived foam cells and frequently associated with the deposition of oxidized LDL. These results support the notion that macrophage-derived LPL in the arterial wall is pro-atherogenic, possibly via the enhancement of foam cell formation during atherogenesis.

High lipoprotein lipase activity increases insulin sensitivity in transgenic rabbits

Lipoprotein lipase (LPL) is the rate-limiting enzyme in the hydrolysis of triglyceride-rich lipoproteins and plays an important role in glucose metabolism. To examine the hypothesis that increased LPL activity may alter insulin sensitivity, we investigated glucose metabolism and insulin sensitivity in transgenic (Tg) rabbits expressing the human LPL gene under the control of a β-actin promoter. An intravenous glucose tolerance test showed that the plasma glucose clearance rate was not significantly different between Tg and non-Tg rabbits; however, the area under the curve for insulin and free fatty acids in Tg rabbits was significantly reduced compared with that of non-Tg rabbits ( P < .05). Using the intravenous insulin tolerance test, we found that the area of under the curve of glucose of Tg rabbits was also significantly reduced ( P < .01). Furthermore, euglycemic-hyperinsulinemic clamp test revealed that the mean glucose infusion rate in Tg rabbits was significantly higher than in non-Tg rabbits ( P < .05). These results demonstrate that systemic overexpression of LPL increases whole-body insulin sensitivity and genetic manipulation of LPL genes may be a potential target for the treatment of diabetic patients.

Advanced glycation end products potentiate the stimulatory effect of glucose on macrophage lipoprotein lipase expression

Lipoprotein lipase (LPL) secreted by macrophages in the arterial wall promotes atherosclerosis. We have shown that macrophages of patients with type 2 diabetes overproduce LPL and that metabolic factors, including glucose, stimulate macrophage LPL secretion. In this study, we determined the effect of advanced glycation end products (AGEs) on LPL expression by macrophages cultured in a high-glucose environment and the molecular mechanisms underlying this effect. Our results demonstrate that AGEs potentiate the stimulatory effect of high glucose on murine and human macrophage LPL gene expression and secretion. Induction of macrophage LPL mRNA levels by AGEs was identical to that elicited by physiologically relevant modified albumin and was inhibited by anti-AGE receptor as well as by antioxidants. Treatment of macrophages with AGEs resulted in protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. Inhibition of these kinases abolished the effect of AGEs on LPL mRNA levels. Finally, exposure of macrophages to AGEs increased the binding of nuclear proteins to the activated protein-1 consensus sequence of the LPL promoter. This effect was inhibited by PKC and MAPK inhibitors. These results demonstrate for the first time that AGEs potentiate the stimulatory effect of high glucose on macrophage LPL expression. This effect appears to involve oxidative stress and PKC/MAPK activation.

Pair-wise detection of eight common DNA polymorphisms of the human lipoprotein lipase gene by PCR-RFLP. Findings of the Olivetti Heart Study

We developed a rapid procedure to analyse simultaneously two different DNA polymorphisms of the human LPL gene by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). The method involves PCR amplification of the gene fragments encompassing two polymorphic sites, direct digestion in the same PCR-tube of the amplification mixture with two restriction enzymes, and the analysis of the resulting DNA fragments by gel electrophoresis. In 422 participants of the 1994 follow-up examination of the Olivetti Heart Study, a total of eight common LPL gene polymorphisms have been analysed in pairs by this procedure: −93 T/G and D9N; V108V and T361T; N291S and PvuII; HindIII and S447X. Two of these polymorphisms (V108V and T361T) were analysed for the first time. This method is suitable for the routine analysis of clinical samples of varying DNA content and practically halves the times and costs of screening for these LPL polymorphisms.

Unusual metabolic characteristics in skeletal muscles of transgenic rabbits for human lipoprotein lipase

BackgroundThe lipoprotein lipase (LPL) hydrolyses circulating triacylglycerol-rich lipoproteins. Thereby, LPL acts as a metabolic gate-keeper for fatty acids partitioning between adipose tissue for storage and skeletal muscle primarily for energy use. Transgenic mice that markedly over-express LPL exclusively in muscle, show increases not only in LPL activity, but also in oxidative enzyme activities and in number of mitochondria, together with an impaired glucose tolerance. However, the role of LPL in intracellular nutrient pathways remains uncertain. To examine differences in muscle nutrient uptake and fatty acid oxidative pattern, transgenic rabbits harboring a DNA fragment of the human LPL gene (hLPL) and their wild-type littermates were compared for two muscles of different metabolic type, and for perirenal fat.ResultsAnalyses of skeletal muscles and adipose tissue showed the expression of the hLPL DNA fragment in tissues of the hLPL group only. Unexpectedly, the activity level of LPL in both tissues was similar in the two groups. Nevertheless, mitochondrial fatty acid oxidation rate, measured ex vivo using [1-14C]oleate as substrate, was lower in hLPL rabbits than in wild-type rabbits for the two muscles under study. Both insulin-sensitive glucose transporter GLUT4 and muscle fatty acid binding protein (H-FABP) contents were higher in hLPL rabbits than in wild-type littermates for the pure oxidative semimembranosus proprius muscle, but differences between groups did not reach significance when considering the fast-twitch glycolytic longissimus muscle. Variations in both glucose uptake potential, intra-cytoplasmic binding of fatty acids, and lipid oxidation rate observed in hLPL rabbits compared with their wild-type littermates, were not followed by any modifications in tissue lipid content, body fat, and plasma levels in energy-yielding metabolites.ConclusionsExpression of intracellular binding proteins for both fatty acids and glucose, and their following oxidation rates in skeletal muscles of hLPL rabbits were not fully consistent with the physiology rules. The modifications observed in muscle metabolic properties might not be directly associated with any LPL-linked pathways, but resulted likely of transgene random insertion into rabbit organism close to any regulatory genes. Our findings enlighten the risks for undesirable phenotypic modifications in micro-injected animals and difficulties of biotechnology in mammals larger than mice.

Pairwise detection of eight common DNA polymorphisms of the human lipoprotein lipase (LPL) gene by PCR-RFLP: Findings of the olivetti prospective heart study

Pairwise detection of eight common DNA polymorphisms of the human lipoprotein lipase (LPL) gene by PCR-RFLP: Findings of the olivetti prospective heart study

Direct detection of single nucleotide polymorphism (SNP) with genomic DNA by the ferrocenylnaphthalene diimide-based electrochemical hybridization assay (FND-EHA).

A ferrocenylnaphthalene diimide-based electrochemical hybridization assay (FND-EHA) was applied to the direct detection of a C-to-G transition in a codon (TCA) for Ser-447 of the human lipoprotein lipase (LPL) gene, which resulted in the termination of the LPL protein there. Either one of two 13-meric oligonucleotide probes, S447 WT and S447X MT, representing sequences complementary to those of the wild type (WT) and mutated (MT) forms, was immobilized on a gold electrode, followed by hybridization with chromosomal DNA extracted from human leukocytes under the condition in which both WT- and MT-type sequences can form a duplex. These two electrodes were soaked in an electrolyte containing FND under a condition [0.1 M HOAc/KOAc (pH 5.6) containing 0.1 KCl and 0.05 mM FND at 40 degrees C], in which only the MT duplex could undergo dissociation. FND was concentrated in proportion to the amount of the duplex formed on the electrode to give rise to a current signal. The electrochemical signal ratios obtained for WT/WT, WT/MT and MT/MT were close to the theoretical 2:1:0 with the S447 WT-modified electrode, and was again close to 0:1:2 with the S447X MT-modified one.

Lipoprotein lipase (LPL) deficiency: a new patient homozygote for the preponderant mutation Gly188Glu in the human LPL gene and review of reported mutations: 75 % are clustered in exons 5 and 6

We have investigated the lipoprotein lipase (LPL) gene of a 2-year-old patient presenting classical features of the familial LPL deficiency including undetectable LPL activity. DNA sequence analysis of exon 5 identified the patient as a homozygote for the Gly188Glu mutation, frequently involved in this disease. A review of cases of LPL deficiency with molecular study of the LPL gene showed a total number of 221 reported mutations involved in this disease. Gly188Glu was involved in 23.5 % of cases and 74.6 % of mutations were clustered in exons 5 and 6. Based on these observations, we propose a method of screening for mutations in this gene.

Induction of LPL gene expression by sterols is mediated by a sterol regulatory element and is independent of the presence of multiple E boxes

Overexpression of the adipocyte differentiation and determination factor-1 (ADD-1) or sterol regulatory element binding protein-1 (SREBP-1) induces the expression of numerous genes involved in lipid metabolism, including lipoprotein lipase (LPL). Therefore, we investigated whether LPL gene expression is controlled by changes in cellular cholesterol concentration and determined the molecular pathways involved. Cholesterol depletion of culture medium resulted in a significant induction of LPL mRNA in the 3T3-L1 preadipocyte cell line, whereas addition of cholesterol reduced LPL mRNA expression to basal levels. Similar to the expression of the endogenous LPL gene, the activity of the human LPL gene promoter was enhanced by cholesterol depletion in transient transfection assays, whereas addition of cholesterol caused a reversal of its induction. The effect of cholesterol depletion upon the human LPL gene promoter was mimicked by cotransfection of expression constructs encoding the nuclear form of SREBP-1a, -1c (also called ADD-1) and SREBP-2. Bioinformatic analysis demonstrated the presence of 3 potential sterol regulatory elements (SRE) and 3 ADD-1 binding sequences (ABS), also known as E-box motifs. Using a combination of in vitro protein-DNA binding assays and transient transfection assays of reporter constructs containing mutations in each individual site, a sequence element, termed LPL-SRE2 (SRE2), was shown to be the principal site conferring sterol responsiveness upon the LPL promoter. These data furthermore underscore the importance of SRE sites relative to E-boxes in the regulation of LPL gene expression by sterols and demonstrate that sterols contribute to the control of triglyceride metabolism via binding of SREBP to the LPL regulatory sequences.

Cladistic structure within the human Lipoprotein lipase gene and its implications for phenotypic association studies.

Haplotype variation in 9.7 kb of genomic DNA sequence from the human lipoprotein lipase (LPL) gene was scored in three populations: African-Americans from Jackson, Mississippi (24 individuals), Finns from North Karelia, Finland (24), and non-Hispanic whites from Rochester, Minnesota (23). Earlier analyses had indicated that recombination was common but concentrated into a hotspot and that recurrent mutations at multiple sites may have occurred. We show that much evolutionary structure exists in the haplotype variation on either side of the recombinational hotspot. By peeling off significant recombination events from a tree estimated under the null hypothesis of no recombination, we also reveal some cladistic structure not disrupted by recombination during the time to coalescence of this variation. Additional cladistic structure is estimated to have emerged after recombination. Many apparent multiple mutational events at sites still remain after removing the effects of the detected recombination/gene conversion events. These apparent multiple events are found primarily at sites identified as highly mutable by previous studies, strengthening the conclusion that they are true multiple events. This analysis portrays the complexity of the interplay among many recombinational and mutational events that would be needed to explain the patterns of haplotype diversity in this gene. The cladistic structure in this region is used to identify four to six single-nucleotide polymorphisms (SNPs) that would provide disequilibrium coverage over much of this region. These sites may be useful in identifying phenotypic associations with variable sites in this gene. Evolutionary considerations also imply that the SNPs in the 3' region should have general utility in most human populations, but the 5' SNPs may be more population specific. Choosing SNPs at random would generally not provide adequate disequilibrium coverage of the sequenced region.

Transgenic rabbits expressing human lipoprotein lipase

To study the functions of lipoprotein lipase (LPL) in lipid and lipoprotein metabolism and the relationship between LPL and atherosclerosis, we generated transgenic rabbits expressing the human LPL gene. A total of 4045 Japanese whiterabbit embryos were microinjected with a 3.8-kb SalI/HindIII fragment containing the chicken beta-actin promoter, human LPL cDNA and rabbit beta-globin with poly (A) signals, and then transplanted into 116 recipient rabbits. Of the 166 pups born, six pups were transgenic as confirmed by Southern blot analysis. ANorthern blot analysis revealed that human LPL was expressed by a number of tissues including the heart, kidney, adrenal gland and intestine. One transgenic rabbit showed up to 3-foldincreased LPL activity in post-heparin plasma compared to thatin nontransgenic rabbits. Human LPL expression in various tissues of transgenic rabbits was further elucidated by in situ hybridization and immunostaining. Since rabbits are superior to mice as a model of atherosclerosis, this transgenicrabbit model should provide a valuable tool for the study of LPL in lipid metabolism and atherosclerosis.