Human Lipoprotein Lipase Research Articles

Abstract 3043: Apolipoprotein A5 Alters The VLDL Proteome To Promote Increased Particle Uptake By Hepatocytes

The levels of circulating triglycerides (TG) carried in TG-rich lipoproteins (TGRL) and their remnants are causally associated with cardiovascular disease (CVD). Liberation of TG from lipoproteins to peripheral tissues is modulated by TGRL-associated proteins including apolipoproteln A5 (ApoA5), which has been reported to profoundly impact TG metabolism. Present at ~150 ng/ml in plasma, it has been thought to work via stimulation (or de-repression) of lipoprotein lipase (LPL), which hydrolyzes TG to deliver fatty acids to cells. To determine whether ApoA5 may alter the proteomic composition of VLDL, we examined the VLDL particle proteome from C57BL/6J WT and C57BL/6J apoA5 KO mice by mass spectrometry. ApoA5 presence was associated with increased VLDL apoE and apoM content and reduced levels of apoA1 and apoD, compared to VLDL from apoA5 KO mice. Presence of apoA5 also tracked with increased levels of the serine protease plasmin and decreased levels of its inhibitor alpha2-microglobulin on the VLDL. Immunoblotting analyses confirmed these results. Despite large differences in apoA5 content, VLDL from WT or KO mice behaved similarly as a substrate for recombinant human LPL in vitro . However, we observed that VLDL from apoA5 KO mice was internalized by cultured mouse hepatocytes less efficiently than WT VLDL (Kd 80±9.0 vs. 35±10 μg/ml). Notably, when recombinant mouse apoA5 was added back to apoA5 KO plasma, levels of VLDL apoE and apoM were rescued, and uptake efficiency was restored (Kd 29±8.0). Our current working model is that ApoA5 alters the proteome of TGRL in ways that affect remnant particle uptake by the liver, perhaps via apoE. ApoA5 may also affect plasmin levels/activity which was recently linked to LPL regulation.

Adaption of a commercial lipase kit to measure bile salt-stimulated lipase in human milk

A highly sensitive, fluorometric method for measuring activities of two major lipases in milk, lipoprotein lipase (LPL) and bile salt-stimulated lipase (BSSL) is described. Human milk LPL showed linear activity from 65 to 347 U/L (U = 1 μmol/min) and the quantification limit was theoretically about 65 U/L. For human milk BSSL, the linear range was from 10 to 267 U/L and the quantification limit was 18 U/L. The mean LPL and BSSL activities were 409 ± 13 U/L and 5263 ± 132 U/L, respectively. This corresponded well to the values reported in literature. The residual LPL and BSSL activities were reduced dramatically to 21%, and 2%, respectively, when the milk was heated to 55 °C; heating to 50 °C caused only nominal reductions of LPL and BSSL (activities remaining ∼82% and 87%, respectively). No BSSL activity was detected in cow, camel, goat milk or in infant formula.

Angiopoietin-Like 3 Protein Inhibition: A New Frontier in Lipid-Lowering Treatment.

Angiopoietin-like 3 protein (ANGPTL3) is an inhibitor of both lipoprotein lipase and endothelial lipase in humans. Population studies indicate a relationship between loss of function mutations in ANGPTL3 and favorable reductions in triglycerides and non- high-density lipoprotein cholesterol. In addition, loss of function mutations is associated with a reduced risk of coronary artery disease. Whereas ANGPTL3's role in human lipid metabolism has yet to be fully clarified, it is unlikely that ANGPTL3 impacts cholesterol uptake via the low-density lipoprotein-receptor, unlike the proprotein convertase subtilisin/kexin9 inhibitors. In contrast to other forms of lipid-lowering therapy, ANGPTL3 inhibition may improve insulin sensitivity. The promise of this new therapy, particularly its independence from the low-density lipoprotein-receptor, has prompted the creation of a monoclonal antibody inhibitor; evinacumab. Evinacumab has shown favorable lipid-lowering action in both human and mouse models. Efficacy trials are currently ongoing and will be completed in the near future. In addition, ANGPTL3 inhibition via an antisense oligonucleotide was performed in healthy human subjects, which resulted in a dose-dependent reduction in circulating ANGPTL3 levels and an antiatherogenic lipid profile. When tested in mouse models, administration of the antisense oligonucleotide caused a reduction in progression of atherosclerosis. Further investigation is required to evaluate the efficacy, safety and net benefit of clinical ANGPTL3 inhibition before it can be accepted into clinical practice.

Structure of lipoprotein lipase in complex with GPIHBP1

Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5-3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1.

Lipoprotein lipase is active as a monomer

Lipoprotein lipase (LPL), the enzyme that hydrolyzes triglycerides in plasma lipoproteins, is assumed to be active only as a homodimer. In support of this idea, several groups have reported that the size of LPL, as measured by density gradient ultracentrifugation, is ∼110 kDa, twice the size of LPL monomers (∼55 kDa). Of note, however, in those studies the LPL had been incubated with heparin, a polyanionic substance that binds and stabilizes LPL. Here we revisited the assumption that LPL is active only as a homodimer. When freshly secreted human LPL (or purified preparations of LPL) was subjected to density gradient ultracentrifugation (in the absence of heparin), LPL mass and activity peaks exhibited the size expected of monomers (near the 66-kDa albumin standard). GPIHBP1-bound LPL also exhibited the size expected for a monomer. In the presence of heparin, LPL size increased, overlapping with a 97.2-kDa standard. We also used density gradient ultracentrifugation to characterize the LPL within the high-salt and low-salt peaks from a heparin-Sepharose column. The catalytically active LPL within the high-salt peak exhibited the size of monomers, whereas most of the inactive LPL in the low-salt peak was at the bottom of the tube (in aggregates). Consistent with those findings, the LPL in the low-salt peak, but not that in the high-salt peak, was easily detectable with single mAb sandwich ELISAs, in which LPL is captured and detected with the same antibody. We conclude that catalytically active LPL can exist in a monomeric state.

New rare genetic variants of LMF1 gene identified in severe hypertriglyceridemia

The LMF1 (lipase maturation factor 1) gene encodes a protein involved in lipoprotein lipase and hepatic lipase maturation. Homozygous mutations in LMF1 leading to severe hypertriglyceridemia (SHTG) are rare in the literature. A few additional rare LMF1 variants have been described with poor functional studies. The aim of this study was to assess the frequency of LMF1 variants in a cohort of 385 patients with SHTG, without homozygous or compound heterozygous deleterious mutations identified in lipoprotein lipase (LPL), apolipoprotein A5 (APOA5), apolipoprotein C2 (APOC2), glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) genes, and to determine their functionality. LMF1 coding variants were screened using denaturing high-performance liquid chromatography followed by direct sequencing. In silico studies of LMF1 variants were performed, followed by invitro functional studies using human embryonic kidney 293T (HEK-293T) cells cotransfected with vectors encoding human LPL and LMF1 cDNA. LPL activity was measured in cell culture medium after heparin addition using human VLDL-TG as substrate. Nineteen nonsynonymous coding LMF1 variants were identified in 65 patients; 10 variants were newly described in SHTG. Invitro, p.Gly172Arg, p.Arg354Trp, p.Arg364Gln, and p.Arg537Trp LMF1 variants decreased LPL activity, and the p.Trp464Ter variant completely abolished LPL activity. We identified a young girl heterozygote for the p.Trp464Ter variant and a homozygote carrier of the p.Gly172Arg variant with a near 50% decreased LPL activity invitro and invivo. The study confirms the rarity of LMF1 variants in a large cohort of patients with SHTG. LMF1 variants are likely to be involved in multifactorial hyperchylomicronemia. Partial LMF1 defects could be associated with intermittent phenotype as described for p.Gly172Arg homozygous and p.Trp464Ter heterozygous carriers.

Abstract 181: The Use of Recombinant Human Lipoprotein Lipase as a Treatment for Hypertriglyceridemia

Pancreatitis affects 270,000 number of people a year in the US and has a mortality rate of approximately 5%. Hypertriglyceridemia is a well established risk factor for acute pancreatitis, accounting for 10% of cases. To investigate recombinant human lipoprotein lipase (rhLPL) as a potential therapy for pancreatitis caused by hypertriglyceridemia, we examined effect of rhLPL produced in CHO cells on lipoproteins in human plasma and hypertriglyceridemic mice. IV injection of 500 μL of 20% Intralipid into mice increased plasma TG levels over 200-fold to about 6000 mg/dL at 15 min. When mice were co-injected with rhLPL (2.5 μg/g BW; IV), TG levels reached 2200 mg/dL and by 3 h were completely normal unlike control animals, which took 24 hours. Plasma TGs were decreased by 81% and 63% compared to control mice when rhLPL was administered IV at a dose 2 μg/g BW and 1 μg/g BW consecutively for mice treated IP with 1 mL of 20% Intralipid. The effect of rhLPL was sustained for 6 hours for high dose rhLPL. Enhanced lipolysis from rhLPL treatment resulted in a 2-fold increase of plasma FFA, but the albumin binding capacity for FFA did not appear to be exceeded because all FFA were bound to either lipoproteins or albumin. Moreover, no pathological changes were observed in the pancreas of treated mice, as assessed by histology. Amylase levels in rhLPL treated mice given Intralipid at 6 h were increased only 1.4-fold, whereas in control mice 2.8-fold. Addition of rhLPL in vitro (2 μg per 250 μL of human plasma) resulted in a significant decrease in lipoprotein particle numbers of all subpopulations of VLDL, small LDL, and small HDL, whereas a significant increase was observed for large LDL and large HDL particle number. The sizes of VLDL shifted towards smaller particles and the opposite was observed for LDL and HDL particles. Overall, there was a 57% reduction of TGs in plasma treated with rhLPL. In summary, rhLPL at relatively low doses accelerates the clearance of TG-rich lipoproteins in mice after a fat load. It does so without causing accumulation of toxic levels of FFA in plasma and reduces hypertriglyceridemia induced markers of pancreatitis. Furthermore, observed changes in human lipoprotein subpopulation distribution after rhLPL treatment are positive in terms of atherosclerosis risk.

Striated muscle gene therapy for the treatment of lipoprotein lipase deficiency.

Excessive circulating triglycerides due to reduction or loss of lipoprotein lipase activity contribute to hypertriglyceridemia and increased risk for pancreatitis. The only gene therapy treatment for lipoprotein lipase deficiency decreases pancreatitis but minimally reduces hypertriglyceridemia. Synthesized in multiple tissues including striated muscle and adipose tissue, lipoprotein lipase is trafficked to blood vessel endothelial cells where it is anchored at the plasma membrane and hydrolyzes triglycerides into free fatty acids. We conditionally knocked out lipoprotein lipase in differentiated striated muscle tissue lowering striated muscle lipoprotein lipase activity causing hypertriglyceridemia. We then crossed lipoprotein lipase striated muscle knockout mice with mice possessing a conditional avian retroviral receptor gene and injected mice with either a human lipoprotein lipase retrovirus or an mCherry control retrovirus. Post-heparin plasma lipoprotein lipase activity increased for three weeks following human lipoprotein lipase retroviral infection compared to mCherry infected mice. Human lipoprotein lipase infected mice had significantly lower blood triglycerides compared to mCherry controls and were comparable to wild-type blood triglyceride levels. Thus, targeted delivery of human lipoprotein lipase into striated muscle tissue identifies a potential therapeutic target for lipoprotein lipase deficiency.

Lipoprotein Lipase: A General Review

Carbohydrates (e.g., glucose) and lipids (e.g., free fatty acids or FFAs) are the most important sources of energy for most organisms, including humans. Lipoprotein lipase (LPL) is an extracellular enzyme (EC 3.1.1.34) that is essential in lipoprotein metabolism. LPL is a glycoprotein that is synthesized and secreted in several tissues (e.g., adipose tissue, skeletal muscle, cardiac muscle, and macrophages). At the luminal surface of the vascular endothelium (site of the enzyme action), LPL hydrolyzes triglyceride-rich lipoproteins (e.g., chylomicrons, very lowdensity lipoproteins), providing FFAs and glycerol for tissue use. Therefore, LPL plays a key metabolic role in providing substrates for lipogenesis and lipid storage, and in supplying immediate energy for different tissues. Knowledge about this enzyme has greatly increased over the past decade. A detailed understanding of the fascinating, although complex, apparatus by which LPL exerts its catalytic activity in the turbulent bloodstream is just one of the examples. Additionally, interest in LPL activity has been reinforced by its pathophysiological relevance in chronic degenerative diseases such as dyslipidemia, obesity, type 2 diabetes mellitus, and Alzheimer's disease, and in other contexts of disordered lipid metabolism such as severe hypertriglyceridemia and the (potentially) associated acute pancreatitis as well as in non-alcoholic fatty liver disease. This work aimed at critically reviewing the current knowledge of historical, terminological, biochemical, pathophysiological, and therapeutic aspects of human LPL activity.

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.

Orlistat response to missense mutations in lipoprotein lipase.

The human lipoprotein lipase (LPL) is a therapeutic target for obesity, and inhibition of LPL with the approved small molecule agent orlistat has been widely used in clinic to treat obesity-related health problems such as diabetes and cardiovascular diseases. However, a variety of missense mutations in LPL protein have been observed, which may cause resistance or sensitization for orlistat, largely limiting the clinical applications of orlistat in obesity therapy. Here, we integrated molecular dynamics simulations and enzyme inhibition to investigate orlistat response to 16 disorder-associated missense mutations in LPL catalytic domain. It was found that most mutations have a modest effect on orlistat binding, and only few can exert strong impact to the binding. Three unfavorable (Trp86Arg, Ile194Thr, and Glu242Lys) and two favorable (His136Arg and Gly188Glu) mutations were identified, which can alter the binding affinity and inhibitory activity of orlistat considerably. Structural and energetic analysis revealed that these potent mutations induce orlistat resistance and sensitization by directly influencing the intermolecular interaction between LPL and orlistat or by indirectly addressing allosteric effect on LPL structure.

Effects of acute hypoxia on human adipose tissue lipoprotein lipase activity and lipolysis.

BackgroundAdipose tissue regulates postprandial lipid metabolism by storing dietary fat through lipoprotein lipase-mediated hydrolysis of exogenous triglycerides, and by inhibiting delivery of endogenous non-esterified fatty acid to nonadipose tissues. Animal studies show that acute hypoxia, a model of obstructive sleep apnea, reduces adipose tissue lipoprotein lipase activity and increases non-esterified fatty acid release, adversely affecting postprandial lipemia. These observations remain to be tested in humans.MethodsWe used differentiated human preadipocytes exposed to acute hypoxia as well as adipose tissue biopsies obtained from 10 healthy men exposed for 6 h to either normoxia or intermittent hypoxia following an isocaloric high-fat meal.ResultsIn differentiated preadipocytes, acute hypoxia induced a 6-fold reduction in lipoprotein lipase activity. In humans, the rise in postprandial triglyceride levels did not differ between normoxia and intermittent hypoxia. Non-esterified fatty acid levels were higher during intermittent hypoxia session. Intermittent hypoxia did not affect subcutaneous abdominal adipose tissue lipoprotein lipase activity. No differences were observed in lipolytic responses of isolated subcutaneous abdominal adipocytes between normoxia and intermittent hypoxia sessions.ConclusionsAcute hypoxia strongly inhibits lipoprotein lipase activity in differentiated human preadipocytes. Acute intermittent hypoxia increases circulating plasma non-esterified fatty acid in young healthy men, but does not seem to affect postprandial triglyceride levels, nor subcutaneous abdominal adipose tissue lipoprotein lipase activity and adipocyte lipolysis.

Effects of Acute Hypoxia on Human Adipose Tissue Lipoprotein Lipase Activity and Lipolysis

ContextAdipose tissue regulates postprandial lipid metabolism by storing dietary fat through lipoprotein lipase (LPL)‐mediated hydrolysis of dietary triglycerides, and by inhibiting non‐esterified fatty acid (NEFA) delivery to nonadipose tissues. Studies conducted in animal showed that acute intermittent hypoxia, a model of obstructive sleep apnea, reduce adipose tissue LPL activity and increase NEFA release, adversely affecting postprandial lipemia. These observations remain to be tested in humans.ObjectiveTo investigate the effects of acute hypoxia on human adipose tissue LPL activity and lipolysis.MethodsDifferentiated human preadipocytes were exposed to hypoxia (3% O2) for 24h and adipose tissue biopsies were obtained from 10 healthy men exposed for 6 hours to either normoxia or intermittent hypoxia following an isocaloric high‐fat meal.ResultsIn differentiated human preadipocytes, acute hypoxia induced a 6‐fold reduction in LPL activity (p<0.001). In humans, the rise in postprandial triglyceride levels did not differ between normoxia and intermittent hypoxia. NEFA levels were significantly higher during intermittent hypoxia session (condition effect, p<0.05). Intermittent hypoxia did not affect subcutaneous abdominal adipose tissue LPL activity. No difference in lipolytic‐induced responses of isolated subcutaneous abdominal adipocytes were observed between normoxia and intermittent hypoxia sessions.ConclusionsAcute hypoxia strongly inhibits LPL activity in differentiated subcutaneous abdominal preadipocytes. Acute intermittent hypoxia increases circulating plasma NEFA in young healthy men, but does not seem to affect postprandial triglyceride levels, nor subcutaneous abdominal adipose tissue LPL activity and adipocyte lipolysis.Support or Funding InformationThe study is dedicated to the memory of our colleague Michaël Babinsky. We are grateful to Mrs. Ann Beninato and Sabrina Ait‐Ouali for their technical assistance. Bimit Mahat is a recipient of a Ontario Graduate scholarship. Étienne Chassé is a recipient of a Institut de recherche de l'Hôpital Montfort scholarship. This study was supported by the Natural Sciences and Engineering Research Council of Canada as well as the University of Ottawa.

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).

Are Common Polymorphisms of the Lipoprotein Lipase and Human Paraoxonase-1 Genes Associated with the Metabolic Syndrome in South African Asian Indians?

A cross-sectional study was performed to determine the possible contribution of the Human Paraoxonase-1 (PON1) and Lipoprotein Lipase (LPL) polymorphisms to the risk of the metabolic syndrome (MetS) in 817 participants of South African Asian Indian ancestry. Demographic and anthropometric data, including fasting blood for analysis of glycaemic and lipid parameters was collected. DNA was isolated from peripheral blood and allelic polymorphisms at positions Q192R, L55M in the PON1 gene and S447X and N291S in the LPL gene were studied using real-time PCR. Melting curve analysis was used to identify homozygotes and heterozygotes. The MetS was classified using the harmonised criteria. The prevalence of the MetS was 47.99%, with the main drivers being the increased waist circumference (96.6%), raised blood pressure (76.8%) and raised triglyceride levels (72.4%). There was no significant difference (p=n/s) in the distribution of the genotypes as well as their alleles in subjects with and without MetS. Increased levels of triglycerides was found in subjects with the MetS who had the QQ (p=0.007; OR=1.19; 95%CI=1.04; 1.36) and QR (p=0.018; OR=1.73; 95% CI=1.12; 2.67) genotypes of the Q192R polymorphisms. Subjects who had both the SX genotype (S447X polymorphism) and the LM genotype (L55M polymorphism) were more likely to have the MetS than those without (p=0.016; OR 2.19; 95% CI: 1.17, 4.06). Interactions involving the PON 1 gene may predispose to the MetS and to its component risk factors such as hypertriglyceridemia in this population. Environmental factors, such as lifestyle behaviour patterns appear to be the main driver contributing to obesity-related MetS.

A new monoclonal antibody, 4-1a, that binds to the amino terminus of human lipoprotein lipase

Lipoprotein lipase (LPL) has been highly conserved through vertebrate evolution, making it challenging to generate useful antibodies. Some polyclonal antibodies against LPL have turned out to be nonspecific, and the available monoclonal antibodies (Mabs) against LPL, all of which bind to LPL's carboxyl terminus, have drawbacks for some purposes. We report a new LPL-specific monoclonal antibody, Mab 4-1a, which binds to the amino terminus of LPL (residues 5–25). Mab 4-1a binds human and bovine LPL avidly; it does not inhibit LPL catalytic activity nor does it interfere with the binding of LPL to heparin. Mab 4-1a does not bind to human hepatic lipase. Mab 4-1a binds to GPIHBP1-bound LPL and does not interfere with the ability of the LPL–GPIHBP1 complex to bind triglyceride-rich lipoproteins. Mab 4-1a will be a useful reagent for both biochemists and clinical laboratories.

Immune Responses to AAV-Vectors, the Glybera Example from Bench to Bedside.

Alipogene tiparvovec (Glybera®) is an adeno-associated virus serotype 1 (AAV1)-based gene therapy that has been developed for the treatment of patients with lipoprotein lipase (LPL) deficiency. Alipogene tiparvovec contains the human LPL naturally occurring gene variant LPLS447X in a non-replicating viral vector based on AAV1. Such virus-derived vectors administered to humans elicit immune responses against the viral capsid protein and immune responses, especially cellular, mounted against the protein expressed from the administered gene have been linked to attenuated transgene expression and loss of efficacy. Therefore, a potential concern about the use of AAV-based vectors for gene therapy is that they may induce humoral and cellular immune responses in the recipient that may impact on efficacy and safety. In this paper, we review the current understanding of immune responses against AAV-based vectors and their impact on clinical efficacy and safety. In particular, the immunogenicity findings from the clinical development of alipogene tiparvovec up to licensing in Europe will be discussed demonstrating that systemic and local immune responses induced by intra-muscular injection of alipogene tiparvovec have no deleterious effects on clinical efficacy and safety. These findings show that muscle-directed AAV-based gene therapy remains a promising approach for the treatment of human diseases.

Skeletal muscle damage and impaired regeneration due to LPL-mediated lipotoxicity

According to the concept of lipotoxicity, ectopic accumulation of lipids in non-adipose tissue induces pathological changes. The most prominent effects are seen in fatty liver disease, lipid cardiomyopathy, non-insulin-dependent diabetes mellitus, insulin resistance and skeletal muscle myopathy. We used the MCK(m)-hLPL mouse distinguished by skeletal and cardiac muscle-specific human lipoprotein lipase (hLPL) overexpression to investigate effects of lipid overload in skeletal muscle. We were intrigued to find that ectopic lipid accumulation induced proteasomal activity, apoptosis and skeletal muscle damage. In line with these findings we observed reduced Musculus gastrocnemius and Musculus quadriceps mass in transgenic animals, accompanied by severely impaired physical endurance. We suggest that muscle loss was aggravated by impaired muscle regeneration as evidenced by reduced cross-sectional area of regenerating myofibers after cardiotoxin-induced injury in MCK(m)-hLPL mice. Similarly, an almost complete loss of myogenic potential was observed in C2C12 murine myoblasts upon overexpression of LPL. Our findings directly link lipid overload to muscle damage, impaired regeneration and loss of performance. These findings support the concept of lipotoxicity and are a further step to explain pathological effects seen in muscle of obese patients, patients with the metabolic syndrome and patients with cancer-associated cachexia.