The absorption, packaging, and delivery of fat to appropriate peripheral tissues is essential for maintaining metabolic homeostasis, and defects or dysregulation of these processes can contribute to metabolic disorders such as diabetes, obesity, and hyperlipidemia. In the intestine, dietary fat is packaged into triglyceride-rich lipoprotein particles and delivered to peripheral tissues through the circulatory system. Lipolysis of lipoprotein triglycerides requires the enzyme lipoprotein lipase (LPL) and takes place on the luminal surface of capillary endothelial cells. Lipolysis by LPL is regulated in part by two proteins, GPIHBP1 and ANGPTL4. GPIHBP1, a GPI-anchored protein of capillary endothelial cells, is responsible for transporting LPL across endothelial cells to the capillary lumen. Without this transport, LPL becomes mislocalized to the interstitial space and cannot access triglyceride-rich lipoproteins, resulting in severe hypertriglyceridemia. Conversely, ANGPTL4 inhibits LPL and ANGPTL4 deficiency results in increased LPL activity and lower plasma triglyceride levels. Our goal is to understand how the interactions between LPL, GPIHBP1, and ANGPTL4 influence the delivery of triglyceride-derived fatty acids to tissues. In this thesis, I (1) use mouse models to elucidate the function of ANGPTL4 in regulating the clearance of diet-derived fat from plasma, (2) describe a mechanism for GPIHBP1-independent plasma triglyceride clearance observed in mice lacking both GPIHBP1 and ANGPTL4, and (3) propose that this GPIHBP1-independent mechanism is also operative in Gpihbp1–/– mice following a high fat diet challenge. The contributions of this thesis are significant because they close a gap in our knowledge of how and where ANGPTL4 functions, as well as indicating that, when ANGPTL4 is suppressed or absent altogether, a GPIHBP1-independent mechanism can function to clear plasma triglycerides.

La Lipoprotéine lipase (LPL) est une enzyme essentielle de la lipolyse intravasculaire dont la régulation est complexe. La découverte des miRs, régulateurs de l'expression posttranscriptionnelle des gènes via leurs interactions avec les régions 3' non traduite (3'UTR), apporte de nouvelles perspectives pour la compréhension de la régulation de la LPL et de ses gènes régulateurs. Nous présentons à travers deux études, l'implication des microARNs (miRs) dans la régulation de la LPL et d'un de ses gènes activateurs APOA5. Dans le premier travail, nous avons mis en évidence la création d'un site de liaison fonctionnel du miR-485-5p par expliquons ainsi le mécanisme potentiellement impliqué dans l'association de ce polymorphisme aux hypertriglycéridémies sévères et modérées en population générale. Dans un second travail, nous avons identifié un haplotype de la LPL, incluant la mutation p.Ser474Ter (rs328) et sept single nucleotide polymorphisme (SNPs) de la région 3'UTR, significativement associés à une diminution des triglycérides (TG) plasmatiques en population générale. Nous avons ensuite démontré la fonctionnalité des sept SNPs de la région 3'UTR par la suppression de sites de liaison de plusieurs miRs. Ainsi ces résultats suggèrent que l'association du variant p.Ser474Ter (rs328) à la triglycéridémie pourrait au moins partiellement être liée à son déséquilibre de liaison avec les sept SNPs fonctionnels de la région 3'UTR. Nos travaux sont parmi les premiers à mettre en évidence l'implication des miRs dans la régulation de la LPL et de ses gènes régulateurs chez l'homme. Ils permettent ainsi d'accroitre la connaissance des mécanismes impliqués dans la régulation de la lipolyse intravasculaire. Enfin, ils éclairent les mécanismes fonctionnels mis en jeu par deux polymorphismes significativement associés à la triglycéridémie / The lipoprotein lipase (LPL) is a key enzyme which regulates plasma triglycerides (TG) intravascular lipolysis involving a complex regulation. The microRNAs (miR) are implicated in gene post-transcriptional regulation through their interaction with the 3’untranslated region (3’UTR). Their discovery provides new insights in the understanding of the LPL regulation and its regulator genes. We present two works regarding the implication of miRs in the regulation of the LPL and one of its activator APOA5. First, we identified a functional miR-485-5p binding site creation induced by the minor C allele of the c.*158C>T (rs22667882) located in APOA5 3’UTR.We therefore provide an explanation of the mechanism potentially involved in this polymorphism association with both mild and severe hypertriglyceridemia in general population. In a second work, we identified a LPL haplotype harboring p.Ser474Ter (rs328) polymorphism and seven single nucleotide polymorphisms (SNPs) located in the 3’UTR. This haplotype is significantly associated with lower plasma triglycerides (TG) concentration in general population. We demonstrated that the SNPs located in the 3’UTR induce several functional miRs binding-site suppressions that could lead to an increase of LPL expression. Finally, p.Ser474Ter association with triglyceridemia could be at least partially explained by its strong linkage disequilibrium with these functional 3’UTR SNPs. These works are amongst the first studies to bright to light the miRs implication in the regulation of LPL or its regulator genes in human. They provide a better knowledge of the mechanisms involved in intravascular lipolysis. Finally, they also explain the functional mechanisms of two polymorphisms, significantly associated with the plasma TG concentration

Dyslipidemia often accompanies metabolic diseases such as obesity and type II diabetes mellitus and represents a risk factor for cardiovascular disease. Clearance of triglycerides from the plasma is mediated by lipoprotein lipase (LPL), which hydrolyzes the triglycerides in chylomicrons and VLDL, liberating fatty acids for tissue uptake. LPL functions in the capillaries of the heart, adipose tissue, and skeletal muscle where LPL is anchored to the capillary wall by its endothelial cell transporter GPIHBP1. LPL activity is regulated by several factors including three members of the angiopoietin-like (ANGPTL) family–ANGPTL3, ANGPTL4, and ANGPTL8. How these proteins interact with LPL, especially in the physiological context of LPL anchored to endothelial cells by GPIHBP1, has not been well characterized. In my studies of ANGPTL4, I found when LPL is bound to GPIHBP1, it is partially, but not completely, protected from inactivation by ANGPTL4. Inactivation of LPL by ANGPTL4 leads to the dissociation of active LPL dimers into inactive monomers and I found that these monomers have a greatly reduced affinity for GPIHBP1. ANGPTL4 can be cleaved in vivo, separating the N-terminal coiled-coil domain from the C-terminal fibrinogen like-domain. I found the N-terminal domain alone is a much more potent LPL inhibitor than the full-length protein, even though both appear to have similar binding affinities for LPL-GPIHBP1 complexes. When I investigated ANGPTL3, I found ANGPTL3 itself is not a potent inhibitor of LPL at physiological concentrations, and unlike ANGPTL4, cleavage of ANGPTL3 does not improve its ability to inhibit LPL. Instead I found that ANGPTL3 forms a complex with ANGPTL8, a complex that only forms efficiently when the two proteins are co-expressed, and that this complex allows ANGPTL3 to bind and inhibit LPL. My data provide new insights into how ANGPTL proteins regulate LPL activity and the delivery of fat to tissues.

Mortality from cardiovascular disease in patients on chronic hemodialysis (HD) is 10 to 20 times greater than in the general population. One major risk factor is renal dyslipidemia, characterised by an impaired catabolism of triglyceride (TG)-rich lipoproteins with accumulation of atherogenic remnant particles. A contributing factor may be derangement of the lipoprotein lipase (LPL) system, the major lipase in the catabolism of TG-rich lipoproteins. The functional pool of LPL is located at vascular surfaces, and is released by heparin into the circulating blood and extracted and degraded by the liver. Unfractionated heparin (UFH) is commonly used during dialysis to avoid clotting in the extracorporeal devices, but is increasingly replaced by various low molecular weight heparin (LMWH) preparations. Plasma LPL activity is usually lower after injection of LMWH which is therefore said to release less LPL and cause less disturbance of lipoprotein metabolism than UFH. However, animal studies have revealed that LMWH is as efficient as UFH in releasing LPL but is less efficient in retarding hepatic uptake. The aim of this study was to explore the effects of UFH and a LMWH (dalteparin) on LPL activity and TG concentrations in HD-patients compared with healthy controls, matched for age and gender. A disturbed LPL system might contribute to an impaired lipoprotein metabolism, and hence, an aggravated cardiovascular condition. An 8-hour primed infusion of UFH to controls gave rise to an initial peak of LPL activity within 30 minutes. The activity then dropped by almost 80% over the next two hours and levelled off to a plateau that corresponded to 15% of the peak level. When UFH was infused to HD-patients the curve for LPL activity resembled that for controls, but was reduced by 50% during the peak, while the plateau activities were comparable. The interpretation was that the functional pool, represented by the initial peak, was impaired in HD-patients, while the production of lipase molecules, reflected by the plateau, was only marginally reduced. During the peak of LPL activity TG decreased in both groups, but less in HD-patients, as was expected from the lower circulating lipase activity. During the plateau phase with low lipase activity, TG increased towards and beyond baseline values. When dalteparin was infused, the same pattern of plasma LPL activity was observed, although remarkably reduced. In controls the peak was only 30% and the subsequent plateau 40% compared with the activities during the UFH infusion. A bolus of UFH given when the LPL activity had levelled off to a plateau brought out about the same amount of activity, regardless of whether dalteparin or UFH had been infused. The conclusion was that both heparin preparations had reduced endothelial LPL to a similar extent, but that dalteparin less efficiently retarded the hepatic uptake of the enzyme. As a consequence to this, TG tended to reach higher levels after the dalteparin infusion. The LPL activities were further reduced in HD-patients during infusion with dalteparin, the peak was only 27% and the plateau 35% compared with the activities when UFH was infused. There was no decrease in TG, but rather a continuous increase, suggesting a profound depletion of functional LPL. In another study in HD-patients, two anticoagulation regimes based on present clinical practice were compared, and the doses were adjusted to the respective manufacturers recommendation. UFH was administered as a primed infusion, whereas dalteparin was given only as a single bolus pre-dialysis, not followed by an infusion. The results were in line with those in the experimental studies and indicate that also in the clinical setting LMWH interferes with the LPL system as least as much as an infusion of UFH does, and temporarily impairs lipolysis of TG. This interference might, in consequence, contribute to an aggravated cardiovascular condition in HD-patients.

La lipoproteïna lipasa (LPL) és un enzim amb un paper central en el metabolisme lipídic que, sotmès a una regulació específica de teixit, modula la distribució dels triacilglicerols circulants entre els diferents teixits de l'organisme. En el nostre grup s'han estudiat els canvis d'activitat LPL associats a una situació d'estrès. En aquest treball descrivim inicialment que l'estrès agut per immobilització en rata provoca un ràpid descens (5 minuts) de l'activitat LPL del teixit adipós blanc retroperitoneal concomitant amb un augment de l'activitat LPL a plasma. Aquesta resposta suggereix que l'alliberament de l'enzim a la sang podria constituir un mecanisme de regulació de l'activitat LPL a teixits, no descrit anteriorment. A més a més, l'augment paral·lel dels nivells de nitrat a plasma durant la immobilització i resultats previs del nostre grup suggereixen que l'òxid nítric podria participar en aquest procés d'alliberament.En aquest context, vàrem recórrer a l'ús d'eines proteòmiques amb l'objectiu d'estudiar una possible modificació post-traduccional de l'LPL produïda per l'òxid nítric o per espècies reactives del nitrogen. L'aplicació d'aquestes eines ens ha portat a demostrar: (i) la inespecificitat de l'anticòs P66, un anticòs policlonal anti-LPL emprat en estudis anteriors, (ii) l'existència d'isoformes de punt isoelèctric (pI) de l'LPL de rata i (iii) la nitració in vivo en tres residus de tirosina de l'LPL de rata en resposta a l'administració de lipopolisacàrid, que podria constituir un nou mecanisme de regulació de l'activitat LPL tissular.El descobriment de l'existència d'isoformes de pI de l'LPL, obre les portes a estudis més exhaustius dirigits a la identificació de les diferències moleculars entre les isoformes i a explorar les seves possibles implicacions funcionals. / Lipoprotein lipase (LPL) is an enzyme that plays a key role in lipid metabolism. LPL is under tissue-specific regulation and modulates the distribution of circulating triacylglycerols between the tissues of the organism. Our research group has studied the changes in LPL activity associated with stress. The present work reports that acute stress by immobilization induces a fast (5 minutes) down-regulation of retroperitoneal white adipose tissue LPL activity and a simultaneous increase in plasma LPL activity. This response suggests that the release of this enzyme from the endothelium to the bloodstream may constitute a fast mechanism of tissue LPL activity regulation that has not previously been studied.In this context, we used proteomic tools to study a potential post-translational modification in LPL induced by nitric oxide or by reactive nitrogen species. The use of such tools has allowed us to demonstrate: (i) the non-specificity of P66 antibody, a polyclonal antibody used in previous studies; (ii) the existence of isoelectric point (pI) isoforms of rat LPL; and (iii) the in vivo nitration of three tyrosine residues in rat LPL in response to lipopolysaccharide administration, which could be a new mechanism of tissue LPL activity regulation.The discovery of LPL pI isoforms opens the door to further studies aimed at identifying the molecular differences between the isoforms and at exploring their potential functional implications.

Indiana University-Purdue University Indianapolis (IUPUI) / The incidence of Metabolic Syndrome (MetS) is increasing worldwide and accompanied by elevated risks for cardiovascular disease (CVD) and other subsequent comorbidities. MetS is associated with increased circulating triglycerides. A key enzyme involved in triglyceride (TG) clearance is lipoprotein lipase (LPL) whose activity is modulated by a variety of factors. Recent literature has identified the importance of angiopoietin-like proteins (ANGPTL) as regulators of LPL activity and has hypothesized a model in which three of these proteins interact with LPL to regulate the partitioning of TG metabolism from adipose to skeletal muscle. The work detailed in this dissertation adds to the model of ANGPTL regulation of LPL by establishing how ANGPTL8 modulates the ability of ANGPTL3 and ANGPTL4 to inhibit LPL activity in the bloodstream and localized environments, respectively. In the updated model, elevated insulin concentrations result in increased hepatic ANGPTL3/8 secretion and increased ANGPTL4/8 in adipose tissue. ANGPTL3/8 works as an endocrine molecule to inhibit skeletal muscle LPL from hydrolyzing circulating TG. Simultaneously, ANGPTL4/8 works in a paracrine mechanism to bind LPL on the endothelial vasculature adjacent to adipose tissue to alleviate ANGPTL4-mediated LPL inhibition and also prevent ANGPTL3/8 inhibition of localized LPL. Thus, in the postprandial state free fatty acids (FFA) from the hydrolysis of TG are directed into adipocytes for storage. Under fasting conditions, ANGPTL8 production is decreased in adipocytes and hepatocytes. This decreased production results in diminished ANGPTL4/8 and ANGPTL3/8 secretion from their respective tissues. As a result, ANGPTL4 inhibits adipocyte localized LPL activity while ANGPTL3 at physiological concentrations has minimal effect on LPL activity. Furthermore, any ANGPTL3/8 which is produced has its LPL-inhibitory ability diminished by the circulating apolipoprotein ApoA5. LPL is more active in skeletal muscle compared to adipose tissue where energy is shunted towards utilization in the muscle and away from storage in adipose tissue. A complete understanding of LPL regulation by ANGPTL proteins can potentially provide therapeutics targets for MetS.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal. / Les patients diabétiques de type 2 développent de manière prématurée et accélérée des lésions athéromateuses. Des études ont démontré que la lipoprotéine lipase (LPL) macrophagique pouvait contribuer, en favorisant l'accumulation de lipides dans les lésions athéromateuses, au développement de l'athérosclérose. Nous avons étudié la régulation de la LPL macrophagique dans le diabète. Nos résultats démontrent que: 1) l'incubation des macrophages murins et humains en présence de concentrations élevées de glucose augmente l'expression génique, la masse immunoréactive et l'activité de la LP. La voie de la protéine kinase C est impliquée dans l'effet modulateur du glucose sur la LPL macrophagique, via la surexpression du facteur de transcription c-fos et la liaison accrue des protéines nucléaires à la séquence AP-1 du promoteur génique de la LPL; 2) le glucose, à des concentrations élevées, stimule in vitro l'expression génique des "peroxisome proliferator-activated receptors" (PP AR) <X et p par les macrophages murins et humains, et diminue celle des PP AR y par les macrophages humains. Une augmentation des niveaux d' ARN messagers macrophagique de PP ARo: et p, et une inhibition de l'expression génique de PP AR y sont aussi observées chez les patients diabétiques de type 2. L'incubation des macrophages murins, en présence de concentrations élevées de glucose, augmente la liaison des protéines nucléaires extraites de ces cellules à l'élément de réponse aux PP ARs situé dans le promoteur du gène de la LPL. Ces données prises dans leur ensemble suggèrent l'implication des PP ARs dans l'effet stimulant du glucose sur l'expression génique de la LPL macrophagique; 3) l'expression, la production et l'activité de LPL macrophagique sont augmentées chez les patients diabétiques et que les facteurs périphériques présents dans le sérum de ces sujets sont en partie responsables de ces anomalies. Une sécrétion macrophagique accrue de facteur de nécrose tumorale-a, tant au niveau basal que stimulé par la LPL, est aussi observée chez les patients diabétiques. En résumé, nos données démontrent une augmentation d'expression de la LPL macrophagique chez le patient diabétique de type 2, au moins partiellement attribuable à l'hyperglycémie. Cette altération de la régulation de la LPL macrophagique associée au diabète pourrait représenter un nouveau mécanisme par lequel l'hyperglycémie favoriserait le développement et la progression de l'athérosclérose, chez le patient diabétique de type 2.

No description available.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease where a significant proportion of patients will develop an aggressive disease. Today, the mutational status of the immunoglobulin heavy variable (IGHV) genes is one of the strongest prognostic markers in CLL, where unmutated IGHV genes correlate with poor outcome. In addition, IGHV3-21 gene usage is associated with poor prognosis independent of mutational status. Recently, several genes were shown to be differently expressed between IGHV mutated and unmutated CLL and were suggested as prognostic markers. The aim of this thesis was to examine the applicability of these RNA-based prognostic markers in CLL. In papers I and II, the prognostic significance of LPL and TCL1A mRNA expression in CLL was investigated in 140 and 144 patients, respectively. High expression was found to be associated with inferior clinical outcome for both markers. However, CLL cases with mutated IGHV3-21 genes displayed low levels of LPL expression, indicating that LPL cannot identify this poor-risk patient group. In contrast, high TCL1A expression was detected in all IGHV3-21 cases. To elucidate the functionality of LPL in CLL, LPL lipase activity was measured in 33 cases. The lipase activity was found to be invariably low, implying an alternative function for LPL in CLL. In paper III, a comprehensive analysis of five RNA-based markers (LPL, TCL1A, ZAP70, CLLU1 and MCL1) was performed in 252 CLL patients. All RNA-based markers except MCL1 predicted clinical outcome, with LPL being the strongest. Moreover, LPL expression independently predicted overall survival when adjusted for established markers. All of the RNA-based markers added additional prognostic information to established markers, e.g. high LPL expression predicted an inferior outcome in patients with mutated IGHV genes or good-risk cytogenetics. For clinical application, over time stability of prognostic markers is crucial. In paper IV, the expression of LPL, TCL1A, ZAP70 and MCL1 was investigated in samples taken at diagnosis and at a follow-up of seven years in 104 CLL patients. LPL was found to be the most stable marker, displaying high correlation between the sequential samples, whereas ZAP70 and MCL1 varied significantly. TCL1A expression increased at follow-up, which may indicate disease progression as TCL1A promotes cell survival. In summary, this thesis highlights the applicability of RNA-based markers in CLL prognostication, both as single markers or in combination with established markers. In particular, LPL was shown to be the strongest RNA-based marker in terms of prognostic strength and stability.

Individuals with high levels of plasma triglycerides are at high risk to develop cardiovascular disease (CVD), currently one of the major causes of death worldwide. Recent epidemiological studies show that loss-of-function mutations in the APOC3 gene lower plasma triglyceride levels and reduce the incidence of coronary artery disease. The APOC3 gene encodes for apolipoprotein (APO) C3, known as an inhibitor of lipoprotein lipase (LPL) activity. Similarly, a common gain-of-function mutation in the LPL gene is associated with reduced risk for CVD. LPL is central for the metabolism of lipids in blood. The enzyme acts at the endothelial surface of the capillary bed where it hydrolyzes triglycerides in circulating triglyceride-rich lipoproteins (TRLs) and thereby allows uptake of fatty acids in adjacent tissues. LPL activity has to be rapidly modulated to adapt to the metabolic demands of different tissues. The current view is that LPL is constitutively expressed and that the rapid modulation of the enzymatic activity occurs by some different controller proteins. Angiopoietin-like protein 4 (ANGPTL4) is one of the main candidates for control of LPL activity. ANGPTL4 causes irreversible inactivation through dissociation of the active LPL dimer to inactive monomers. Other proteins that have effects on LPL activity are the APOCs which are surface components of the substrate TRLs. APOC2 is a well-known LPL co-factor, whereas APOC1 and APOC3 independently inhibit LPL activity. Given the important role of LPL for triglyceride homeostasis in blood, the aim of this thesis was to find small molecules that could increase LPL activity and serve as lead compounds in future drug discovery efforts. Another aim was to investigate the molecular mechanisms for how APOC1 and APOC3 inhibit LPL activity. Using a small molecule screening library we have identified small molecules that can protect LPL from inactivation by ANGPTL4 during incubations in vitro. Following a structure-activity relationship study we have synthesized lead compounds that more efficiently protect LPL from inactivation by ANGPTL4 in vitro and also have dramatic triglyceride-lowering properties in vivo. In a separate study we show that low concentrations of fatty acids possess the ability to prevent inactivation of LPL by ANGPTL4 under in vitro conditions. With regard to APOC1 and APOC3 we demonstrate that when bound to TRLs, these apolipoproteins prevent binding of LPL to the lipid/water interface. This results in decreased lipolysis and in an increased susceptibility of LPL to inactivation by ANGPTL4. We demonstrate that hydrophobic amino acid residues that are centrally located in the APOC3 molecule are critical for attachment of this protein to lipid emulsion particles and consequently for inhibition of LPL activity. In summary, this work has identified a lead compound that protects LPL from inactivation by ANGPTL4 in vitro and lowers triglycerides in vivo. In addition, we propose a molecular mechanism for inhibition of LPL activity by APOC1 and APOC3.