Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism. The enzyme catalyzes hydrolysis of triacylglycerols (TG) of chylomicrons and of very low density lipoproteins (VLDL). However, the mechanisms involved in the regulation of this protein are not fully understood yet. Therefore, the aim of the theses is to study selected aspects of LPL activity regulation. Recently discovered apolipoprotein A-V (apo A-V) substantially affects triglyceridemia and it is presumed that it may function as LPL activator. However, its concentration in the blood is extremely low and we therefore investigated whether most of apo A-V could be bound to the heparan sulfate proteoglycan (HSPG) of vascular wall similarly to LPL. Intravenous heparin application in healthy volunteers resulted in an expected increase in LPL activity but apo A-V concentration did not change. Our results do not support the hypothesis that most of apo A-V is bound to HSPG of the capillary endothelium. An alcohol consumption plays also a role in LPL regulation - the long-term moderate alcohol consumption is known to increase enzyme activity; on the contrary, it is presumed that LPL activity is inhibited immediately after alcohol consumption. However, the direct evidence for such a premise is missing. The other aim of the theses was to...

Une augmentation de l’hypertrophie et l'hyperplasie des adipocytes est au coeur du développement de l'obésité. Nous avons déjà constaté que 14-3-3zeta (14-3-3ζ), une protéine d’échafaudage moléculaire, a plusieurs rôles essentiels dans l'adipogenèse. Cependant, les contributions de 14-3-3ζ dans la fonction des adipocytes matures ne sont pas connues. Les cellules 3T3-L1 et souris dépourvues de 14-3-3ζ dans les adipocytes (adi14-3-3ζKO) ont été utilisés pour examiner le rôle de 14-3-3ζ dans la lipolyse. L’élimination de 14-3-3ζ dans les cellules 3T3-L1 par l’ARNi a réduit significativement la lipolyse stimulée par l'isoprotérénol (un agoniste bêta adrénergique), la forskoline (un activateur de l’adénylate cyclase) et le dibutyryl AMPc (dbcAMP). Les analyses par qPCR ont démontré des réductions significatives d’adipose triglyceride lipase (Atgl) et lipase hormonsensible (Hsl) au niveau transcriptionnel. De plus, une réduction au niveau des substrats de la PKA phosphorylés et totaux tels que HSL et CREB, a été détectée par Western Blot dans les 3T3-L1 appauvris en 14-3-3ζ. Ces résultats in vitro ont été récapitulés in vivo, car des diminutions des taux phosphorylés et totaux de HSL ont été observés dans le tissu adipeux gonadique des souris adi14-3-3ζKO. Les souris adi14-3-3ζKO et les explants gonadiques ont également montré une lipolyse affaiblie après des injections i.p de l’agoniste bêta 3-adrénergique CL-316,243 et un traitement de l’isoprotérénol respectivement. De manière intéressante, une diminution de l’expression de 14-3-3ζ dans les cellules 3T3-L1 et les souris adi14-3-3ζKO a mené à une diminution des caractéristiques des adipocytes matures telles que les niveaux d’ARNm de Pparg, Lpl et Fabp4, les niveaux de PPARγ, le contenu en triglycérides et l'incorporation de Oil Red-O. Collectivement, ces résultats démontrent que 14-3-3ζ joue un rôle essentiel en facilitant la lipolyse et en déterminant la maturité des adipocytes. / Altered hypertrophy and hyperplasia of adipocytes lie at the core of the development of obesity. We previously demonstrated that the molecular scaffold 14-3-3zeta (14-3-3ζ) had essential roles in adipogenesis. However, the contributions of 14-3-3ζ to mature adipocyte function are not known. 3T3-L1 cells and tamoxifen-inducible adipocyte-specific 14-3-3ζ knockout mice (adi14-3-3ζKO) models were used to examine the roles of 14-3-3ζ in lipolysis. siRNA-mediated knockdown of 14-3-3ζ impaired lipolysis in 3T3-L1 cells stimulated by the beta-adrenergic agonist isoproterenol (Iso), forskolin (an activator of adenylyl cyclase) and dibutyryl cAMP (dbcAMP). qPCR analyses revealed significant reductions in lipase transcript levels (Atgl and Hsl). Furthermore, reductions in the phosphorylated and total levels of PKA substrates such as HSL and CREB were detected in 14-3-3ζ-depleted 3T3-L1 lysates by immunoblotting. These findings were recapitulated in vivo, as reductions in phosphorylated and total HSL levels were detected in the gonadal adipose tissue of adi14-3-3ζKO mice. adi14-3-3ζKO mice and gonadal explants also displayed impaired lipolysis following i.p CL-316,243 (a beta-3 adrenergic agonist) injections and Iso treatment respectively. Interestingly, decreased 14-3-3ζ expression in 3T3-L1 cells and mice revealed reductions in characteristics of a mature adipocyte, such as Pparg, Lpl, and Fabp4 transcript levels, PPARγ levels, triglyceride content, and Oil Red O (ORO) incorporation. Collectively, these results demonstrate that 14-3-3ζ has essential roles in facilitating lipolysis and determining adipocyte maturity.

Indications that existing parameter sets of extended Linear Interaction Energy (LIE) models are transferable between lipases from Rhizomucor Miehei and Thermomyces Lanigunosus in complex with a small set of vinyl esters are demonstrated. By calculat- ing energy terms that represents the cost of forming cavities filled by the ligand and the complex we can add them to a LIE model with en established parameter set. The levels of precision attained will be comparable to those of an optimal fit. It is also demonstrated that the Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) methods are in- applicable to the problem of calculating absolute binding energies, even when the largest source of variance has been reduced.

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.

Pankreatit anses vara en vanligt förekommande sjukdom hos hundar och katter och kan diagnostiseras genom mätningar av koncentrationen Canine Pancreas-specific Lipase (cPL) respektive Feline Pancreas-specific Lipase (fPL) i serum. Utifrån dessa koncentrationer graderas patienten enligt normalvärde, gråzon eller indikation på pankreatit. Golden standardmetoden för att analysera cPL/fPL är Spec cPL respektive Spec fPL. Nya metoder har utvecklats för kvantitativ mätning av pankreas-specifik lipas såsom Vcheck V200, ett instrument, som analyserar cPL/fPL med en fluorescerande immunoassay. Syftet med studien var att utvärdera analys av cPL/fPL på Vcheck V200 samt jämföra resultatet från detta instrument med värdena från ett referenslaboratorium i Tyskland för att se om det fanns en signifikant skillnad mellan metoderna. Koncentrationen cPL i hundserum (n=37) och koncentrationen fPL i kattserum (n=29) analyserades på Vcheck V200. Dessa prover skickades även till referenslaboratoriet där analysen Spec cPL respektive Spec fPL utfördes. Spridningen var stor kring bias i Bland-Altman diagram för både cPL och fPL och jämförelsen mellan metoderna för de specifika koncentrationerna av cPL/fPL bedömdes vara statistiskt signifikant (p<0,05). 27% av hundproverna graderas olika enligt de båda metoderna och skillnaden var signifikant (p<0,05). 24% av kattproverna graderades olika men skillnaden var inte signifikant (p=0,257). Studien tyder på att jämförelsen mellan de båda metoderna var signifikant förutom vid graderingen av kattproverna. Beaktande detta och det faktum att kvalitetssäkringen brister vid analys av fPL på grund av avsaknad av kontroller kan i dagsläget inte cPL/fPL på Vcheck V200 ersätta den nuvarande golden standardmetoden, trots att ingen signifikant skillnad sågs vid gradering av kattprover. / Pancreatitis is a common disease in canine and felines and can be diagnosed by measuring the concentration of Canine Pancreas-specific Lipase (cPL) or Feline Pancreas-specific Lipase (fPL) in serum. Based on the concentration of cPL/fPL, the patient is then classified in different diagnostic categories (normal value, gray zone or indication of pancreatitis). Spec cPL and Spec fPL is currently the golden standard method for analysis of cPL and fPL. New methods have been developed for the quantitative measurement of pancreatic lipases. Vcheck V200, being one example, utilizing a fluorescent immunoassay for quantification of the lipase. The aim of this study was to evaluate the cPL and fPL analysis on the Vcheck V200 and to examine if there was a significant difference (p≤0,05) when comparing the result from Vcheck V200 with the results from a reference laboratory. The concentration of cPL (n=37) and fPL (n=29) in serum from canine or felines were analyzed using Vcheck V200. The samples were also sent to the reference laboratory where Spec cPL and Spec cPL were performed. A Bland-Altman plot comparison between the two methods showed a large spread for both analysis of cPL and fPL. Comparison of the specific values for analysis of cPL and fPL between the two methods revealed a significant difference (p<0,05). 27% of the dog samples were categorized differently according to the two methods and this difference was significant (p<0,05). 24% of the cat samples were categorized differently and no significant difference were observed (p=0,257). This study indicates that the difference between the two methods was significant, besides the classification of cat samples. Considering this and the lack of quality assurance regarding analysis of fPL due to lack of controls, the cPL/fPL analysis on Vcheck V200 cannot replace the Spec cPL or Spec fPL at present.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment development for the purpose of preventing and managing the disease. The most widely distributed vaccine against TB is Bacillus Calmette-Gue´rin (BCG). Apart from being ineffective in certain individuals, BCG offers only a limited timeframe of protection, is unable to serve as a booster for extending this timeframe and due to the intradermal route of administration requires costly refrigeration and syringes. LipY protein, a M. tuberculosis cell wall lipase, may play a potential role as not only a drug target but a potential vaccine antigen. LipY is known to be up-regulated during both active infection and dormancy. In a previous study, sera from TB patients had shown an IgG and IgM response against it. In this study transplastomic Lactuca sativa and Nicotiana tabacum plants were generated by transforming the chloroplasts through the particle delivery system with pLsDv-LipY and pLD-LipY vectors respectively. The vectors were flanked by the native trnI and trnA gene sequence to facilitate homologous recombination into the chloroplast genome. The vector also contained the 16S rRNA promoter, the selectable marker gene, aadA for specitinomycin resistance, the rbcL untranslated region, the LsPpsbA (PpsbA in N. tabacum) promoter, and LsTpsbA (tpsbA in N. tabacum) untranslated region. iv Site specific integration of the LipY gene into the chloroplast genome was confirmed by PCR. Homoplasmy of transplastomic plants was confirmed by Southern blot analysis. These plants showed normal growth and were fertile, producing seeds. Once germinated, these seeds did not show Mendelian segregation of the transgene. Immunoblot analysis was performed to analyze the expression of the LipY protein. A 40kDa protein was produced in E.coli, and a 25kDa protein was produced in chloroplasts; a cleaved product in chloroplasts is still valuable as an antigen for vaccine production. Future studies will include testing this chloroplast derived antigen in animal models for vaccine development.

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