The effect of acute exercise on blood lipids and lipoproteins has been examined and their response is believed to reflect the changes that occur as a result of chronic exercise training. The cross sectional differences between trained and untrained individuals and the results of longitudinal investigations suggest LDL particle size and distribution may be altered after acute exercise. Moreover, the response of the lipid and lipoprotein profile of Black men may be different than that of White men, owing to initial concentration differences or a genetic predisposition. PURPOSE: To examine the effects of expending 300 and 600 calories through treadmill walking at a moderate intensity (65% VO2max) on blood lipids and lipoproteins as compared to a non-exercise, control trial over the course of 72 hours. METHODS: Ten White and ten Black men participated in this study after being screened for strict inclusion criteria (including: parents/grandparents either Black or White and from the United States; sedentary, but otherwise healthy; VO2max 30-45ml⋅kg⋅min-1; and body fatness ¡Ü25%). Analysis of variance with repeated measures design was used (Group x Trial x Time). Blood was collected at Baseline (0hr), 12hr, 24hr, 48hr and 72hr for determination of blood lipid concentrations, LDL particle size, LDL distribution and CETPa. RESULTS: Black men of this investigation had higher HDL-C (49.4 vs 41.8 mg⋅dL-1), HDL3-C (35.1 vs 30.8 mg⋅dL-1) and CETPa (82.1 vs 52.8 pmol⋅ml-1⋅3hr-1) and lower TC (148.9 vs 177.1 mg⋅dL- 1), LDL-C (83.4 vs 116.3 mg⋅dL-1) and TC to HDL ratios (3.06 vs 4.46) than the White men (p<0.05) over the control period. There were no group differences detected in LDL particle size (White 25.63nm vs Black 25.79nm), LDL distribution (Zone 1, White 58.19% vs Black 64.71%; Zone 2, White 20.16% vs Black 16.31%; Zone 3, White 21.65% vs Black 18.98%), HDL2-C (White 11.5 vs Black 14.2 mg⋅dL-1), or TG (White 95.0 vs Black 79.4 mg⋅dL-1). Black and White men responded to acute exercise in similar fashion for these variables and therefore our statistical model collapsed the data into a single group by trial and over time. Exercise did not statistically alter TC, LDL-C, LDL particle size, LDL distribution, HDL2-C or CETPa in the 72 hours following each exercise bout. However, HDL-C was increased 6.2%, 10.1% and 5.8%, HDL3-C was increased 9.8%, 13.8% and 9.8%, while TG were reduced 24.8%, 27.3% and 22.4% at 12hr, 24hr and 48hr, respectively from the Base value of the 600 Kcal Trial. Additionally, TG were reduced 18.0% at 12hr from the baseline value of the 300 Kcal Trial. CONCLUSION: These results are the first to indicate that acute treadmill walking at moderate intensity of sufficient caloric expenditure modified HDL-C, HDL3-C and TG in Black men. No differences were determined in the manner in which Black and White American men statistically increased HDL-C and HDL3-C and reduced TG after acute exercise. In addition, there was no impact of acute exercise on TC, LDL-C, HDL2-C, CETPa, LDL particle size or LDL distribution in either group. However, these data suggest that Black/White differences exist in CETPa, where Black men have greater activity of this enzyme / Dissertation / PhD

A nanoemulsão LDE tem composição lipídica semelhante à da LDL natural e é utilizada para estudos do metabolismo da LDL. Estudos anteriores mostraram que a LDE é captada pelas células pelo LDL-r, porém outros receptores podem estar envolvidos nesta captação, como LRP-1, CD36 e CD68. Os objetivos deste estudo foram: investigar a captação da LDE por células endoteliais, fibroblastos, monócitos, macrófagos e H292, identificar os receptores envolvidos na captação da LDE pelas mesmas células e avaliar os efeitos da modificação química da LDE sobre a estabilidade, captação celular, lipoperoxidação celular e citotoxicidade. A LDE marcada com [3H]-colesterol livre e [14C]-éster de colesterol foi incubada por 4 horas com as linhagens celulares. Após a incubação, foram realizados os testes de captação da LDE e competição da LDE com a LDL natural. A expressão dos receptores LDL-r, LRP, CD36 e CD68 foi avaliada pelos métodos de imunocitoquímica, citometria de fluxo e PCR real time. Para investigar os efeitos da modificação da LDE (LDE-CO), o éster de colesterololeato de colesterol (monoinsaturado), foi substituído por linoleato de colesterol (LDE-CL) (poliinsaturado) e por estearato de colesterol (LDE-CE) (saturado). Estas nanoemulsões foram submetidas a testes de estabilidade (tamanho, polidispersão, pH e peroxidação), captação celular, peroxidação lipídica celular e citotoxicidade. Nos resultados, foi observado que todas as células estudadas internalizaram o colesterol livre e éster de colesterol proporcionalmente às concentrações de LDE-CO incubadas com diferença de saturação entre elas, sendo o colesterol livre mais captado que o éster de colesterol da LDE-CO por todas as células estudadas. Além disso, os monócitos (THP-1) demonstraram maior captação de LDE-CO que as demais células. No estudo de competição com a LDL natural ocorreu uma diminuição da captação (r2-0,73), sugerindo que as duas partículas competem pelo mesmo receptor. A LDE-CO foi capaz de inibir a expressão protéica dos receptores LDL em HUVEC (3,98 vezes), monócito (6,25 vezes) e fibroblasto (3,70 vezes) e a expressão gênica em monócito e HUVEC. Por citometria de fluxo, a expressão protéica do LDL-r em H292 e fibroblasto diminuiu. Em HUVEC a LDE-CO aumentou a expressão protéica em 3,57 vezes, já em monócito, a LDE-CO diminuiu a expressão gênica e protéica (3,15 vezes) do LRP-1. Em macrófago e em H292, a LDE-CO aumentou a expressão gênica do LRP-1. A LDE-CO foi capaz de aumentar a expressão gênica e protéica (3,1 vezes) do CD36 em HUVEC, diminuir a expressão protéica (4,34 vezes) em macrófago e diminuir a expressão gênica e protéica (2,94 vezes) em H292. A LDE foi capaz de aumentar a expressão protéica (2,09 vezes) do CD68 em H292, e aumentar a expressão gênica em monócito e macrófago. A linhagem celular que apresentou maior taxa de sobrevivência na presença da LDE-CO foi o fibroblasto. Nas análises dos efeitos da modificação química da LDE, a LDE-oleato apresentou o tamanho e a lipoperoxidação menores que a LDE-linoleato e LDE-estearato. Nenhuma das LDEs apresentou modificação da estabilidade antes de 30 dias. As células apresentaram maior lipoperoxidação na presença de LDECL quando comparada à presença de LDE-CO e LDE-CE. A captação de [3H]-colesterol livre foi maior que de éster de colesterol das três LDEs por todas as células estudadas. A composição da LDE com oleato de colesterol foi a que apresentou características mais favoráveis em termos de tamanho de partículas e susceptibilidade à peroxidação. A captação celular do colesterol livre foi maior do que a do éster de colesterol em todas as linhagens estudadas das três LDEs, sugerindo que o colesterol livre possa dissociar-se da LDE e ser captado pelas células por vias não específicas. Os dados obtidos neste trabalho ajudam na compreensão dos mecanismos de captação e da influência da composição na estabilidade e adequação do sistema LDE e outros similares às suas potenciais aplicações terapêuticas ou diagnósticas. / With fat composition similar to natural LDL, the LDE nanoemulsion can be used to study the metabolism of LDL. Other studies have shown that LDE is uptaken by cells by LDL-r receptors. Other receptors such as LRP-1, CD36 and CD38 may also be involved in the uptake. The objectives of this study were to investigate the uptake of LDE by endothelial and tumor cells, fibroblasts, monocytes and macrophages, to identify those receptors involved in this process and to evaluate the effects on LDE uptake by changing its chemical composition. A labeled LDE with [3H]-cholesterol and [14C]- cholesteryl ester was incubated for 4 hours with cells, after which LDE uptake and competition tests were evaluated. LDL-r, LRP, CD36 and CD38 were evaluated by using immunocytochemistry methods, cytometric flow and real time PCR. To investigate the effects of LDE chemical composition modifications, cholesteryl oleate (LDE-CO) was replaced with cholesteryl linoleate (LDE-CL) and cholesterol stearate (LDE-CE). These were then tested for stability, cellular uptake, lipoperoxidation and citotoxitity. Results showed that all cells internalized [3H]-cholesterol and [14C]-cholesteryl ester proportionally to incubated LDE-CO concentrations albeit with some saturation differences. LDE-CO lipid uptake had a higher cholesterol uptake than the cholesteryl ester uptake. Furthermore, monocytes (THP-1) had a higher LDE-CO uptake than other cells. LDE-CO uptake decreased (r2 -0.73) in the presence of natural LDL, suggesting that these two particles may be competing for the same receptors. LDE-CO appeared to inhibit LDL protein receptor expression in HUVEC (3.98 times), in monocytes (6.25 times) and in fibroblasts (3.70 times), as well as the gene expression in monocytes and HUVEC. A decrease in LDL-r expression in both H292 and fibroblasts was also observed. LDE-CO increased the protein expression in HUVEC 3.75 times while in monocytes, it was able to decrease gene and protein expression of LRP-1, 3.15 times. In macrophages and H292, there was an increase in genetic expression of LRP-1. LDE-CO increased the CD36 in HUVEC gene and protein expressions 3.1 times, decreased the macrophage protein expression 4.34 times and decreased the H292 gene and protein expression 2.94 times. LDE increased protein expression 2.09 times in CD68 in H292 and increase gene expression in both monocytes and macrophages. Fibroblasts presented the highest survival rate in the presence of LDE-CO of all cells studied. The LDE chemical modification effect studies, presented smaller sized LDE-CO and less lipoperoxidation than LDE-CL and LDE-CE presented no stability modifications in less than 30 days. Cells presented higher lipoperoxidation in the presence of LDE-CL when compared to the presence of LDE-CO and LDE-CE. [3H]-cholesterol was greater than cholesteryl ester for all three LDE types in all the studied cells. LDE-CO presented favorable characteristics in terms of particle size and susceptibility to peroxidation. Cholesterol cell uptake was higher than that of cholesteryl ester for all LDEs of all the studied cells which suggests that that cholesterol may be capable of disassociating itself from LDE and being uptaken by cells through non-specific pathways. The results of this study can help to better understand the mechanisms of uptake by cells, the effects of stability and LDE system adequation for therapeutic and diagnostic applications.

A aterosclerose é uma doença crônico-inflamatória multifatorial com o envolvimento do sistema imunológico, sendo o resultado da interação de diferentes elementos celulares. A lipoproteína de baixa densidade eletronegativa [LDL(-)], capaz de induzir o acúmulo de ésteres de colesterol em macrófagos e a subsequente formação de células espumosas, desempenha um papel-chave na doença. Anticorpos recombinantes têm sido gerados nas últimas décadas, como o scFv (single chain fragment variable), com o intuito de serem utilizados como uma novas alternativas de prevenção para o surgimento da lesão. Diante do papel da LDL(-) na aterosclerose, este projeto avaliou o efeito da imunização passiva de camundongos LDLr-/-- com scFv anti-LDL(-) em solução e scFv anti-LDL(-) conjugado a nanocápsulas, em relação ao desenvolvimento e progressão da aterosclerose. Após obtenção do scFv e sua conjugação à nanocápsulas (NC-scFv), ensaios in vitro determinaram a diminuição da captação de LDL(-) em macrófagos tratados com o scFv 2C7 anti-LDL(-) em solução. No entanto, o tratamento com NC-scFv promoveu o aumento da internalização de LDL(-) em relação ao controle, possivelmente por um mecanismo de endocitose mediada por receptor específico. Estudos in vivo determinaram que camundongos LDLr-/- com idade entre 2 e 3 meses tratados com o scFv em solução apresentaram menor área de lesão aterosclerótica (p<0,05) quando comparados ao controle e que animais com 3 a 4 meses de idade tratados com NC-scFv demonstraram uma tendência à diminuição do mesmo parâmetro. Na análise da expressão de proteínas por imunohistoquímica, ambos os grupos tratados com scFv 2C7 anti-LDL(-) em solução e NC-scFv demostraram redução significativa da expressão dos receptores CD14 e TLR-4 no local da lesão. Esse achado tem grande importância, uma vez que dados da literatura apresentam ambos os receptores como possíveis candidatos ao reconhecimento da LDL(-). Diante dos dados obtidos, o estudo evidenciou a eficácia do scFv 2C7 anti-LDL(-) em solução e da formulação NC-scFv no contexto da aterosclerose, possibilitando a sua utilização como estratégias terapêuticas na intervenção precoce para prevenir o desenvolvimento e a progressão da doença. / Atherosclerosis is a chronic inflammatory multifactorial disease related to the immune system and being the result of interaction of different cellular elements. The electronegative LDL, since the changes undergone by this particle are able to induce the accumulation of cholesterol esters in macrophages and the subsequent formation of foam cells, plays a key role in atherosclerosis. Recombinant antibodies have been generated in recent decades, such as scFv, (single chain fragment variable), and they may be used as a new alternative treatment for atherosclerosis treatment or prevention. Considering the role of LDL(-) in atherosclerosis, this project evaluated the effects of the treatment with anti-LDL(-) scFv 2C7 solution and anti-LDL(-) scFv conjugated to nanocapsules as a passive immunization strategy on atherosclerosis induced in LDL receptor knockout mice. After obtaining the anti-LDL(-) scFv 2C7 solution and its conjugation to nanocapsules (NC-scFv), in vitro tests led to the decrease in LDL(-) uptake in macrophages treated with anti-LDL(-) scFv 2C7. However, the treatment of macrophages with NC-scFv promoted increased internalization of LDL(-) as compared to control, possibly due to a mechanism of specific receptor-mediated endocytosis. In vivo studies have determined that LDLR-/- mice aged 2 and 3 months treated with anti-LDL(-) scFv 2C7 solution showed less atherosclerotic lesion area (p <0.05) compared to control and animals aged 3 to 4 months treated with NC-scFv showed a decreasing tendency of the same parameter. In the analysis of protein expression by immunohistochemistry, both groups treated with anti-LDL(-) scFv 2C7 solution and NC-scFv showed significant reduction of CD14 receptor expression and TLR-4 at the lesion site. This finding is of great importance, since the literature has both receptors as candidates for recognition of the LDL(-). From the data obtained, the study demonstrated the efficacy of treatments anti-LDL(-) scFv 2C7 in solution and NC-scFv in the context of atherosclerosis, enabling their use as therapeutic strategies in the early intervention to prevent the development and progression of the disease.

Introduction There has been much discussion on personalised medicine; however use of genotype in risk prediction for coronary heart disease (CHD) has not resulted in appreciable improvements over non-genetic risk factors. The primary aim was to determine whether candidate single nucleotide polymorphisms (SNPs) identified from genome-wide association studies improved prediction of CHD over conventional risk factors (CRF). The secondary aim was to determine whether the use of apolipoproteins or lipoprotein(a) improved risk prediction of CHD. Methods Analyses used the Edinburgh Heart Disease Prevention Study (EHDPS), with 1592 men aged 30-59 and follow-up after 20 years; and the Edinburgh Artery Study (EAS), with 1592 men and women aged 54-75 and 15 years of follow-up. Candidate SNPs were identified by systematic literature reviews. CHD status was evaluated as severe (myocardial infarction or coronary revascularisation), and any (severe CHD, angina or non-specified ischaemic heart disease). Cox proportional hazards models were used to evaluate addition of candidate SNPs or lipids to models containing CRF. Results A group of genome-wide significant SNPs resulted in a non-significant improvement in C-index for severe CHD (0.038, p=0.082), and a significant improvement in C-index for any CHD (0.042, p=0.016); the associated net reclassification improvements (NRI) were 20.5% and 18.7%, respectively. Regression trees identified SNPs that were predictive of the remaining variance after adjusting for CRF; this resulted in a significant improvement in C-index for any CHD (0.031, p=0.008). The NRI were 11.0% and 9.6% for severe and any CHD, respectively. When compared with HDL cholesterol/total cholesterol, apolipoprotein AI/total cholesterol yielded a NRI of 3.3% for severe CHD. Lipoprotein(a) improved prediction of severe CHD, with a non-significant improvement in C-index (0.020, p=0.087), and NRI of 11.8%. Conclusion The results of this study indicate that a well selected group of candidate SNPs can improve risk prediction for CHD over-and-above CRF. The inclusion of lipoprotein(a), along with CRF, appeared to improve prediction of severe CHD, but not any CHD.

Christopher John Haines. / Thesis (M.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 239-279). / LIST OF TABLES --- p.xviii / LIST OF FIGURES --- p.xxi / LIST OF ABBREVIATIONS --- p.xxii / Chapter CHAPTER1 --- INTRODUCTION --- p.1 / Problems related to the menopause / Research plan / Chapter CHAPTER2 --- OVERVIEW --- p.15 / Introduction / Atherosclerosis and the lipid profile / Coronary artery disease and lipid abnormalities in women / "Exogenous oestrogens, progestogens and coronary artery disease " / Lipoprotein (a) / Chapter CHAPTER3 --- GENERAL METHODOLOGY --- p.134 / Recruitment of cases / Pharmacokinetics / Data collection and analysis of samples / Ethical considerations / Chapter CHAPTER4 --- STUDY I -THE SHORT TERM EFFECTS OF ORAL OESTROGEN --- p.157 / Crossover analysis of effects of oral oestrogen on lipoprotein (a) and other lipoproteins / Relationship between lipoprotein (a) and other lipids and lipoproteins / Chapter CHAPTER5 --- STUDY II -THE SUSTAINED EFFECTS OF ORAL OESTROGEN --- p.186 / Analysis of prolonged effects of oral oestrogen on lipoprotein (a) and other lipids and lipoproteins / Chapter CHAPTER6 --- STUDY III -THE EFFECTS OF COMBINED CYCLICAL HORMONE REPLACEMENT THERAPY --- p.196 / Analysis of effect of combined cyclical hormone replacement therapy on lipoprotein (a) and other lipids and lipoproteins / Comparison between sampling during oestrogen alone and combined phase of treatment / Chapter CHAPTER7 --- STUDY IV -THE EFFECTS OF PERCUTANEOUS OESTROGEN --- p.214 / Analysis of effect of percutaneous on lipoprotein (a) and other lipids and lipoproteins / Chapter CHAPTER8 --- SUMMARY AND CONCLUSIONS --- p.228 / BIBLIOGRAPHY --- p.239

Receptors that transport vitellogenin into oocytes are of vital importance to egg-laying species because they promote oocyte development. In this study, we describe the cloning of the first hymenopteran vitellogenin receptor (VgR) cDNA. Using reverse transcription polymerase chain reaction (RT-PCR) and both 5’- and 3’- rapid amplification of cDNA ends (RACE), cDNA fragments encompassing the entire coding region of a putative VgR from fire ant (= SiVgR) were cloned and sequenced. The complete SiVgR cDNA has a length of 5764 bp encoding a 1782-residue protein with a predicted molecular mass of 201.3 kDa. The deduced amino acid sequence of the SiVgR revealed that it encoded a protein belonging to the low-density lipoprotein receptor superfamily. The number and arrangement of modular domains of SiVgR are the same as those of mosquito and fruit fly VgRs, except there are only four Class A cysteine-rich repeats in the first ligand binding domain of SiVgR compared to five in the mosquito and fruit fly. The deduced amino acid sequence of the SiVgR exhibited 35% and 31% identity to those of the mosquito and fruit fly VgRs, respectively. Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in ovaries of reproductive females − both alates (virgins) and queens (mated) and was more abundant in alates. The developmental profile of transcriptional expression was determined by semiquantitative RT-PCR. It showed that the SiVgR transcript increased 6-fold from 0- to 10-days after mating, then remained constant through 30 days. It also showed that the SiVgR transcripts increased with age in alate virgin females. The transcriptional expression of the SiVgR was up-regulated more than two-fold by methoprene, a juvenile hormone analog, as determined by using an in vitro system. This suggested the SiVgR gene is JH regulated.

Cardiovascular disease is a major cause of mortality and morbidity in patients on chronic haemodialysis (HD). One main contributing factor is renal dyslipidaemia, characterized by an impaired catabolism of triglyceride (TG)-rich lipoproteins with accumulation of atherogenic remnant particles. The enzyme lipoprotein lipase (LPL) is a key molecule in the lipolysis of TG-rich lipoproteins into free fatty acids. The activity of LPL is reduced in HD-patients. This study was performed to elucidate various conditions and factors that may have an impact on LPL-related lipid metabolism. I. The functional pool of LPL is located at the vascular surface. The enzyme is released by heparin and low molecular weight heparins (LMWH) into the circulating blood and extracted and degraded by the liver. Heparin and LMWH are used for anticoagulation during HD to avoid clotting in the extracorporeal devices. This raises a concern that the LPL system may become exhausted by repeated administration of LMWH in patients on HD. In a randomized cross over designed study twenty patients on chronic HD were switched from a primed infusion of heparin to a single bolus of LMWH (tinzaparin).  The LPL activity in blood was higher on HD with LMWH at 40 minutes but lower at 180 minutes compared to HD with heparin. These values did not change during the 6-month study period. With heparin a significant TG reduction was found at 40 minutes and a significantly higher TG value at 180 and 210 minutes than at start. TG was higher during the HD-session with tinzaparin than with heparin. Our data demonstrate that repeated HD with heparin or with LMWH does not exhaust the LPL-system in the long term but does disturb the LPL system and TG metabolism during every HD session. II. In this study HD patients were compared with patients on peritoneal dialysis (PD) in a case control fashion. PD patients showed the same reaction of the LPL system to LMWH as HD patients. This confirmed that both HD and PD patients had the same, reduced, heparin-releasable LPL pool. The main difference was that in PD patients the TG continued to be cleared effectively even at 180 minutes after the bolus of LMWH injection. This may be due to a slower removal of the released LPL by the liver in PD patients.  III. In recent years, citrate (Citrasate) in the dialysate has been used in Sweden as a local anticoagulant for chronic HD. We performed a randomized cross over study that included 23 patients (16 men and 7 women) to investigate if citrate in the dialysate is safe and efficient enough as anticoagulant. The study showed that citrate anticoagulation eliminated the need of heparin or LMWH as anticoagulation for HD in half of the patients. However, individual optimization of doses of anticoagulants used together with citrate have to be made. IV. Recently angiopoietin-like proteins, ANGPTL3 and 4 have emerged as important modulators of lipid metabolism as potent inhibitors of LPL. Twenty-three patients on chronic HD and 23 healthy persons were included as case and controls to investigate the levels of these proteins in plasma of HD-patients and to evaluate if HD may alter these levels. The data showed that plasma levels of ANGPTL3 and 4 were increased in patients with kidney disease compared to controls. This may lead to inactivation of LPL. High flux-HD, but not low flux-HD, reduced the levels of ANGPTL4, while the levels of ANGPTL3 were not significantly influenced.  On HD with local citrate as anticoagulant, no LPL activity was released into plasma during dialysis in contrast to the massive release of LPL with heparin (LMWH). Citrate HD was not associated with a significant drop in plasma TG at 40 minutes, while both HD with citrate and heparin resulted in significantly increased TG levels at 180 minutes compared to the start values. Conclusions:  Citrate as a local anticoagulant during haemodialysis eliminates the need of heparin or LMWH in about half of the HD patients. Citrate does not induce release of LPL from its endothelial binding sites. We have shown that although HD with heparin causes release of the endothelial pool of LPL during each dialysis session, the basal pool is similarly low in PD patients that do not receive heparin. This indicates that the LPL pool is lowered as a consequence of the uraemia, per se. One explanation could be the increased levels of ANGPTL3 and 4. HD with high flux filters can temporarily lower the levels of ANGPTL4. Further studies are, however, needed to understand why LPL activity is low in patients with kidney disease.

Lipoprotein lipase (LPL) is a central enzyme in lipid metabolism. It is a non-covalent, homodimeric and N-glycosylated protein, which is regulated in a tissue-specific manner and is dependent on an activator protein, apolipoprotein CII. Dissociation of active LPL dimers to monomers leads to loss of activity. This was previously found to be an important event in the rapid regulation of LPL in tissues. The mechanisms involved in the processing of LPL to active dimers, as well as in LPL inactivation through monomerization, were unknown. We have investigated the folding properties of the LPL protein, in particular the requirements for LPL to attain its active quaternary structure and to remain in the native conformation. On expression of LPL in insect cells we found that most of the LPL protein was synthesized in an inactive monomeric form. By co-expression of LPL with human molecular haperones, especially with calreticulin (CRT), the activity of LPL increased greatly, both in the cells and in the media. The effect of CRT on LPL activity was not due to increased levels of the LPL protein, but was due to an increased proportion of active dimeric LPL. Co-immunoprecipitation experiments showed direct interaction between LPL and CRT supporting the idea that this ER-based molecular chaperone supports the formation of active LPL dimers. We showed that, bis-ANS, the aromatic hydrophobic probe 1,1.-bis(aniline)-4,4.- bis(naphthalene)-8,8.disulfonate, can be used to obtain specific information about the interaction of LPL with lipid substrates and with apoCII. Bis-Ans was found to be a potent inhibitor of LPL activity, but apoCII prevented the inhibition. Our results suggest that bis-Ans binds to three exposed hydrophobic sites, of which one is at or close to the binding site(s) for apoCII. In studies of the mechanisms responsible for the spontaneous inactivation of LPL, we showed that active LPL is a dynamic dimer in which the subunits rapidly exchange partners. The rapid equilibrium between dimers and monomers exists even under conditions where LPL is relatively stable. This supports the idea that the dimer is in equilibrium with dimerization-competent, possibly active monomers. This dimerization-competent intermediate was also implicated in studies of the inactivation kinetics. The inactive LPL monomer was found to have a stable, defined conformation irrespective of how it was formed. The main differences in conformation between the inactive monomer and the active dimer were located in the middle part of the LPL subunit. Experiments with bis-Ans demonstrated that more hydrophobic regions were exposed in the inactive monomer, indicating a molten globule conformation. We concluded that the middle part of the LPL subunit is most likely engaged in the formation of the active LPL dimer. The dimerization-competent LPL monomer is a hypothetical conformational state, because it has not been possible to isolate it. To study complete refolding of LPL we used fully denatured LPL and were able to demonstrate that the recovery of LPL activity was about 40% when the denaturant was diluted by a buffer containing 20% human serum and 2M NaCl. Further studies identified calcium as the component in serum that was crucial for the reactivation of LPL. The refolding of LPL was shown to involve at least two steps, of which the first one was rapid and resulted in folded, but inactive monomers. The second step, from inactive monomers to active dimers, was slow and calcium-dependent. Also inactive monomers isolated from human tissue were able to recover activity under the influence of calcium. We proposed that calcium-dependent control of LPL dimerization might be involved in the normal post-translational regulation of LPL activity. In conclusion, LPL is a relatively unstable enzyme under physiological conditions due to its noncovalent dimeric structure. The energy barrier for folding to the active dimer is high and requires the presence of calcium ions and molecular chaperones to be overcome. The dimeric arrangement is probably essential to accomplish rapid down-regulation of LPL activity according to metabolic demand, e.g. in adipose tissue on fasting.

Apolipoprotein (apo) A-V is the most recently discovered member of a protein family responsible for the structure and metabolic fate of plasma lipoproteins. While most of the apolipoproteins are well characterized with regard to structure, interactions and function, the role of apoA-V is not well understood. ApoA-V is synthesized only in liver and is present in blood at much lower concentration than the other apolipoproteins. Although apoA-V is firmly established as an important determinant for plasma triacylglycerol (TG) metabolism, the mechanism is unclear. ApoA-V has been suggested to act through 1) an intracellular mechanism affecting lipoprotein assembly and secretion, 2) direct or indirect activation of lipoprotein lipase (LPL), or 3) interaction with endocytotic lipoprotein receptors. Two other novel players involved in the clearance of lipoproteins are angiopoietin-like protein (ANGPTL) 3 and 4. Previous studies have shown that the coiled-coil domain (ccd) of ANGPTL3 and -4 can inactivate lipoprotein lipase (LPL). The functional site of action of LPL is at the capillary endothelium, but the enzyme is synthesized mostly in adipocytes and myocytes and has to be transported by trancytosis to the luminal side of endothelial cells. Both ANGPTLs are present in tissues and in the circulating blood, but it is not known were the inactivation of LPL normally takes place. The aim of this thesis was to investigate the mechanism by which apoA-V exerts its effect on TG metabolism and to investigate in further detail how ANGPTLs act on the LPL system. Binding of apoA-V to receptors involved in lipoprotein metabolism was investigated by surface plasmon resonance technique (SPR). ApoA-V was found to bind to the LDL receptor related protein 1 (LRP1) and to the mosaic type 1 receptor sorLA. Binding could be competed by receptor associated protein (RAP) or by heparin, and was calcium dependent. We concluded that apoA-V binds to the LA-repeats of these receptors. In further experiments apoA-V was shown to increase binding of TG-rich chylomicrons to the receptors. This demonstrated a possible mechanism for the TG-lowering effect of apoA-V in vivo. A putative binding region in apoA-V for heparin and receptors was investigated by site-directed mutagenesis. Two positively charged amino acid residues were changed (Arg210Glu/Lys211Gln), resulting in decreased binding to heparin and to LRP1 and thus the localization of one important functional region in apoA-V. Since the receptor sorLA also contains a Vsp10p domain, another Vsp10p domain family member, sortilin, was investigated. ApoA-V was found to interact also with this receptor. In experiments with human embryonic kidney cells transfected with sorLA or sortilin, apoA-V was found to bind to cell surfaces and to be rapidly internalized while co-localized with the receptors on the way to lysosomes for degradation. Additional apoA-V mutants, identified in patients with severe hypertriglyceridemia, were investigated with regard to effects in vitro on LPL activity and receptor binding. The most severe mutants displayed null binding to LRP1, whereas the effect on LPL activity was retained. These results suggest that lack of receptor interaction mirrors the loss of biological function in a better way than the in vitro effect on LPL activity. We noted that ccd-ANGPTL3 and -4 did not prevent the LPL-mediated uptake of chylomicron-like lipoproteins in primary murine hepatocytes. Therefore LPL activity was measured after pre-incubation with ccd-ANGPTL3 or 4 in the presence or absence of TG-rich lipoproteins. Physiological concentrations of lipoproteins were found to protect LPL from inactivation by ccd-ANGPTLs. Investigation by SPR demonstrated that the ccd-ANGPTLs did not bind to the lipoproteins. Other experiments showed that less than 1% of ANGPTL4 in human serum was bound to TG-rich lipoproteins. This implies that the known binding of LPL to TG-rich lipoproteins stabilizes the enzyme and protects it from inactivation by ANGPTLs. We conclude that the normal levels of ANGPTLs in plasma are too low to affect the LPL-system and that inactivation of the enzyme by ANGPTLs is more likely to occur locally in the extracellular interstitium of tissues where LPL is en route to its endothelial binding sites and where the concentrations of the TG-rich lipoproteins are low.

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.