Dyslipidemia can be a comorbidity of both insulin-resistance and atherosclerosis. Hypertriglyceridemia is common in hyperglycemia and is associated with hypoalphalipoproteinemia (low HDL) and with altered nucleotide or purinergic signaling. We therefore hypothesized that extracellular nucleotides may affect hepatic lipoprotein metabolism. Our studies confirm this view and show that nucleotides regulate cellular proteolytic pathways in liver cells and thereby control lipoprotein secretion and their metabolism by hepatic lipase (HL). Treatment of liver cells with the nucleotide, adenosine diphosphate (ADP), stimulates VLDL-apoB100 and apoE secretion, but blocks HDL-apoA-I and HL secretion. ADP functions like a proteasomal inhibitor to block proteasomal degradation and stimulate apoB100 secretion. Blocking the proteosome is known to activate autophagic pathways. The nucleotide consequently stimulates autophagic degradation in liver cells and increases cellular levels of the autophagic proteins, LC3 and p62. Confocal studies show that ADP increases cellular LC3 levels and promotes co-localization of LC3 and apoA-I in an autophagosomal degradation compartment. ADP acts through the G-protein coupled receptor, P2Y13, to stimulate autophagy and block both HDL and HL secretion. Overexpression of P2Y13 increases cellular LC3 levels and blocks the induction of both HDL and HL secretion, while P2Y13 siRNA reduce LC3 protein levels and cause up to a ten-fold stimulation in HDL and HL secretion. P2Y13 gene expression regulates autophagy through the insulin receptor (IR-β). A reduction in P2Y13 expression increases the phosphorylation of IR-β and protein kinase B (Akt) >3-fold, while increasing P2Y13 expression inhibits the activation of IR-β and Akt. Experiments with epitope-labeled apoA-I and HL show that activation of purinergic pathways has no effect on the internalization and degradation of extracellular apoA-I and HL, which confirms the view that nucleotides primarily impact intracellular protein transport and degradation. In conclusion, elevated blood glucose levels may promote dyslipidemia by stimulating purinergic signaling through P2Y13 and IR-β and perturbing the intracellular degradation and secretion of both HDL and VLDL.

[Truncated abstract] The low density lipoprotein receptor-related protein (LRP), a member of the low-density lipoprotein (LDL) receptor gene family is involved in numerous biological processes including lipoprotein metabolism. This thesis concerns investigations into some aspects of LRP metabolism/regulation and possible roles in coronary artery disease (CAD). Specific aims were: to investigate the association between polymorphisms in the LRP gene and in its associated protein, the lipoprotein receptor-associated protein (RAP), with the risk of CAD; to extensively examine the influence of the LRP exon 22 C200T polymorphism on lipid metabolism; to develop and characterise assays for the mRNA expression of LRP and 2 other genes relevant to lipid metabolism, the LDL receptor (LDLR), and HMG CoA reductase (HMGCR); and finally, to apply the latter techniques to studies on the influence of genetic variation in LRP, and dietary and drug interventions, on LRP, LDLR and HMGCR mRNA expression in nucleated blood cells from healthy human subjects. Six hundred CAD subjects and 700 similarly aged controls were genotyped for 8 LRP gene polymorphisms as well as for the RAP V311M polymorphism. ... In the final phase of my studies, I examined the influence of 4 weeks therapy with a cholesterol lowering drug, an HMGCR inhibitor, atorvastatin (20mg daily), on the mRNA expression of LDLR, LRP and HMGCR in human nucleated blood cells. Twelve normal Caucasian male subjects aged 49 ? 5 (SD) years were studied. Plasma total cholesterol and LDL-C decreased by averages of 29 % and 41 % after the 4 week period. This was accompanied by an elevation in LDLR mRNA expression by approximately 30 35 %. In contrast, there was no significant effect on LRP and HMGCR mRNA expression. In conclusion, the original findings in this thesis included: demonstration of a strong influence of the LRP exon 22 C200T polymorphism on coronary artery disease and LDLR expression, but without a clear effect on fasting or postprandial lipid levels; data on the biological variation in LDLR and LRP gene expression in nucleated blood cells from normal subjects; the influence of an oral fat load on the expression viii of these genes, finding that LDLR was significantly depressed; and finally, the observation that statin therapy upregulated LDLR in nucleated blood cells.

Dyslipidemia can be a comorbidity of both insulin-resistance and atherosclerosis. Hypertriglyceridemia is common in hyperglycemia and is associated with hypoalphalipoproteinemia (low HDL) and with altered nucleotide or purinergic signaling. We therefore hypothesized that extracellular nucleotides may affect hepatic lipoprotein metabolism. Our studies confirm this view and show that nucleotides regulate cellular proteolytic pathways in liver cells and thereby control lipoprotein secretion and their metabolism by hepatic lipase (HL). Treatment of liver cells with the nucleotide, adenosine diphosphate (ADP), stimulates VLDL-apoB100 and apoE secretion, but blocks HDL-apoA-I and HL secretion. ADP functions like a proteasomal inhibitor to block proteasomal degradation and stimulate apoB100 secretion. Blocking the proteosome is known to activate autophagic pathways. The nucleotide consequently stimulates autophagic degradation in liver cells and increases cellular levels of the autophagic proteins, LC3 and p62. Confocal studies show that ADP increases cellular LC3 levels and promotes co-localization of LC3 and apoA-I in an autophagosomal degradation compartment. ADP acts through the G-protein coupled receptor, P2Y13, to stimulate autophagy and block both HDL and HL secretion. Overexpression of P2Y13 increases cellular LC3 levels and blocks the induction of both HDL and HL secretion, while P2Y13 siRNA reduce LC3 protein levels and cause up to a ten-fold stimulation in HDL and HL secretion. P2Y13 gene expression regulates autophagy through the insulin receptor (IR-β). A reduction in P2Y13 expression increases the phosphorylation of IR-β and protein kinase B (Akt) >3-fold, while increasing P2Y13 expression inhibits the activation of IR-β and Akt. Experiments with epitope-labeled apoA-I and HL show that activation of purinergic pathways has no effect on the internalization and degradation of extracellular apoA-I and HL, which confirms the view that nucleotides primarily impact intracellular protein transport and degradation. In conclusion, elevated blood glucose levels may promote dyslipidemia by stimulating purinergic signaling through P2Y13 and IR-β and perturbing the intracellular degradation and secretion of both HDL and VLDL.

Wichtige Marker in der klinischen Routine für die Risikoabschätzung von kardiovaskulären Erkrankungen (CVD) sind Blutcholesterinwerte auf Basis von Lipoproteinklassen wie ''schlechtes'' LDL oder ''gutes'' HDL. Dies vernachlässigt, dass jede Lipoproteinklasse eine nicht-homogene Population von Lipoproteinpartikeln unterschiedlicher Zusammensetzung aus Lipiden und Proteinen bildet. Studien zeigen zudem, dass solche Sub-populationen von Lipoproteinen im Stoffwechsel als auch im Beitrag zu CVD unterschiedlich sind. Mehrwert und routinemäßiger Einsatz einer detaillierteren Auftrennung von Lipoproteinen sind jedoch umstritten, da die experimentelle Fraktionierung und Analyse aufwendig, zeit- und kostenintensiv sind. Die vorliegende Arbeit ''Computational Lipidology'' präsentiert einen neuartigen Modellierungsansatz für die Berechnung von Lipoproteinverteilungen (Lipoproteinprofil) im Blutplasma, wobei erstmals individuelle Lipoproteinpartikel anstelle von Lipoproteinklassen betrachtet werden. Das Modell berücksichtigt elementare Bestandteile (Lipide, Proteine) und Prozesse des Stoffwechsel von Lipoproteinen. Stochastische wie deterministische Simulationen errechnen auf Basis aller Lipoproteinpartikel im System deren Dichteverteilung. Die Modellberechnungen reproduzieren erfolgreich klinisch gemessene Lipoproteinprofile von gesunden Patienten und zeigen Hauptmerkmale von pathologischen Situationen, die durch Störung eines der zugrundeliegenden molekularen Prozesse verursacht werden. Hochaufgelöste Lipoproteinprofile zeigen die Verteilung von sogenannten ''high-resolution density sub-fractions'' (hrDS) innerhalb von Hauptlipoproteinklassen. Die Ergebnisse stimmen mit klinischen Beobachtungen sehr gut überein, was die Arbeit als einen signifikanten Schritt in Richtung Analyse von individuellen Unterschieden, patienten-orientierte Diagnose von Fettstoffwechselstörungen und Identifikation neuer Sub-populationen von potentiell klinischer Relevanz qualifiziert. / Monitoring the major lipoprotein classes, particularly low-density lipoproteins (''bad'' LDL) and high-density lipoproteins (''good'' HDL) for characterizing risk of cardiovascular disease (CVD) is well-accepted and routine in clinical practice. However, it is only one-half of the truth as lipoprotein classes comprise non-homogeneous populations of lipoprotein particles varying significantly in their composition of lipids and apolipoproteins. Various studies have shown differing metabolic behavior and contribution to CVD of individual lipoprotein sub-populations. Nevertheless, the superiority of more detailed lipoprotein fractionation is still a matter of debate because experimental separation and analysis is an elaborate, time-consuming and expensive venture and not yet worthwhile for routine measurements. The present work ''Computational Lipidology'' aims at establishing a novel modeling approach to calculate the distribution of lipoproteins (lipoprotein profile) in blood plasma being the first that settles on individual lipoprotein complexes instead of common lipoprotein classes. Essential lipoprotein constituents and processes involved in the lipoprotein metabolism are taken into account. Stochastic as well as deterministic simulations yield the distribution of lipoproteins over density based on the set of individual lipoprotein complexes in the system. The model calculations successfully reproduce lipoprotein profiles measured in healthy subjects and show main characteristics of pathological situations elicited by disorder in one of the underlying molecular processes. Moreover, the model reveals the distribution of high-resolution lipoprotein sub-fractions (hrDS) within major density classes. The results show satisfactory agreement with clinical observations which qualifies the work as a significant step towards analyzing inter-individual variability, patient-oriented diagnosis of lipid disorders and identifying new sub-fractions of potential clinical relevance.

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