Apolipoproteins have important roles in the transport of lipids and the regulation of lipoprotein metabolism as cofactors for enzymes and ligands for receptor-binding. Their function and metabolism are closely related to the development of many diseases. This dissertation describes the investigation of the distribution and metabolism of apoE and glycated apoE in diabetes, obesity and hyperlipidaemia in comparison with healthy people. In order to carry out the research, I developed several robust laboratory methods and techniques for the isolation and measurement of apoE and glycated apoE. These included (1) a modified in-house ultracentrifugation for isolation of lipoprotein fractions (2) high sensitivity sandwich enzyme-linked immunosorbent assay (ELISA) for apoE and (3) m-aminophenylboronate affinity chromatography for the separation of glycated and non- glycated apoE.In healthy people the apoE concentration in different lipoprotein fractions is influenced by age, gender and apoE genotype. The effect of atorvastatin on serum apoE concentration in patients with type 2 diabetes with nephropathy was dependent on the dose of atorvastatin and apoE genotype and was strongly correlated with the reduction in triglycerides (TG) in very low density lipoprotein (VLDL).The effect of bariatric surgery on obese patients with and without diabetes demonstrated that after bariatric surgery, VLDL-apoE increased and apoE in low density lipoprotein (LDL), high density lipoprotein (HDL) and d>1.21g/ml fractions decreased; both glycated LDL-apoE and glycated HDL-apoE decreased. Total apoE and glycated apoE concentrations in plasma decreased to levels comparable to those of healthy controls. However, the distribution within the lipoprotein fractions was very different. The effect of niacin/laropiprant (LRPT) on lipoproteins in hyperlipidaemia patients was assessed in a blind crossover trial. Niacin/LRPT slightly decreased VLDL-apoE and LDL-apoE. It had no effect on apoE in HDL. Glycated apoE did not change in hyperlipidaemia. These results show that, compared with healthy people, the apoE distribution in obese and hyperlipidaemia patients is abnormal despite no change in total apoE concentration in some cases. The results also demonstrate that glycated apoE originates preferentially from VLDL. Various mechanisms for these results and relationships with other lipids are discussed. Furthermore, I suggest several potential directions, especially in vitro, for further research on apoE function and metabolism.

Alzheimer's disease (AD) is the most prevalent form of age-associated dementia. Cholesterol dysregulation is linked with AD onset. Besides age, the most important risk factor associated with AD is the inheritance of the epsilon-4 allele of apolipoprotein E, a cholesterol transporter. In addition, while hypercholesterolemia has been shown to be an independent risk factor for AD, the nature of the cholesterol-AD link is still not clear. This gap in our understanding is partly due to a lack of knowledge about cholesterol metabolism in the central nervous system (CNS). The low-density lipoprotein receptor (LDLR) is the main receptor of apoE and a central regulator of serum cholesterol levels. Therefore, we sought to characterize the potential participation of LDLR in AD pathogenesis and/or progression. Previous reports with similar aims came to contradictory conclusions. Such studies assessed potential changes in AD in the absence of LDLR by utilizing the LDLR-/- mouse model and crossing it to AD mouse models. Initially we evaluated LDLR-/- mice as a suitable model to study AD. We found that LDLR-/- mice overexpressed a functional splice-variant of LDLR, LDLRDelta4. Moreover, its protein localized in similar regions as the LDLR did in control mice. Finally, we determined that LDLRDelta4 bound apoE, which underscores the impact of the isoform's function in the CNS. We then focused on characterizing changes to LDLR in AD models. We found that APP overexpression in cells increased LDLR mRNA and protein. APP overexpression and Abeta treatment shifted LDLR localization. An AD mouse model showed increased LDLR in hippocampus. Conversely, LDLR levels were decreased in APP-/- mice. Finally, we found that microtubules were affected in cells overexpressing APP. In conclusion, the data presented argue for the importance of LDLR-mediated regulation of cholesterol during AD progression. Also, LDLR may participate in the initial pathogenic insults leading to amyloid deposition, which make it a potential therapeutic target to treat AD. Finally, we propose that APP/Abeta overexpression disrupts microtubule formation; this alteration affects protein trafficking. One of the proteins affected is LDLR, the repercussions of which may ultimately result in cholesterol dysregulation.

The relationship between hyperlipidemia and sensorineural hearing loss remains obscure. In this study, we elucidate for the first time the cochlear morphological and auditory alterations and their relationships with hyperlipidemia, atherosclerosis, and endothelial dysfunction in apolipoprotein-E knockout (ApoE-KO) mice. Ten-week-old ApoE-KO mice were fed either atherosclerotic diet (1.25% cholesterol) or normal diet. Wild type mice (C57BL/6J) served as normal controls. Fourteen weeks later, marked hyperlipidemia, atherosclerosis, endothelial dysfunction, and hearing impairment, especially in the high frequencies, had developed in ApoE-KO mice as compared with C57BL/6J mice (P < 0.001). A high positive correlation between hearing loss and the extent of atherosclerosis and plasma total cholesterol levels was found. Hearing loss, especially at high frequencies, was detected in all ApoE-KO mice. Hair cell loss mainly at the base turn, thickening of vascular intima, and lumen stenosis of the spiral modiolar artery (SMA) in cochlea were also found; these histological changes were exacerbated by the atherosclerotic diet. Furthermore, endothelial nitric oxide synthase (eNOS) in aortic wall and cochlea was distinctly reduced in ApoE-KO mice. These results demonstrate that hyperlipidemia and atherosclerosis can induce alterations in cochlear morphology and function. The stenosis of SMA, which may cause cochlear ischemia and hypoxia, endothelial dysfunction, and low eNOS activity, may contribute to hearing loss.

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