title: Insulin resistance and the ability of high-density lipoprotein to provide vascular protection
Creators: name: Beazer, Jack David
Publisher: name: University of Glasgow
Year: 2024

description: The worldwide prevalence of obesity and its metabolic consequences continues to rapidly increase. Obesity leads to metabolic derangements in insulin signalling and secretion, glucose control, and plasma lipid distribution, resulting in insulin resistance and dyslipidaemia. Over time, this phenotype can lead to pancreatic beta cell failure and type 2 diabetes mellitus (T2DM). The deleterious metabolic changes associated with obesity are causes of vascular dysfunction and are associated with an increased risk of cardiovascular disease (CVD). Notably, obesity-induced dyslipidaemia reduces plasma high-density lipoprotein cholesterol (HDL-C) concentration. HDL-C is well established as an inverse predictor of CVD risk due to its reverse cholesterol transport function, however, pharmacotherapies aimed at raising HDL-C concentration have thus far failed to reduce CVD risk. Attention has therefore turned towards understanding HDL composition and vascular functions, including anti-inflammatory, antioxidant, vasodilatory and anti-thrombotic actions. HDL composition and function is known to be compromised in obesity and T2DM, however, a thorough investigation of HDL composition and vascular function through the full insulin resistance spectrum has not yet been performed. This thesis sought to measure HDL composition and vascular function in a number of previously performed cross-sectional and longitudinal studies including otherwise healthy participants with varying degrees of insulin resistance. HDL composition was assessed using quantitative microplate assays for HDL total protein and cholesterol content and immunosorbent assays for the predominant HDL protein apolipoprotein AI (apoAI). The entire HDL proteome was measured using nano-liquid chromatography mass spectrometry. HDL size was measured using native gel electrophoresis. HDL vascular protective function was assessed by measuring its antioxidant function through paraoxonase-1 (PON-1) activity and its anti-inflammatory activity was measured by assessing the ability of HDL to reduce inflammatory cytokine-mediated adhesion molecule expression in endothelial cells.

This thesis first described the establishment of an assay of HDL anti-inflammatory function in human dermal microvascular endothelial cells (HMEC-1). Healthy control HDL dosed onto HMEC-1 at a concentration of 300 μg/mL apoAI was able to reproducibly reduce vascular cell adhesion molecule 1 (VCAM-1) expression in the face of maximally inflammatory tumour necrosis factor alpha (TNFα) by 60%, with a coefficient of variation of 14.6%. To ensure that remnant salts from the sodium bromide sequential density ultracentrifugation method for the isolation of HDL was not interfering with the cell assay, a comparison of HDL composition, size and function was performed between this technique and biologically inert iodixanol density gradient ultracentrifugation. This thesis described an altered profile of HDL composition between the two isolation techniques, with iodixanol density gradient isolated HDL fractions containing a higher protein content than sodium bromide sequential density ultracentrifugation (10.14 ± 1.49 mg/mL compared to 3.18 ± 1.10 mg/mL respectively, p &lt; 0.001, mean ± SD) and a reduction in the proteomic detection of key HDL proteins such as apoAI, apolipoprotein AII and PON-1. HDL size could not be determined in the iodixanol isolated HDL fractions due to contaminating plasma proteins. Though vascular anti-inflammatory function was higher in iodixanol isolated HDL (77.5 ± 4.4 % compared to 58.3 ± 4.8%, mean ± SD, p = 0.014) this may have been due to the presence of confounding plasma proteins. Sodium bromide sequential density ultracentrifugation was therefore employed for the remainder of this thesis.

Previous work in this laboratory focussed on HDL composition through healthy gestation and preeclampsia. Pregnancy is characterised by early insulin sensitivity and late insulin resistance, while preeclampsia is associated with increased insulin resistance and inflammation. Despite insulin resistance, in healthy gestation maternal vascular function is improved whereas preeclampsia is characterised by vascular dysfunction. This thesis completed this study of HDL by adding measures of size and anti-inflammatory function. The proportion of large HDL increased at 16 weeks (38.6 ± 5.1 %, mean ± SD), 25 weeks (41.3 ± 5.0 %) and 35 weeks of gestation (40.4 ± 7.8 %) compared to pre-pregnancy (25.4 ± 9.6 %, p = &lt;0.0001). This was unchanged in preeclampsia. Surprisingly, once corrected for HDL protein content, HDL anti-inflammatory function was decreased at 16 weeks of gestation (0.5 ± 0.3 %/μg protein, p &lt;0.001), 25 weeks of gestation (0.6 ± 0.3 %/μg, p &lt; 0.001), 35 weeks of gestation (0.6 ± 0.3 %/μg, p &lt; 0.001) and 13-weeks post-partum (0.8 ± 0.5 %/μg, p = 0.015) compared to pre-pregnancy (1.9 ± 1.1 %/μg). HDL anti-inflammatory function was not different in preeclampsia compared to healthy gestation. An assessment of HDL composition and function in pregnancies complicated by gestational diabetes mellitus (GDM) followed. HDL did not differ between healthy third trimester pregnancy and GDM pregnancy by any metric, suggesting that the effect of metabolic changes in gestation override those associated with insulin resistant complications of pregnancy.

A cross sectional comparison of healthy middle-aged men to those with impaired glucose regulation (IGR) and those who performed regular endurance exercise revealed a number of changes in HDL composition. ApoAI was highest in the endurance athletes (1.65 ± 0.62 mg/mL) and lowest in IGR men (0.63 ± 0.18 mg/mL) compared to controls (1.21 ± 0.34 mg/mL), while the acute phase reactant serum amyloid A was highest in IGR men (1.77 [1.15, 2.98] μg/mg HDL protein, median [IQR]) compared to both controls and endurance athletes (0.75 [0.37, 2.40] and 0.75 [0.30, 2.40] μg/mg HDL protein respectively, both p = 0.042). Endurance athletes had the highest proportion of HDL 2b, the largest of the HDL subfractions, but HDL size distribution was unchanged between healthy and IGR men. Proteomic analysis uncovered distinct changes in HDL protein composition between the three groups. HDL from IGR men was enriched in inflammation and coagulation related proteins, while endurance athlete HDL was enriched in apolipoprotein AII, apolipoprotein D and the thyroid hormone transporter transthyretin. This suggested that HDL composition reflects underlying systemic physiology. In terms of HDL function, both antioxidant and anti-inflammatory functions were impaired in IGR men but not improved in endurance athletes.

Finally, a comparison of the response to weight gain and subsequent weight loss was performed between healthy young European and South Asian men. South Asians are diagnosed with T2DM on average a decade before their European counterparts and at a lower body mass index, suggesting an ethnic difference in metabolic physiology. Weight gain and weight loss had no major impact on HDL composition or function in Europeans. Compared to Europeans, South Asian HDL had a higher abundance of inflammatory and coagulation related proteins irrespective of weight gain, while a number of lipid handling proteins such as apolipoprotein CIII and apolipoprotein F were increased in South Asian HDL after weight gain. Both HDL antioxidant and anti-inflammatory functions were not impaired in South Asians despite the changes in HDL composition. This suggested that HDL composition reflects altered lipid handling in South Asians.

Taken together, this thesis suggested that HDL composition reflects the systemic environment in which it resides through its scavenger activities, which can sometimes affect its vascular function. It appeared that though HDL function could be reduced, as was the case in IGR men and late pregnancy, improvement in insulin resistance through weight loss or endurance exercise did not improve HDL vascular protective function which suggested a ceiling of HDL function. Given the failure of HDL raising therapies, this thesis suggested that treatment of the underlying pathophysiology in obesity and its complications is likely to improve HDL function.