Lifestyle factors, adiposity and cardiometabolic risk factors in South Asian women living in Scotland

South Asians migrating to the Western world have a 3 to 5-fold higher risk of developing type 2 diabetes and double the risk of cardiovascular disease (CVD) than the background population of White European descent, without exhibiting a proportional higher prevalence of conventional cardiometabolic risk factors. Notably, women of South Asian descent are more likely to be diagnosed with type 2 diabetes as they grow older compared with South Asian men and, in addition, they have lost the cardio-protective effects of being females. Despite South Asian women in Western countries being a high risk group for developing future type 2 diabetes and CVD, they have been largely overlooked. The aims of this thesis were to compare lifestyle factors, body composition and cardiometabolic risk factors in healthy South Asian and European women who reside in Scotland, to examine whether ethnicity modifies the associations between modifiable environmental factors and cardiometabolic risks and to assess whether vascular reactivity is altered by ethnicity or other conventional and novel CVD risks. I conducted a cross-sectional study and recruited 92 women of South Asian and 87 women of White European descent without diagnosed diabetes or CVD. Women on hormone replacement therapy or hormonal contraceptives were excluded too. Age and body mass index (BMI) did not differ between the two ethnic groups. Physical activity was assessed and with self-reported questionnaires and objectively with the use of accelerometers. Cardiorespiratory fitness was quantified with the predicted maximal oxygen uptake (VO2 max) during a submaximal test (Chester step test). Body composition was assessed with skinfolds measured at seven body sites, five body circumferences, measurement of abdominal subcutaneous (SAT) and visceral adipose tissue (VAT) with the use of magnetic resonance imaging (MRI) and liver fat with the use MR spectroscopy. Dietary density was assessed with food frequency questionnaires. Vascular response was assessed by measuring the response to acetylcholine and sodium nitroprusside with the use of Laser Doppler Imaging with Iontophoresis (LDI-ION) and the response to shear stress with the use of Peripheral Arterial Tonometry (EndoPAT). The South Asian women exhibited a metabolic profile consistent with the insulin resistant phenotype, characterised by greater levels of fasting insulin, lower levels of high density lipoprotein (HDL) and higher levels of triglycerides (TG) compared with their European counterparts. In addition, the South Asians had greater levels of glycated haemoglobin (HbA1c) for any given level of fasting glucose. The South Asian women engaged less time weekly with moderate to vigorous physical activity (MVPA) and had lower levels of cardiorespiratory fitness for any given level of physical activity than the women of White descent. In addition, they accumulated more fat centrally for any given BMI. Notably, the South Asians had equivalent SAT with the European women but greater VAT and hepatic fat for any given BMI. Dietary density did not differ among the groups. Increasing central adiposity had the largest effect on insulin resistance in both ethic groups compared with physical inactivity or decreased cardiorespiratory fitness. Interestingly, ethnicity modified the association between central adiposity and insulin resistance index with a similar increase in central adiposity having a substantially larger effect on insulin resistance index in the South Asian women than in the Europeans. I subsequently examined whether ethnic specific thresholds are required for lifestyle modifications and demonstrated that South Asian women need to engage with MVPA for around 195 min.week-1 in order to equate their cardiometabolic risk with that of the Europeans exercising 150 min.week-1. In addition, lower thresholds of abdominal adiposity and BMI should apply for the South Asians compared with the conventional thresholds. Although the South Asians displayed an adverse metabolic profile, vascular reactivity measured with both methods did not differ among the two groups. An additional finding was that menopausal women with hot flushing of both ethnic groups showed a paradoxical vascular profile with enhanced skin perfusion (measured with LDI-ION) but decreased reactive hyperaemia index (measured with EndoPAT) compared with asymptomatic menopausal women. The latter association was independent of conventional CVD risk factors. To conclude, South Asian women without overt disease who live in Scotland display an adverse metabolic profile with steeper associations between lifestyle risk factors and adverse cardiometabolic outcomes compared with their White counterparts. Further work in exploring ethnic specific thresholds in lifestyle interventions or in disease diagnosis is warranted.

MicroRNA modulation of aldosterone production in the adrenal gland

Hypertension is the major risk factor for coronary disease worldwide. Primary hypertension is idiopathic in origin but is thought to arise from multiple risk factors including genetic, lifestyle and environmental influences. Secondary hypertension has a more definite aetiology; its major single cause is primary aldosteronism (PA), the greatest proportion of which is caused by aldosteroneproducing adenoma (APA), where aldosterone is synthesized at high levels by an adenoma of the adrenal gland. There is strong evidence to show that high aldosterone levels cause adverse effects on cardiovascular, cerebrovascular, renal and other systems. Extensive studies have been conducted to analyse the role that regulation of CYP11B2, the gene encoding the aldosterone synthase enzyme plays in determining aldosterone production and the development of hypertension. One significant regulatory factor that has only recently emerged is microRNA (miRNA). miRNAs are small non-coding RNAs, synthesized by a series of enzymatic processes, that negatively regulate gene expression at the posttranscriptional level. Detection and manipulation of miRNA is now known to be a viable method in the treatment, prevention and prognosis of certain diseases. The aim of the present study was to identify miRNAs likely to have a role in the regulation of corticosteroid biosynthesis. To achieve this, the miRNA profile of APA and normal human adrenal tissue was compared, as was the H295R adrenocortical cell line model of adrenocortical function, under both basal conditions and following stimulation of aldosterone production. Key differentially-expressed miRNAs were then identified and bioinformatic tools used to identify likely mRNA targets and pathways for these miRNAs, several of which were investigated and validated using in vitro methods. The background to this study is set out in Chapter 1 of this thesis, followed by a description of the major technical methods employed in Chapter 2. Chapter 3 presents the first of the study results, analysing differences in miRNA profile between APA and normal human adrenal tissue. Microarray was implemented to detect the expression of miRNAs in these two tissue types and several miRNAs were found to vary significantly and consistently between them. Furthermore, members of several miRNA clusters exhibited similar changes in expression pattern between the two tissues e.g. members of cluster miR-29b-1 (miR-29a-3p and miR-29b-3p) and of cluster miR-29b-2 (miR-29b-3p and miR-29c- 3p) are downregulated in APA, while members of cluster let-7a-1 (let-7a-5p and let-7d-5p), cluster let-7a-3 (let-7a-5p and let-7b-5p) and cluster miR-134 (miR- 134 and miR-382) are upregulated. Further bioinformatic analysis explored the possible biological function of these miRNAs using Ingenuity® Systems Pathway Analysis software. This led to the identification of validated mRNAs already known to be targeted by these miRNAs, as well as the prediction of other mRNAs that are likely targets and which are involved in processes relevant to APA pathology including cholesterol synthesis (HMGCR) and corticosteroidogenesis (CYP11B2). It was therefore hypothesised that increases in miR-125a-5p or miR- 335-5p would reduce HMGCR and CYP11B2 expression. Chapter 4 describes the characterisation of H295R cells of different strains and sources (H295R Strain 1, 2, 3 and HAC 15). Expression of CYP11B2 was assessed following application of 3 different stimulants: Angio II, dbcAMP and KCl. The most responsive strain to stimulation was Strain 1 at lower passage numbers. Furthermore, H295R proliferation increased following Angio II stimulation. In Chapter 5, the hypothesis that increases in miR-125a-5p or miR-335-5p reduces HMGCR and CYP11B2 expression was tested using realtime quantitative RT-PCR and transfection of miRNA mimics and inhibitors into the H295R cell line model of adrenocortical function. In this way, miR-125a-5p and miR-335-5p were shown to downregulate CYP11B2 and HMGCR expression, thereby validating certain of the bioinformatic predictions generated in Chapter 3. The study of miRNA profile in the H295R cell lines was conducted in Chapter 6, analysing how it changes under conditions that increase aldosterone secretion, including stimulation Angiotensin II, potassium chloride or dibutyryl cAMP (as a substitute for adrenocorticotropic hormone). miRNA profiling identified 7 miRNAs that are consistently downregulated by all three stimuli relative to basal cells: miR-106a-5p, miR-154-3p, miR-17-5p, miR-196b-5p, miR-19a-3p, miR-20b- 5p and miR-766-3p. These miRNAs include those derived from cluster miR-106a- 5p/miR-20b-5p and cluster miR-17-5p/miR-19a-3p, each producing a single polycistronic transcript. IPA bioinformatic analysis was again applied to identify experimentally validated and predicted mRNA targets of these miRNAs and the key biological pathways likely to be affected. This predicted several interactions between miRNAs derived from cluster miR-17-5p/miR-19a-3p and important mRNAs involved in cholesterol biosynthesis: LDLR and ABCA1. These predictions were investigated by in vitro experiment. miR-17-5p/miR-106a-p and miR-20b-5p were found to be consistently downregulated by stimulation of aldosterone biosynthesis. Moreover, miR-766-3p was upregulation throughout. Furthermore, I was able to validate the downregulation of LDLR by miR-17 transfection, as predicted by IPA. In summary, this study identified key miRNAs that are differentially-expressed in vivo in cases of APA or in vitro following stimulation of aldosterone biosynthesis. The many possible biological actions these miRNAs could have were filtered by bioinformatic analysis and selected interactions validated in vitro. While direct actions of these miRNAs on steroidogenic enzymes were identified, cholesterol handling also emerged as an important target and may represent a useful point of intervention in future therapies designed to modulate aldosterone biosynthesis and reduce its harmful effects.