Insulin mediated upregulation of the renin angiotensin system in human subcutaneous adipocytes is reduced by Rosiglitazone

Background: Obesity associated hypertension is likely to be due to multiple mechanisms. Identification of the renin-angiotensin system (RAS) within adipose tissue does, however, suggest a potential causal role for it in obesity-associated hypertension. Obese patients are often hyperinsulinaemic, but mechanisms underlying insulin upregulation of the RAS in adipose tissue are unclear. TNFα, an inducer of angiotensinogen in hepatocytes, is elevated in hyperinsulinaemic, obese individuals, and may provide a link in mediating insulin upregulation of the RAS in adipose tissue. Further, thiazolidinediones lower blood pressure in vivo and downregulation of the RAS in adipose tissue may contribute to this effect. We therefore examined the effect of rosiglitazone (RSG), on the insulin mediated upregulation of the RAS. Methods and Results: Sera were obtained from the arterial circulation and from venous blood draining subcutaneous abdominal adipose tissue. Isolated human abdominal subcutaneous adipocytes (n=12) were treated with insulin (1-1000nM) and insulin in combination with RSG (10nM), and RSG (10nM) alone to determine angiotensinogen expression, angiotensin II, bradykinin and TNFα secretion. Subcutaneous adipocytes were also treated with TNFα (10-100ng/mL) to examine the direct effect on angiotensinogen expression and angiotensin II secretion. The findings showed that the arterio-venous difference in angiotensin II levels was significant (↑23%; p<0.001). Insulin increased TNFα secretion in a concentration-dependent manner (p<0.01) whilst RSG (10nM) significantly reduced the insulin mediated rise in TNFα (p<0.001), as well as AGT and angiotensin II. TNFα also increased angiotensinogen and angiotensin II in isolated adipocytes. Conclusions: Our in vivo data suggest that human subcutaneous adipose tissue is a significant source of angiotensin II. This study also demonstrates a potential TNFα mediated mechanism through which insulin may stimulate the RAS and may contribute to explain obesity associated hypertension. RSG downregulates the RAS in subcutaneous adipose tissue and this effect may contribute to the long-term effect of RSG on blood pressure

Human epicardial adipose tissue expresses a pathogenic profile of adipocytokines in patients with cardiovascular disease

Introduction: Inflammation contributes to cardiovascular disease and is exacerbated with increased adiposity, particularly omental adiposity; however, the role of epicardial fat is poorly understood. Methods: For these studies the expression of inflammatory markers was assessed in epicardial fat biopsies from coronary artery bypass grafting (CABG) patients using quantitative RT-PCR. Further, the effects of chronic medications, including statins, as well as peri-operative glucose, insulin and potassium infusion, on gene expression were also assessed. Circulating resistin, CRP, adiponectin and leptin levels were determined to assess inflammation. Results: The expression of adiponectin, resistin and other adipocytokine mRNAs were comparable to that in omental fat. Epicardial CD45 expression was significantly higher than control depots (p < 0.01) indicating significant infiltration of macrophages. Statin treated patients showed significantly lower epicardial expression of IL-6 mRNA, in comparison with the control abdominal depots (p < 0.001). The serum profile of CABG patients showed significantly higher levels of both CRP (control: 1.28 ± 1.57 μg/mL vs CABG: 9.11 ± 15.7 μg/mL; p < 0.001) and resistin (control: 10.53 ± 0.81 ng/mL vs CABG: 16.8 ± 1.69 ng/mL; p < 0.01) and significantly lower levels of adiponectin (control: 29.1 ± 14.8 μg/mL vs CABG: 11.9 ± 6.0 μg/mL; p < 0.05) when compared to BMI matched controls. Conclusion: Epicardial and omental fat exhibit a broadly comparable pathogenic mRNA profile, this may arise in part from macrophage infiltration into the epicardial fat. This study highlights that chronic inflammation occurs locally as well as systemically potentially contributing further to the pathogenesis of coronary artery disease

Changes in endotoxin levels in T2DM subjects on anti-diabetic therapies

Introduction Chronic low-grade inflammation is a significant factor in the development of obesity associated diabetes. This is supported by recent studies suggesting endotoxin, derived from gut flora, may be key to the development of inflammation by stimulating the secretion of an adverse cytokine profile from adipose tissue. Aims The study investigated the relationship between endotoxin and various metabolic parameters of diabetic patients to determine if anti-diabetic therapies exerted a significant effect on endotoxin levels and adipocytokine profiles. Methods Fasting blood samples were collected from consenting Saudi Arabian patients (BMI: 30.2 ± (SD)5.6 kg/m2, n = 413), consisting of non-diabetics (ND: n = 67) and T2DM subjects (n = 346). The diabetics were divided into 5 subgroups based on their 1 year treatment regimes: diet-controlled (n = 36), metformin (n = 141), rosiglitazone (RSG: n = 22), a combined fixed dose of metformin/rosiglitazone (met/RSG n = 100) and insulin (n = 47). Lipid profiles, fasting plasma glucose, insulin, adiponectin, resistin, TNF-α, leptin, C-reactive protein (CRP) and endotoxin concentrations were determined. Results Regression analyses revealed significant correlations between endotoxin levels and triglycerides (R2 = 0.42; p < 0.0001); total cholesterol (R2 = 0.10; p < 0.001), glucose (R2 = 0.076; p < 0.001) and insulin (R2 = 0.032; p < 0.001) in T2DM subjects. Endotoxin showed a strong inverse correlation with HDL-cholesterol (R2 = 0.055; p < 0.001). Further, endotoxin levels were elevated in all of the treated diabetic subgroups compared with ND, with the RSG treated diabetics showing significantly lower endotoxin levels than all of the other treatment groups (ND: 4.2 ± 1.7 EU/ml, RSG: 5.6 ± 2.2 EU/ml). Both the met/RSG and RSG treated groups had significantly higher adiponectin levels than all the other groups, with the RSG group expressing the highest levels overall. Conclusion We conclude that sub-clinical inflammation in T2DM may, in part, be mediated by circulating endotoxin. Furthermore, that whilst the endotoxin and adipocytokine profiles of diabetic patients treated with different therapies were comparable, the RSG group demonstrated significant differences in both adiponectin and endotoxin levels. We confirm an association between endotoxin and serum insulin and triglycerides and an inverse relationship with HDL. Lower endotoxin and higher adiponectin in the groups treated with RSG may be related and indicate another mechanism for the effect of RSG on insulin sensitivity

The in vitro effects of resistin on the innate immune signaling pathway in isolated human subcutaneous adipocytes

Context: Obesity-associated inflammation is a contributory factor in the pathogenesis of type 2 diabetes mellitus (T2DM); the mechanisms underlying the progression to T2DM are unclear. The adipokine resistin has demonstrated pro-inflammatory properties in relation to obesity and T2DM. Objective: To characterize resistin expression in human obesity and address the role of resistin in the innate immune pathway. Furthermore, examine the influence of lipopolysaccharide, recombinant human resistin (rhResistin), insulin and rosiglitazone in human adipocytes. Finally, analyze the effect of rhResistin on the expression of components of the NF-κB pathway and insulin signaling cascade. Methods: Abdominal subcutaneous adipose tissue was obtained from patients undergoing elective liposuction surgery (n = 35, aged: 36-49 yr; BMI: 26.5 ± 5.9 kg/m2). Isolated adipocytes were cultured with rhResistin (10-50 ng/ml). The level of cytokine secretion from isolated adipocytes was examined by ELISA. The effect of rhResistin on protein expression of components of the innate immune pathway was examined by Western blot. Results: In-vitro studies demonstrated that antigenic stimuli increase resistin secretion (P < 0.001) from isolated adipocytes. Pro-inflammatory cytokine levels were increased in response to rhResistin (P < 0.001); this was attenuated by rosiglitazone (P < 0.01). When examining components of the innate immune pathway, rhResistin stimulated Toll-like receptor-2 protein expression. Similarly, mediators of the insulin signaling pathway, phosphospecific JNK1 and JNK2, were upregulated in response to rhResistin. Conclusion: Resistin may participate in more than one mechanism to influence pro-inflammatory cytokine release from human adipocytes; potentially via the integration of NF-κB and JNK signaling pathways

Quantifying single nucleotide variant detection sensitivity in exome sequencing

BACKGROUND: The targeted capture and sequencing of genomic regions has rapidly demonstrated its utility in genetic studies. Inherent in this technology is considerable heterogeneity of target coverage and this is expected to systematically impact our sensitivity to detect genuine polymorphisms. To fully interpret the polymorphisms identified in a genetic study it is often essential to both detect polymorphisms and to understand where and with what probability real polymorphisms may have been missed. RESULTS: Using down-sampling of 30 deeply sequenced exomes and a set of gold-standard single nucleotide variant (SNV) genotype calls for each sample, we developed an empirical model relating the read depth at a polymorphic site to the probability of calling the correct genotype at that site. We find that measured sensitivity in SNV detection is substantially worse than that predicted from the naive expectation of sampling from a binomial. This calibrated model allows us to produce single nucleotide resolution SNV sensitivity estimates which can be merged to give summary sensitivity measures for any arbitrary partition of the target sequences (nucleotide, exon, gene, pathway, exome). These metrics are directly comparable between platforms and can be combined between samples to give “power estimates” for an entire study. We estimate a local read depth of 13X is required to detect the alleles and genotype of a heterozygous SNV 95% of the time, but only 3X for a homozygous SNV. At a mean on-target read depth of 20X, commonly used for rare disease exome sequencing studies, we predict 5–15% of heterozygous and 1–4% of homozygous SNVs in the targeted regions will be missed. CONCLUSIONS: Non-reference alleles in the heterozygote state have a high chance of being missed when commonly applied read coverage thresholds are used despite the widely held assumption that there is good polymorphism detection at these coverage levels. Such alleles are likely to be of functional importance in population based studies of rare diseases, somatic mutations in cancer and explaining the “missing heritability” of quantitative traits

Increased circulating ANG II and TNF-α represents important risk factors in obese Saudi adults with hypertension irrespective of diabetic status and BMI

Central adiposity is a significant determinant of obesity-related hypertension risk, which may arise due to the pathogenic inflammatory nature of the abdominal fat depot. However, the influence of pro-inflammatory adipokines on blood pressure in the obese hypertensive phenotype has not been well established in Saudi subjects. As such, our study investigated whether inflammatory factors may represent useful biomarkers to delineate hypertension risk in a Saudi cohort with and without hypertension and/or diabetes mellitus type 2 (DMT2). Subjects were subdivided into four groups: healthy lean controls (age: 47.9±5.1 yr; BMI: 22.9±2.1 Kg/m2), non-hypertensive obese (age: 46.1±5.0 yr; BMI: 33.7±4.2 Kg/m2), hypertensive obese (age: 48.6±6.1 yr; BMI: 36.5±7.7 Kg/m2) and hypertensive obese with DMT2 (age: 50.8±6.0 yr; BMI: 35.3±6.7 Kg/m2). Anthropometric data were collected from all subjects and fasting blood samples were utilized for biochemical analysis. Serum angiotensin II (ANG II) levels were elevated in hypertensive obese (p<0.05) and hypertensive obese with DMT2 (p<0.001) compared with normotensive controls. Systolic blood pressure was positively associated with BMI (p<0.001), glucose (p<0.001), insulin (p<0.05), HOMA-IR (p<0.001), leptin (p<0.01), TNF-α (p<0.001) and ANG II (p<0.05). Associations between ANG II and TNF-α with systolic blood pressure remained significant after controlling for BMI. Additionally CRP (p<0.05), leptin (p<0.001) and leptin/adiponectin ratio (p<0.001) were also significantly associated with the hypertension phenotype. In conclusion our data suggests that circulating pro-inflammatory adipokines, particularly ANG II and, TNF-α, represent important factors associated with a hypertension phenotype and may directly contribute to predicting and exacerbating hypertension risk

A multi-modal MRI study of the central response to inflammation in rheumatoid arthritis

Special thanks to the patient community who participated in this research effort. Thanks to Mariella D’Allesandro for efforts towards recruitment.Peer reviewedPublisher PD

Access to justice during the COVID-19 pandemic in the UK

The COVID-19 pandemic has presented many challenges for our legal system. The courts have had to adapt to the unprecedented change in circumstances. One of the most dramatic changes to occur was the introduction of remote court hearings. This report, jointly authored by the Open University and Northumbria University, seeks to explore this transition and the impact it has had on access to justice. The Open University Policy Clinic is part of the Open Justice Centre. Open Justice provides free legal advice and education to members of the public in a variety of legal areas. In 2019, Open Justice established the Policy Clinic to undertake research on behalf of organisations and charities, aiming to influence policy and law reform. The Northumbria Student Law Office was set up in 1981 and offers free legal advice to members of the public on a range of different areas. The Policy Clinic within the Student Law Office was set up in the 2018/2019 academic year with the aim to undertake research influencing policy and law reform. We began our research by gathering data from a sample of 80 Crown Courts and 77 County Courts. In addition to this, we conducted fieldwork in the Court of Protection. This involved all members of the team requesting access as public observers to remote hearings. In total, 25 hearings were attended. There was also a review of existing literature surrounding remote hearings. This report will identify the themes arising from our research that have a direct influence on access to justice. We will discuss the findings from our research alongside the existing data, and comparisons will be made between these two data sets. We believe this report is particularly important as we enter a post-COVID-19 world and questions are being raised as to whether the changes that have occurred during the pandemic should be implemented permanently. The aim of this report is to gain an insight into the effect of the pandemic and to assess the practicality of online courts

Virological failure and development of new resistance mutations according to CD4 count at combination antiretroviral therapy initiation

Objectives: No randomized controlled trials have yet reported an individual patient benefit of initiating combination antiretroviral therapy (cART) at CD4 counts > 350 cells/μL. It is hypothesized that earlier initiation of cART in asymptomatic and otherwise healthy individuals may lead to poorer adherence and subsequently higher rates of resistance development. Methods: In a large cohort of HIV-positive individuals, we investigated the emergence of new resistance mutations upon virological treatment failure according to the CD4 count at the initiation of cART. Results: Of 7918 included individuals, 6514 (82.3%), 996 (12.6%) and 408 (5.2%) started cART with a CD4 count ≤ 350, 351-499 and ≥ 500 cells/μL, respectively. Virological rebound occurred while on cART in 488 (7.5%), 46 (4.6%) and 30 (7.4%) with a baseline CD4 count ≤ 350, 351-499 and ≥ 500 cells/μL, respectively. Only four (13.0%) individuals with a baseline CD4 count > 350 cells/μL in receipt of a resistance test at viral load rebound were found to have developed new resistance mutations. This compared to 107 (41.2%) of those with virological failure who had initiated cART with a CD4 count < 350 cells/μL. Conclusions: We found no evidence of increased rates of resistance development when cART was initiated at CD4 counts above 350 cells/μL. HIV Medicin