Vascular endothelial PDPK1 plays a pivotal role in the maintenance of pancreatic beta cell mass and function in adult male mice It has been brought to our attention that Fig. 5a showing the vasculature in islets of control flox mice is not in fact an endocrine cell but rather exocrine tissue.

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Extracellular vesicles in metabolic disease Abstract Extracellular vesicles (EVs) are submicron-sized lipid envelopes that are produced and released from a parent cell and can be taken up by a recipient cell. EVs are capable of mediating cellular signalling by carrying nucleic acids, proteins, lipids and cellular metabolites between cells and organs. Metabolic dysfunction is associated with changes in plasma concentrations of EVs as well as alterations in their EV cargo. Since EVs can act as messengers between parent and recipient cells, they could be involved in cell-to-cell and organ-to-organ communication in metabolic diseases. Recent literature has shown that EVs are produced by cells within metabolic tissues, such as adipose tissue, pancreas, muscle and liver. These vesicles have therefore been proposed as a novel intercellular communication mode in systemic metabolic regulation. In this review, we will describe and discuss the current literature that investigates the role of adipose-derived EVs in the regulation of obesity-associated metabolic disease. We will particularly focus on the EV-dependent communication between adipocytes, the vasculature and immune cells in type 2 diabetes.

White coat hypertension in early pregnancy in women with pre-existing diabetes: prevalence and pregnancy outcomes Abstract Aims/hypothesis Hypertensive disorders are prevalent among pregnant women with pre-existing diabetes, but the prevalence and impact of white coat hypertension are unknown. Measurement of home BP before initiation of antihypertensive treatment is necessary to identify white coat hypertension since international guidelines recommend that white coat hypertension is left untreated. The aim of this study, conducted among women with pre-existing diabetes, was therefore to examine the prevalence of white coat hypertension in early pregnancy, and pregnancy outcome in women with white coat hypertension in early pregnancy. Methods A prospective cohort study was undertaken involving women with pre-existing diabetes from a geographically well-defined area. Based on office BP in early pregnancy and home BP measured for 3 days, women were categorised in three groups: (1) white coat hypertension, defined as office BP ≥ 135/85 mmHg and mean home BP < 130/80 mmHg; (2) chronic hypertension, defined as pre-pregnancy hypertension including newly detected office BP ≥ 135/85 mmHg with home BP ≥ 130/80 mmHg; and (3) normotension. Office BP was measured every 2 weeks and, if ≥ 135/85 mmHg, home BP measurements were performed. White coat hypertension was left untreated, and tight antihypertensive treatment was initiated when both office BP ≥ 135/85 mmHg and home BP ≥ 130/80 mmHg. Pregnancy-induced hypertensive disorders were defined as office BP ≥ 140/90 mmHg with home BP ≥ 130/80 mmHg when available, with onset after 20 weeks of gestation. Results In total, 32 out of 222 women with pre-existing diabetes had newly detected office BP ≥ 135/85 mmHg in early pregnancy. White coat hypertension was present in 84% (27/32) of these women, representing 12% (95% CI 8%, 17%) of the whole cohort. Chronic hypertension was present in 14% (n = 32) and normotension in 74% (n = 163). Women with white coat hypertension were characterised by higher pre-pregnancy BMI (p = 0.011), higher home BP (p < 0.001) and higher prevalence of type 2 diabetes (p = 0.009), but similar HbA1c (p = 0.409) compared to women with normotension. Regarding pregnancy outcome, pregnancy-induced hypertensive disorders developed in 44% (12/27) of women with white coat hypertension in comparison with 22% (36/163) among initially normotensive women (p = 0.013), while the prevalence of preterm delivery was comparable (p = 0.143). The adjusted analysis, performed post hoc, suggested approximately double the risk of developing pregnancy-induced hypertensive disorders (OR 2.43 [CI 0.98, 6.05]) if white coat hypertension was present in early pregnancy, independently of pre-pregnancy BMI and parity. Conclusions/interpretation White coat hypertension is prevalent in women with pre-existing diabetes and may indicate a high risk of later development of pregnancy-induced hypertensive disorders. To distinguish between persistent white coat hypertension and onset of pregnancy-induced hypertension, repeated home BP monitoring is recommended when elevated office BP is detected. The study was registered at ClinicalTrials.gov (ID: NCT02890836).

Deficiency in AIM2 induces inflammation and adipogenesis in white adipose tissue leading to obesity and insulin resistance Abstract Aims/hypothesis Absent in melanoma 2 (AIM2) is a cytosolic sensor for double-stranded DNA and a tumour suppressor. Binding of double-stranded DNA to AIM2 forms the AIM2 inflammasome, leading to activation of caspase-1 and production of IL-1β and IL-18. Although inflammasome-independent effects of AIM2 have been reported, its role in energy metabolism is unknown. We aimed to evaluate the effect of AIM2 in energy metabolism and glucose homeostasis. Methods Male and female whole body Aim2 knockout (Aim2−/−) mice were used in the current study. Body weight, food intake, body composition, energy expenditure, fasting blood glucose levels, GTT and body temperature were measured at indicated time points. RNA sequencing was carried out on gonadal white adipose tissue (gWAT) in 14-month-old female mice. mRNA and protein levels in tissues were analysed by quantitative real-time PCR and immunoblot. Immune cell infiltration in gWAT was examined by flow cytometry. Stromal vascular fractions isolated from gWAT were used to investigate adipocyte differentiation. Results Male and female Aim2−/− mice were obese compared with wild-type controls from 7 weeks of age until 51 weeks of age, with increased adiposity in both subcutaneous and visceral fat depots. While there were no differences in food intake, Aim2−/− mice demonstrated decreased energy expenditure and impaired brown adipose tissue function compared with wild-type controls. Fasting glucose and insulin levels were elevated, and Aim2−/− mice were glucose intolerant on intraperitoneal GTT. RNA sequencing revealed marked upregulation of the IFN-inducible gene Ifi202b, which encodes protein 202 (p202) and elevated inflammatory signalling in gWAT of Aim2−/− mice. Increased infiltration of total and Ly6Clow monocytes was noted at 8 weeks of age in gWAT, before the onset of obesity and insulin resistance. Ifi202b knockdown blocked adipogenesis in stromal vascular fractions and reduced inflammation in bone marrow-derived macrophages, demonstrating a key role of p202 in mediating the increased adipogenesis and inflammation in Aim2−/− mice. Conclusions/interpretation These results demonstrate a fundamental role for AIM2 in energy metabolism, inflammation and insulin resistance. Our studies establish a novel link between the innate immunity proteins, AIM2 and p202, and metabolism.

Induced pluripotent stem cell macrophages present antigen to proinsulin-specific T cell receptors from donor-matched islet-infiltrating T cells in type 1 diabetes Abstract Aims/hypothesis Type 1 diabetes is an autoimmune disorder characterised by loss of insulin-producing beta cells of the pancreas. Progress in understanding the cellular and molecular mechanisms underlying the human disease has been hampered by a dearth of appropriate human experimental models. We previously reported the characterisation of islet-infiltrating CD4+ T cells from a deceased organ donor who had type 1 diabetes. Methods Induced pluripotent stem cell (iPSC) lines derived from the above donor were differentiated into CD14+ macrophages and tested for their capacity to present antigen to T cell receptors (TCRs) derived from islet-infiltrating CD4+ T cells from the same donor. Results The iPSC macrophages displayed typical macrophage morphology, surface markers (CD14, CD86, CD16 and CD11b) and were phagocytic. In response to IFNγ treatment, iPSC macrophages upregulated expression of HLA class II, a characteristic that correlated with their capacity to present epitopes derived from proinsulin C-peptide to a T cell line expressing TCRs derived from islet-infiltrating CD4+ T cells of the original donor. T cell activation was specifically blocked by anti-HLA-DQ antibodies but not by antibodies directed against HLA-DR. Conclusions/interpretation This study provides a proof of principle for the use of iPSC-derived immune cells for modelling key cellular interactions in human type 1 diabetes.

Mechanisms of hyperinsulinaemia in apparently healthy non-obese young adults: role of insulin secretion, clearance and action and associations with plasma amino acids Abstract Aims/hypothesis This study aimed to examine the metabolic health of young apparently healthy non-obese adults to better understand mechanisms of hyperinsulinaemia. Methods Non-obese (BMI < 30 kg/m2) adults aged 18–35 years (N = 254) underwent a stable isotope-labelled OGTT. Insulin sensitivity, glucose effectiveness and beta cell function were determined using oral minimal models. Individuals were stratified into quartiles based on their insulin response during the OGTT, with quartile 1 having the lowest and quartile 4 the highest responses. Results Thirteen per cent of individuals had impaired fasting glucose (IFG; n = 14) or impaired glucose tolerance (IGT; n = 19), allowing comparisons across the continuum of insulin responses within the spectrum of normoglycaemia and prediabetes. BMI (~24 kg/m2) was similar across insulin quartiles and in those with IFG and IGT. Despite similar glycaemic excursions, fasting insulin, triacylglycerols and cholesterol were elevated in quartile 4. Insulin sensitivity was lowest in quartile 4, and accompanied by increased insulin secretion and reduced insulin clearance. Individuals with IFG had similar insulin sensitivity and beta cell function to those in quartiles 2 and 3, but were more insulin sensitive than individuals in quartile 4. While individuals with IGT had a similar degree of insulin resistance to quartile 4, they exhibited a more severe defect in beta cell function. Plasma branched-chain amino acids were not elevated in quartile 4, IFG or IGT. Conclusions/interpretation Hyperinsulinaemia within normoglycaemic young, non-obese adults manifests due to increased insulin secretion and reduced insulin clearance. Individual phenotypic characterisation revealed that the most hyperinsulinaemic were more similar to individuals with IGT than IFG, suggesting that hyperinsulinaemic individuals may be on the continuum toward IGT. Furthermore, plasma branched-chain amino acids may not be an effective biomarker in identifying hyperinsulinaemia and insulin resistance in young non-obese adults.

Adipocyte-specific disruption of ATPase copper transporting α in mice accelerates lipoatrophy Abstract Aims/hypothesis ATPase copper transporting α (ATP7A), also known as Menkes disease protein, is a P-type ATPase that transports copper across cell membranes. The critical role of ATP7A-mediated copper homeostasis has been well recognised in various organs, such as the intestine, macrophages and the nervous system. However, the importance of adipocyte ATP7A-mediated copper homeostasis on fat metabolism is not well understood. Here, we sought to reveal the contribution of adipose ATP7A to whole-body fat metabolism in mice. Methods We generated adipocyte-specific Atp7a-knockout (ASKO) mice using the Cre/loxP system, with Cre expression driven by the adiponectin promoter. ASKO mice and littermate control mice were aged on a chow diet or fed with a high-fat diet (HFD); body weight, fat mass, and glucose and insulin metabolism were analysed. Histological analysis, transmission electron microscopy and RNA-sequencing (RNA-Seq) analysis of white adipose tissue (WAT) were used to understand the physiological and molecular changes associated with loss of copper homeostasis in adipocytes. Results Significantly increased copper concentrations were observed in adipose tissues of ASKO mice compared with control mice. Aged or HFD-fed ASKO mice manifested a lipoatrophic phenotype characterised by a progressive generalised loss of WAT. Dysfunction of adipose tissues in these ASKO mice was confirmed by decreased levels of both serum leptin and adiponectin and increased levels of triacylglycerol and insulin. Systemic metabolism was also impaired in these mice, as evidenced by a pronounced glucose intolerance, insulin resistance and hepatic steatosis. Moreover, we demonstrate a significant induction of lipolysis and DNA-damage signalling pathways in gonadal WAT from aged and HFD-fed ASKO mice. In vitro studies suggest that copper overload is responsible for increased lipolysis and DNA damage. Conclusions/interpretation Our results show a previously unappreciated role of adipocyte Atp7a in the regulation of ageing-related metabolic disease and identify new metallophysiologies in whole-body fat metabolism. Data availability The datasets generated during the current study are available in the Genome Sequence Archive in BIG Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession number CRA001769 (http://bigd.big.ac.cn/gsa).

Short-term strength and balance training does not improve quality of life but improves functional status in individuals with diabetic peripheral neuropathy: a randomised controlled trial Abstract Aims/hypothesis The aim of this study was to test the effectiveness of a structured strength and balance training intervention in improving health-related quality of life (HRQoL) and functional status in individuals with diabetic peripheral neuropathy (DPN). Methods The study was a single-blind parallel-group randomised controlled trial comparing 2 months of once-weekly home-based strength and balance training against standard medical therapy. Participants were patients with physician-diagnosed type 2 diabetes and neuropathy recruited from five public sector institutions in Singapore between July 2014 and October 2017. Participants were block-randomised to intervention or control arms. Outcomes were assessed at baseline, 2 months and 6 months by a trained assessor blinded to group assignment. Primary outcomes were change in physical component summary (PCS) score of SF-36v2 (a 36-item generic HRQoL instrument that has been validated for use in Singapore) and EQ-5D-5L index score (derived from a five-item generic HRQoL instrument [EQ-5D-5L]) over 6 months. Secondary outcomes were change in functional status (timed up-and-go [TUG], five times sit-to-stand [FTSTS], functional reach, static balance, ankle muscle strength and knee range of motion) and balance confidence over 6 months. Mean differences in scores between groups were compared using mixed models. Results Of the 143 participants randomised (intervention, n = 70; control, n = 73), 67 participants were included in each arm for the final intention-to-treat analysis. The two groups were similar, except in terms of sex. There were no significant differences between groups on the primary outcomes of PCS score (mean difference [MD] 1.56 [95% CI −1.75, 4.87]; p = 0.355) and EQ-5D-5L index score (MD 0.02 [95% CI −0.01, 0.06]; p = 0.175). There were significant improvements in TUG test performance (MD −1.14 [95% CI −2.18, −0.1] s; p = 0.032), FTSTS test performance (MD −1.31 [95% CI −2.12, −0.51] s; p = 0.001), ankle muscle strength (MD 4.18 [95% CI 0.4, 7.92] N; p = 0.031), knee range of motion (MD 6.82 [95% CI 2.87, 10.78]°; p = 0.001) and balance confidence score (MD 6.17 [95% CI 1.89, 10.44]; p = 0.005). No adverse events due to study participation or study intervention were reported. Conclusions/interpretation Short-term structured strength and balance training did not influence HRQoL but produced sustained improvements in functional status and balance confidence at 6 months. More intensive interventions may be needed to influence HRQoL in these individuals. However, this intervention may be a useful treatment option for individuals with DPN to reduce the risk of falls and injuries. Trial registration ClinicalTrials.gov NCT02115932 Funding This work was supported by the National Medical Research Council, Singapore.

Neutrophil elastase contributes to the pathological vascular permeability characteristic of diabetic retinopathy Abstract Aims/hypothesis Levels of neutrophil elastase, a serine protease secreted by neutrophils, are elevated in diabetes. The purpose of this study was to determine whether neutrophil elastase (NE) contributes to the diabetes-induced increase in retinal vascular permeability in mice with streptozotocin-induced diabetes, and, if so, to investigate the potential role of IL-17 in this process. Methods In vivo, diabetes was induced in neutrophil elastase-deficient (Elane−/−), Il-17a−/− and wild-type mice. After 8 months of diabetes, Elane−/− mice and wild-type age-matched control mice were injected with FITC-BSA. Fluorescence microscopy was used to assess leakage of FITC-BSA from the retinal vasculature into the neural retina. The level of NE in Il-17a−/− diabetic retina and sera were determined by ELISA. In vitro, the effect of NE on the permeability and viability of human retinal endothelial cells and the expression of junction proteins and adhesion molecules were studied. Results Eight months of diabetes resulted in increased retinal vascular permeability and levels of NE in retina and plasma of wild-type animals. All of these abnormalities were significantly inhibited in mice lacking the elastase. The diabetes-induced increase in NE was inhibited in mice lacking IL-17. In vitro, NE increased retinal endothelial cell permeability, which was partially inhibited by a myeloid differentiation primary response 88 (MyD88) inhibitor, NF-κB inhibitor, and protease-activated receptor (PAR)2 inhibitor. NE degraded vascular endothelial-cadherin (VE-cadherin) in a concentration-dependent manner. Conclusions/interpretation IL-17 regulates NE expression in diabetes. NE contributes to vascular leakage in diabetic retinopathy, partially through activation of MyD88, NF-κB and PAR2 and degradation of VE-cadherin.