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We assessed the levels of blood circulating immune checkpoint molecules (ICMs) at diagnosis of type 1 diabetes, and determined their association with the risk of developing an additional autoimmune disorder over time. Children with new-onset type 1 diabetes (n = 143), without biological and/or clinical signs of additional autoimmune disorders, and healthy children (n = 75) were enrolled, and blood circulating levels of 14 ICMs were measured. The children with type 1 diabetes were divided into two groups on the basis of the development of an additional autoimmune disease in the 5 years after diabetes onset. Differences in soluble ICM levels between the groups were assessed, and a Cox regression analysis was used to evaluate their association with the risk of development of an additional autoimmune disease over time. To validate the data, circulating ICMs were measured in an independent cohort of 60 children with new-onset type 1 diabetes stratified into two groups. We found that the levels of circulating ICMs were significantly higher in children with new-onset diabetes compared with healthy children. Further, we observed that children with type 1 diabetes who developed a second autoimmune disease over time (T1D-AAD+ children) had higher levels of soluble ICMs than children with type 1 diabetes who did not (T1D-AAD− children). Cox regression models revealed that high circulating levels of CD137/4-1BB and PD-1 molecules at diabetes diagnosis were associated with the risk of developing an additional autoimmune disease in both type 1 diabetes cohorts. Our findings suggest that soluble CD137/4-1BB and PD-1 molecules may be used as prognostic biomarkers in children with type 1 diabetes, and may pave the way for novel immunological screening at diabetes onset, allowing early identification of children at higher risk of developing other autoimmune conditions over time.

Diabetes mellitus causes a progressive loss of functional efficacy in stem cells, including cardiac progenitor cells (CPCs). The underlying molecular mechanism is still not known. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate genes at the post-transcriptional level. We aimed to determine if diabetes mellitus induces dysregulation of miRNAs in CPCs and to test if in vitro therapeutic modulation of miRNAs would improve the functions of diabetic CPCs. CPCs were isolated from a mouse model of type 2 diabetes (db/db), non-diabetic mice and human right atrial appendage heart tissue. Total RNA isolated from mouse CPCs was miRNA profiled using Nanostring analysis. Bioinformatic analysis was employed to predict the functional effects of altered miRNAs. MS analysis was applied to determine the targets, which were confirmed by western blot analysis. Finally, to assess the beneficial effects of therapeutic modulation of miRNAs in vitro and in vivo, prosurvival miR-30c-5p was overexpressed in mouse and human diabetic CPCs, and the functional consequences were determined by measuring the level of apoptotic cell death, cardiac function and mitochondrial membrane potential (MMP). Among 599 miRNAs analysed in mouse CPCs via Nanostring analysis, 16 miRNAs showed significant dysregulation in the diabetic CPCs. Using bioinformatics tools and quantitative real-time PCR (qPCR) validation, four altered miRNAs (miR-30c-5p, miR-329-3p, miR-376c-3p and miR-495-3p) were identified to play an important role in cell proliferation and survival. Diabetes mellitus significantly downregulated miR-30c-5p, while it upregulated miR-329-3p, miR-376c-3p and miR-495-3p. MS analysis revealed proapoptotic voltage-dependent anion-selective channel 1 (VDAC1) as a direct target for miR-30c-5p, and cell cycle regulator, cyclin-dependent protein kinase 6 (CDK6), as the direct target for miR-329-3p, miR-376c-3p and miR-495-3p. Western blot analyses showed a marked increase in VDAC1 expression, while CDK6 expression was downregulated in diabetic CPCs. Finally, in vitro and in vivo overexpression of miR-30c-5p markedly reduced the apoptotic cell death and preserved MMP in diabetic CPCs via inhibition of VDAC1. Our results demonstrate that diabetes mellitus induces a marked dysregulation of miRNAs associated with stem cell survival, proliferation and differentiation, and that therapeutic overexpression of prosurvival miR-30c-5p reduced diabetes-induced cell death and loss of MMP in CPCs via the newly identified target for miR-30c-5p, VDAC1.

A method has been developed for the simultaneous measurement of insulin release and electrical activity in single micro-dissected mouse islets of Langerhans. The effects of D-glucose have been studied in individual islets. Each islet was exposed to 0, 5.6, 11.1, 16.7, 22.2, 27.8 and 33.3 mmol/l glucose in a stepwise fashion. The minimum glucose concentration required to elicit spike activity is lower than that required to stimulate insulin release above basal levels and the maximum spike frequency occurs at a lower glucose concentration than does maximum insulin release. Following a reduction in glucose from 27.8 (or 33.3) to 5.6 mmol/l, membrane potentials returned to resting values within 2 min whereas insulin returned to basal values after 20 min. Increasing glucose from 5.6 to 27.8 mmol/l induced spike activity within 10 s; the insulin response was detected within 40 s. Thus, it is possible to use the single mouse islet for simultaneous measurements of insulin release and electrical activity.

Per-Arnt-Sim kinase (PASK) is a nutrient-regulated domain-containing protein kinase previously implicated in the control of insulin gene expression and glucagon secretion. Here, we explore the roles of PASK in the control of islet hormone release, by generating mice with selective deletion of the Pask gene in pancreatic beta or alpha cells. Floxed alleles of Pask were produced by homologous recombination and animals bred with mice bearing beta (Ins1 Cre; PaskBKO) or alpha (Ppg Cre [also known as Gcg]; PaskAKO) cell-selective Cre recombinase alleles. Glucose homeostasis and hormone secretion in vivo and in vitro, gene expression and islet cell mass were measured using standard techniques. Ins1 Cre-based recombination led to efficient beta cell-targeted deletion of Pask. Beta cell mass was reduced by 36.5% (p < 0.05) compared with controls in PaskBKO mice, as well as in global Pask-null mice (38%, p < 0.05). PaskBKO mice displayed normal body weight and fasting glycaemia, but slightly impaired glucose tolerance, and beta cell proliferation, after maintenance on a high-fat diet. Whilst glucose tolerance was unaffected in PaskAKO mice, glucose infusion rates were increased, and glucagon secretion tended to be lower, during hypoglycaemic clamps. Although alpha cell mass was increased (21.9%, p < 0.05), glucagon release at low glucose was impaired (p < 0.05) in PaskAKO islets. The findings demonstrate cell-autonomous roles for PASK in the control of pancreatic endocrine hormone secretion. Differences between the glycaemic phenotype of global vs cell type-specific null mice suggest important roles for tissue interactions in the control of glycaemia by PASK.

Aims/hypothesis. The World Health Organisation Consultation recommended new diagnostic criteria for diabetes mellitus including: lowering of the diagnostic fasting plasma glucose to 7.0 mmol/l and introduction of a new category: impaired fasting glycaemia. The diagnostic 2-h glucose concentrations for diabetes and for impaired glucose tolerance were unchanged. This study identifies fasting plasma glucose concentrations predicting a diabetic 2-h plasma glucose of 11.1 mmol/l or more, analyses the sensitivity and specificity of different screening strategies for diabetes and describes the cardiovascular risk profile in people with impaired fasting glycaemia. Methods. European population based studies (n = 17) or large, representative samples of employees (n = 3) with both fasting and 2-h post load glucose concentrations following 75-g oral glucose tolerance tests were included (18 918 men and 10 190 women). The Iceland study (8881 men and 9407 women) is presented separately as a 50-g glucose load was used. Results. The fasting plasma glucose predicting a 2-h plasma glucose of 11.1 mmol/l or more with optimal sensitivity and specificity was a) 5.8 mmol/l in women and 6.4 mmol/l in men; b) independent of age; c) increased with obesity. Fasting plasma glucose of 7.0/7.8 mmol/l or more predicted a diabetic 2-h plasma glucose with sensitivities of 49.0/29.8 % and specificities of 98.2/99.7 %, respectively. Conclusion/interpretation. If fasting glucose is used alone, the 31 % of diabetic subjects with a non-diabetic fasting glucose but a diabetic 2-h glucose, will not be diagnosed; impaired fasting glycaemia and impaired glucose tolerance do not identify the same people; the risk profile of people with impaired fasting glycaemia depends on 2-h glucose concentrations. Obesity is the main confounder in the association between fasting and 2-h glucose. [Diabetologia (1999) 42: 647–654]