Low expression and secretion of circulating soluble CTLA-4 in peripheral blood mononuclear cells and sera from type 1 diabetic children.

BACKGROUND: High levels of soluble cytotoxic T-lymphocyte antigen 4 (soluble CTLA-4), an alternative splice form of the regulatory T-cell (Treg) associated CTLA-4 gene, have been associated with type 1 diabetes (T1D) and other autoimmune diseases, such as Grave's disease and myasthenia gravis. At the same time, studies have shown soluble CTLA-4 to inhibit T-cell activation through B7 binding. This study aimed to investigate the role of soluble CTLA-4 in relation to full-length CTLA-4 and other Treg-associated markers in T1D children and in individuals with high or low risk of developing the disease. METHODS: T1D children were studied at 4 days, 1 and 2 years after diagnosis in comparison to individuals with high or low risk of developing the disease. Isolated peripheral blood mononuclear cells were stimulated with the T1D-associated glutamic acid decarboxylase 65 and phytohaemagglutinin. Subsequently, soluble CTLA-4, full-length CTLA-4, FOXP3 and TGF-ß mRNA transcription were quantified and protein concentrations of soluble CTLA-4 were measured in culture supernatant and sera. RESULTS AND CONCLUSIONS: Low protein concentrations of circulating soluble CTLA-4 and a positive correlation between soluble CTLA-4 mRNA and protein were seen in T1D, in parallel with a negative correlation in healthy subjects. Further, low levels of mitogen-induced soluble CTLA-4 were accompanied by low C-peptide levels. Interestingly, low mitogen-induced soluble CTLA-4 mRNA and low TGF-ß mRNA expression were seen in high risk individuals, suggesting an alteration in activation and down-regulating immune mechanisms during the pre-diabetic phase.

Differential glucose-regulation of microRNAs in pancreatic islets of non-obese type 2 diabetes model Goto-Kakizaki rat.

BACKGROUND: The Goto-Kakizaki (GK) rat is a well-studied non-obese spontaneous type 2 diabetes (T2D) animal model characterized by impaired glucose-stimulated insulin secretion (GSIS) in the pancreatic beta cells. MicroRNAs (miRNAs) are short regulatory RNAs involved in many fundamental biological processes. We aim to identify miRNAs that are differentially-expressed in the pancreatic islets of the GK rats and investigate both their short- and long term glucose-dependence during glucose-stimulatory conditions. METHODOLOGY/PRINCIPAL FINDINGS: Global profiling of 348 miRNAs in the islets of GK rats and Wistar controls (females, 60 days, N = 6 for both sets) using locked nucleic acid (LNA)-based microarrays allowed for the clear separation of the two groups. Significant analysis of microarrays (SAM) identified 30 differentially-expressed miRNAs, 24 of which are predominantly upregulated in the GK rat islets. Monitoring of qPCR-validated miRNAs during GSIS experiments on isolated islets showed disparate expression trajectories between GK and controls indicating distinct short- and long-term glucose dependence. We specifically found expression of rno-miR-130a, rno-miR-132, rno-miR-212 and rno-miR-335 to be regulated by hyperglycaemia. The putative targets of upregulated miRNAs in the GK, filtered with glucose-regulated mRNAs, were found to be enriched for insulin-secretion genes known to be downregulated in T2D patients. Finally, the binding of rno-miR-335 to a fragment of the 3'UTR of one of known down-regulated exocytotic genes in GK islets, Stxbp1 was shown by luciferase assay. CONCLUSIONS/SIGNIFICANCE: The perturbed miRNA network found in the GK rat islets is indicative of a system-wide impairment in the regulation of genes important for the normal functions of pancreatic islets, particularly in processes involving insulin secretion during glucose stimulatory conditions. Our findings suggest that the reduced insulin secretion observed in the GK rat may be partly due to upregulated miRNA expression leading to decreased production of key proteins of the insulin exocytotic machinery.

Differences in islet-enriched miRNAs in healthy and glucose intolerant human subjects.

Many microRNAs (miRNAs) are known to be cell-type specific and are implicated in development of diseases. We investigated the global expression pattern of miRNAs in human pancreatic islets compared to liver and skeletal muscle, using bead-based technology and quantitative RT-PCR. In addition to the known islet-specific miR-375, we also found enrichment of miR-127-3p, miR-184, miR-195 and miR-493∗ in the pancreatic islets. The expression of miR-375, miR-127-3p, miR-184 and the liver-enriched miR-122 is positively correlated to insulin biosynthesis, while the expression of miR-127-3p and miR-184 is negatively correlated to glucose-stimulated insulin secretion (GSIS). These correlations were absent in islets of glucose intolerant donors (HbA1c ≥ 6.1). We suggest that the presence of an islet-specific miRNA network, which consists of at least miR-375, miR-127-3p and miR-184, potentially involved in insulin secretion. Our results provide new insight into miRNA-mediated regulation of insulin secretion in healthy and glucose intolerant subjects.

Beta-cell specific deletion of Dicer1 leads to defective insulin secretion and diabetes mellitus.

Mature microRNAs (miRNAs), derived through cleavage of pre-miRNAs by the Dicer1 enzyme, regulate protein expression in many cell-types including cells in the pancreatic islets of Langerhans. To investigate the importance of miRNAs in mouse insulin secreting ß-cells, we have generated mice with a ß-cells specific disruption of the Dicer1 gene using the Cre-lox system controlled by the rat insulin promoter (RIP). In contrast to their normoglycaemic control littermates (RIP-Cre(+/-) Dicer1(Δ/wt)), RIP-Cre(+/-)Dicer1(flox/flox) mice (RIP-Cre Dicer1(Δ/Δ)) developed progressive hyperglycaemia and full-blown diabetes mellitus in adulthood that recapitulated the natural history of the spontaneous disease in mice. Reduced insulin gene expression and concomitant reduced insulin secretion preceded the hyperglycaemic state and diabetes development. Immunohistochemical, flow cytometric and ultrastructural analyses revealed altered islet morphology, marked decreased ß-cell mass, reduced numbers of granules within the ß-cells and reduced granule docking in adult RIP-Cre Dicer1(Δ/Δ) mice. ß-cell specific Dicer1 deletion did not appear to disrupt fetal and neonatal ß-cell development as 2-week old RIP-Cre Dicer1(Δ/Δ) mice showed ultrastructurally normal ß-cells and intact insulin secretion. In conclusion, we have demonstrated that a ß-cell specific disruption of the miRNAs network, although allowing for apparently normal ß-cell development, leads to progressive impairment of insulin secretion, glucose homeostasis and diabetes development.